TRS 979 62nd Report

979
W H O Te c h n i c a l R e p o r t S e r i e s
979
WHO Expert Committee on Biological Standardization

This report presents the recommendations of a WHO expert
committee commissioned to coordinate activities leading
to the adoption of international recommendations for the
production and control of vaccines and other biologicals
and the establishment of international biological reference
materials.
Following a brief introduction, the report summarizes a
number of general issues brought to the attention of the
Committee. The next part of the report, of particular relevance
to manufacturers and national regulatory authorities, sets out
WHO Expert Committee
revised WHO Guidelines on the quality, safety and efficacy
of candidate dengue tetravalent vaccines (live, attenuated),
along with revised WHO Recommendations in relation to the
on Biological
production and quality control of bacille Calmette–Guérin
(BCG) vaccines and of acellular pertussis vaccines. In addition,
a generic protocol for the calibration of seasonal and pandemic
Standardization
influenza antigen working reagents is included. Revised WHO
Guidelines for thromboplastins and plasma used to control
oral anticoagulant therapy with vitamin K antagonists are then
presented. Finally, new WHO assessment criteria for national Sixty-second report
blood regulatory systems are provided.
Subsequent sections of the report then provide information on
the current status and proposed development of international
reference materials in the areas of antibiotics; biotherapeutics
WHO Technical Report Series

other than blood products; blood products and related
substances; in vitro diagnostic device reagents; and vaccines
and related substances.
A series of annexes are then presented which include an
updated list of WHO Recommendations, Guidelines and
other documents on biological substances used in medicine
(Annex 1), followed by a series of WHO Recommendations
and Guidelines adopted on the advice of the Committee
(Annexes 2–7). All additions made during the meeting to
the list of International Standards and Reference Reagents
for biological substances maintained by WHO are then
summarized in Annex 8, and are also available at: http://www.
who.int/biologicals.
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W H O Te c h n i c a l R e p o r t S e r i e s
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WHO Expert Committee

on Biological
Standardization
Sixty-second report
This report contains the collective views of an international group of experts and
does not necessarily represent the decisions or the stated policy of the World Health Organization
WHO Library Cataloguing-in-Publication Data
WHO Expert Committee on Biological Standardization, sixty-second report.
(WHO technical report series ; no. 979)
1. Biological products - standards. 2. Vaccines - standards. 3. Reference standards.
4. Guidelines. I.World Health Organization. II.WHO Expert Committee on Biological
Standardization (2011: Geneva, Switzerland). III.Series.
ISBN 978 92 4 120979 3 (NLM classification: QW 800)
ISBN 978 92 4 069113 1 (e-book)
ISSN 0512-3054
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Printed in Italy
Contents
Abbreviations xi
1. Introduction 1
2. General 2
2.1 Current directions 2
2.1.1 Strategic directions in biological standardization 2
2.1.2 Vaccines and biological therapeutics: recent and planned activities in
biological standardization 3
2.1.3 Blood products and related in vitro diagnostics: recent and planned activities
in biological standardization 5
2.2 Reports 6
2.2.1 Report of the WHO Blood Regulators Network 6
2.2.2 Reports from international laboratories and WHO collaborating centres for
biological standards 7
2.3 Issues 10
2.3.1 Scientific issues identified by users of WHO biological Reference Preparations 10
2.3.2 Issues shared with the WHO Expert Committee on Specifications for
Pharmaceutical Preparations 11
3. International Recommendations, Guidelines and other matters
related to the manufacture and quality control of biologicals 13
3.1 Vaccines and related substances 13
3.1.1 Guidelines on the quality, safety and efficacy of dengue tetravalent vaccines
(live, attenuated) 13
3.1.2 Recommendations to assure the quality, safety and efficacy of BCG vaccines 13
3.1.3 Recommendations to assure the quality, safety and efficacy of acellular
pertussis vaccines 14
3.1.4 Assessing risk when a potential adventitious agent is found 15
3.1.5 Calibration of seasonal and pandemic influenza antigen working reagents
by WHO essential regulatory laboratories 16
3.1.6 Proposed new projects for developing or updating written standards 16
3.2 Blood products and related substances 17
3.2.1 Guidelines for thromboplastins and plasma used to control oral
anticoagulant therapy with vitamin K antagonists 17
3.2.2 Assessment criteria for national blood regulatory systems 18
4. International reference materials – vaccines and related substances 20
4.1 WHO International Standards and Reference Reagents – vaccines and related
substances 20
4.1.1 First WHO International Standard for meningococcal serogroup C
polysaccharide 20
4.1.2 First WHO International Standard for anti-pneumococcal antibodies
in serum 20
4.1.3 Third WHO International Standard for trivalent inactivated polio vaccine 21
4.2 Proposed new projects – vaccines and related substances 21
4.2.1 Replacement of the First WHO International Standard for antibody to
influenza A virus subtype H1N1pdm 21
iii
4.3 Ongoing stability monitoring – vaccines and related substances 22
4.3.1 Inactivated hepatitis A vaccine 22
4.4 Progress reports – vaccines and related substances 23
4.4.1 Characteristics of an improved potency assay for inactivated influenza
vaccines 23
4.4.2 Hepatitis B vaccine 23
5. International reference materials – blood products and related
substances 24
5.1 WHO International Standards and Reference Reagents – blood products and
related substances 24
5.1.1 Assignment of a value for von Willebrand factor propeptide to the
Sixth WHO International Standard for factor VIII/VWF plasma 24
5.1.2 Third WHO International Standard for fibrinogen (plasma) 24
5.1.3 First WHO International Reference Reagent for anti-human neutrophil
antigen-1a antibody 25
5.1.4 First WHO International Reference Reagents for blood group genotyping 26
5.1.5 Revision of the instructions for use of the First WHO International
Reference Repository for platelet transfusion relevant bacterial strains 26
5.1.6 Apolipoprotein B 27
5.2 Proposed new projects – blood products and related substances 28
5.2.1 Replacement of the Second WHO International Reference Preparation
for serum IgE 28
5.2.2 Replacement of the Third WHO International Standard for plasmin 28
5.2.3 Haemoglobin A2 29
6. International reference materials – in vitro diagnostic device reagents 30
6.1 WHO International Standards and Reference Reagents – in vitro diagnostic device
reagents 30
6.1.1 Third WHO International Standard for HIV-1 for NAT-based assays 30
6.1.2 Third WHO International Standard for hepatitis B virus for NAT-based assays 30
6.1.3 First WHO International Reference Panel for hepatitis B virus genotype
for HBsAg assays 31
6.1.4 Fourth WHO International Standard for hepatitis C virus for
NAT-based assays 32
6.1.5 First WHO International Standard for hepatitis E virus for NAT-based assays 33
6.1.6 First WHO International Reference Standard for anti-Trypanosoma cruzi
antibodies 34
6.1.7 First WHO International Standard for Epstein–Barr virus for NAT-based assays 35
6.2 Proposed new projects – in vitro diagnostic device reagents 36
6.2.1 Third Meeting of WHO Collaborating Centres for Biological Standards 36
6.2.2 Replacement of the First WHO International Standard for hepatitis A virus
for NAT-based assays 37
6.2.3 Replacement WHO International Standard for hepatitis B e-antigen 37
6.2.4 Replacement WHO International Standard for hepatitis B e-antibodies 38
6.2.5 First WHO International Reference Panel for hepatitis E virus genotypes
for NAT-based assays 38
6.2.6 Replacement of the First WHO International Reference Panel for
HIV-1 subtypes 39
6.2.7 First WHO International Reference Panel for HIV-1 circulating
recombinant forms 39
iv
6.3 Ongoing stability monitoring 39
6.3.1 Prostate-specific antigen 39
7. International reference materials – biotherapeutics other than
blood products 41
7.1 WHO International Standards and Reference Reagents – biotherapeutics other
than blood products 41
7.1.1 First WHO International Standard for transforming growth factor beta-3 41
7.2 Proposed new projects – biotherapeutics other than blood products 42
7.2.1 Replacement of the First WHO International Standard for interleukin-2 42
7.2.2 Replacement of the Second WHO International Standard for recombinant
human erythropoietin 42
7.2.3 Replacement of the Fourth WHO International Standard for urinary
follicle-stimulating hormone and urinary luteinizing hormone 43
7.2.4 Replacement of the Second WHO International Standard for luteinizing
hormone (human, pituitary) 43
8. International reference materials – antibiotics 45
8.1 WHO International Standards and Reference Reagents – antibiotics 45
8.1.1 Third WHO International Standard for dihydrostreptomycin 45
8.2 Proposed new projects – antibiotics 45
8.2.1 Replacement of the First WHO International Standard for neomycin B 45
8.2.2 Replacement of the Second WHO International Standard for neomycin 46
Annex 1
WHO Recommendations, Guidelines and other documents related to the manufacture
and quality control of biological substances used in medicine 47
Annex 2
Guidelines on the quality, safety and efficacy of dengue tetravalent vaccines
Annex 3
Recommendations to assure the quality, safety and efficacy of BCG vaccines 137
Annex 4
Recommendations to assure the quality, safety and efficacy of acellular pertussis vaccines 187
Annex 5
Generic protocol for the calibration of seasonal and pandemic influenza antigen
working reagents by WHO essential regulatory laboratories 261
Annex 6
Guidelines for thromboplastins and plasma used to control oral anticoagulant therapy
with vitamin K antagonists 271
Annex 7
Assessment criteria for national blood regulatory systems 317
Annex 8
Biological substances: WHO International Standards and Reference Reagents 365
v
WHO Expert Committee on Biological Standardization Sixty-second report

17 to 21 October 2011
Members1
Dr J. Epstein, Director, Office of Blood Research and Review, Center for Biologics Evaluation
and Research, Food and Drug Administration, Rockville, MD, United States of America
(USA)
Dr E. Griffiths, Director-General, Biologics and Genetic Therapies, Health Canada, Ottawa,
Ontario, Canada (Chairman)
Mrs T. Jivapaisarnpong, Director, Institute of Biological Products, Department of Medical
Sciences, Ministry of Public Health, Bangkok, Thailand
Dr H. Klein, Chief, Department of Transfusion Medicine, National Institutes of Health,
Bethesda, MD, USA
Dr P. Minor, Head, Division of Virology, National Institute for Biological Standards and
Control, Potters Bar, England
Dr F.M. Moftah, Director-General, National Blood Transfusion Service, Ministry of Health,
Giza, Egypt
Dr J. Petricciani, Palm Springs, CA, USA (Rapporteur)
Dr L.S. Slamet,2 Deputy for Therapeutic Products, Narcotic, Psychotropic and Addictive
Substance Control, Directorate General of Food and Drug Control of the Republic of
Indonesia, Jakarta, Indonesia
Dr P. Strengers, Medical Director, Division of Plasma Products, Sanquin, Amsterdam, the
Netherlands
Professor H. Yin, Deputy Director, Center for Drug Evaluation, State Food and Drug
Administration, Beijing, China
WHO Technical Report Series, No. 979, 2013
Representatives of other organizations

Chinese Pharmacopoeia Commission
Dr X. Hong, Biologics Division, Chinese Pharmacopoeia Commission, Beijing, China
Council of Europe, European Department for the Quality of Medicines & Healthcare
Dr K.H. Buchheit, Department of Biological Standardisation, OMCL Network and
HealthCare, Strasbourg, France
1
The decisions of the Committee were taken in closed session with only members of the Committee
present. Each Committee member had completed a declaration of interests form prior to the meeting.
These were assessed by the WHO Secretariat and no declared interests were considered to be a conflict for
full participation in the meeting.
2
Unable to attend.
vi
Dr E. Charton, Department of Biological Standardisation, OMCL Network and HealthCare,

Strasbourg, France
Developing Country Vaccine Manufacturers’ Network
Dr S. Jadhav, Executive Director, Quality Assurance & Regulatory Affairs, Serum Institute
of India Limited, Pune, India
European Diagnostic Manufacturers Association
Mrs C. Betremieux, Scientific Affairs Manager, Immuno Hematology Division, Bio-Rad
Laboratories, Cressier, Switzerland
European Medicines Agency
Dr N. Gate, Principal Scientist, European Medicines Agency, London, England
International Alliance of Biologicals
Professor J. Löwer, International Alliance of Biologicals, Dreieich, Germany
International Federation of Clinical Chemistry and Laboratory Medicine
Professor P. Gillery, Vice-chair, IFCC Scientific Division, Reims, France
International Federation of Pharmaceutical Manufacturers Associations
Dr A. Sabouraud, Head of Pharmaceutical Affairs & Responsible Pharmacist, Sanofi Pasteur
SA, Marcy l'Étoile, France
Dr C. Saillez, Senior Manager, Worldwide Vaccine Registration, GlaxoSmithKline Biologicals,
Wavre, Belgium
International Society of Blood Transfusion
Dr G. Daniels, ISBT Secretary General, Head of Molecular Diagnostics, Bristol Institute for
Transfusion Science & IBGRL, National Health Service Blood and Transplant, Bristol,
England
International Society on Thrombosis and Haemostasis
Professor K. Mertens, Head, Department of Plasma Proteins, Sanquin Research, Amsterdam,
the Netherlands
Plasma Protein Therapeutics Association
Dr I. von Hoegen, Senior Director, Quality and Safety, PPTA Europe, Brussels, Belgium
United States Pharmacopeia
Dr T.S. Morris, Vice President, Biologics and Biotechnology, United States Pharmacopeial
Convention, Rockville, MD, USA
Secretariat
Dr A.S. Alquezar, National Program of Quality Control in the Brazilian Society of Clinical
Laboratories, Rio de Janeiro, Brazil (Temporary Adviser)
Dr M. Baca-Estrada, Chief, Bacterial and Combination Vaccines Division, Health Canada,
Ottawa, Ontario, Canada (Temporary Adviser)
vii
Professor A.D.T. Barrett, Director, Sealy Center for Vaccine Development, University of
Texas Medical Branch, Galveston, TX, USA (Temporary Adviser)
Dr D.A. Bleijs, Policy Officer, Expertise Centre for Substances, National Institute for Public
Health and the Environment, Bilthoven, the Netherlands (Temporary Adviser)
Dr A. Bristow, Head, Technology Development and Infrastructure, National Institute for
Biological Standards and Control, Potters Bar, England (Temporary Adviser)
Dr M. Chudy, Division of Virology, Paul-Ehrlich-Institute, Langen, Germany (Temporary
Adviser)
Professor K. Cichutek, President, Paul-Ehrlich-Institute, Langen, Germany (Temporary
Adviser)
Dr P.A. Diop, Director, Department of Pharmacy and Medicines, Ministry of Health and
Medical Prevention, Dakar, Senegal (Temporary Adviser)
Dr S. Fakhrzadeh, Senior Expert, Vaccine Office, Pharmaceutical & Narcotic Affairs Division,
Food and Drug Organization, Ministry of Health & Medical Education of the Islamic
Republic of Iran, Tehran, Islamic Republic of Iran (Temporary Adviser)
Dr P. Ganz, Director, Centre for Blood and Tissues Evaluation, Health Canada, Ottawa,
Ontario, Canada (Temporary Adviser)
Dr I. Hamaguchi, Director, Department of Safety Research on Blood and Biological
Products, National Institute of Infectious Diseases, Tokyo, Japan (Temporary Adviser)
Dr K. Haslov, Vice President, Vaccine Quality Control, Statens Serum Institute, Copenhagen,
Denmark (Temporary Adviser)
Dr M. Heiden, Haematology and Transfusion Medicine, Paul-Erhlich-Institute, Langen,
Germany (Temporary Adviser)
Dr M.M. Ho, Division of Bacteriology, National Institute for Biological Standards and
Control, Potters Bar, England (Temporary Adviser)
Dr A. Hubbard, Division of Haematology, National Institute for Biological Standards and

Dr S. Inglis, Director, National Institute for Biological Standards and Control, Potters Bar,
England (Temporary Adviser)
Dr H. Jung Oh, Senior Scientific Officer and Reviewer, Biopharmaceuticals and
Herbal Medicine Evaluation Department, Korea Food and Drug Administration,
Chungcheongbuk-do, Republic of Korea (Temporary Adviser)
Dr M. Jutzi, Clinical Reviewer Haemovigilance, Swissmedic, Berne, Switzerland (Temporary
Adviser)
Dr A. Kato, Chief, Department of Virology III, National Institute of Infectious Diseases,
Tokyo, Japan (Temporary Adviser)
viii
Dr I. Knezevic, Team Leader, Essential Medicines and Pharmaceutical Policies, WHO,

Geneva, Switzerland
Dr K.M. Lee, Senior Scientific Officer, Biologics Research Division, Korea Food and Drug
Administration, Seoul, Republic of Korea (Temporary Adviser)
Dr L. Markoff,3 Laboratory Chief, Division of Virology, Center for Biologics Evaluation and
Research, Food and Drug Administration, Bethesda, MD, USA (Temporary Adviser)
Dr B. Meade, Consultant, Meade Biologics, Hillsborough, NC, USA (Temporary Adviser)
Dr N.E. Metwalli, Medical Officer, Blood Safety, Laboratory & Imaging, WHO Regional
Office for the Eastern Mediterranean, Cairo, Egypt
Dr G. Michaud, Deputy Director, Office of Blood Research and Review, Center for
Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD, USA
(Temporary Adviser)
Dr T. Montag-Lessing,3 Head, Section of Microbial Safety, Paul-Ehrlich-Institute, Langen,
Germany (Temporary Adviser)
Dr S. Morgeaux, Head of Viral Control Unit, Agence Française de Sécurité Sanitaire
des Produits de Santé, Direction des Laboratoires et des Contrôles, Lyons, France
(Temporary Adviser)
Dr C. Morris, Division of Retrovirology, National Institute for Biological Standards and
Dr M. Nübling, Division of Virology, Paul-Ehrlich-Institute, Langen, Germany (Temporary
Adviser)
Dr M. Ochiai, Senior Research Scientist, Division of Quality Assurance, National Institute
of Infectious Diseases, Tokyo, Japan (Temporary Adviser)
Dr V. Oeppling, Head of Section for Microbiological Vaccines, Paul-Ehrlich-Institute,
Langen, Germany (Temporary Adviser)
Dr M. Otani, Quality Control Department, Fundaçao Pró Sangue Hemocentro de São
Paulo, São Paulo, Brazil (Temporary Adviser)
Dr A.M. Padilla Marroquin, Scientist, Quality Assurance and Safety: Medicines, WHO,
Geneva, Switzerland
Dr M. Powell, Medicines and Healthcare Products Regulatory Agency, London, England
(Temporary Adviser)
Dr I. Sainte-Marie, Department for Evaluation of Biological Products, Saint Denis, France
(Temporary Adviser)
3
For specific agenda items only – by telephone.
ix
Professor R. Seitz, Head, Division of Haematology and Transfusion Medicine, Paul-Ehrlich-

Institute, Langen, Germany (Temporary Adviser)
Mr S. Shani, Deputy Drugs Controller, Central Drugs Standard Control Organization,
Under Directorate General of Health Services, Ministry of Health & Family Welfare,
New Delhi, India (Temporary Adviser)
Dr G. Smith, Director, Blood and Tissues Unit, Therapeutic Goods Administration, Woden,
Australia (Temporary Adviser)
Dr Y. Sohn, Director General, Biopharmaceuticals and Herbal Medicine Evaluation
Department, Korea Food and Drug Administration, Chungcheongbuk-do, Republic of
Korea (Temporary Adviser)
Dr J. Southern, Adviser to Medicines Control Council of South Africa, Ministry of Health,
Cape Town, South Africa (Temporary Adviser)
Dr R. Thorpe, Division of Immunobiology, National Institute for Biological Standards and
Dr D.W. Trent, Director, Regulatory and Scientific Affairs, Office of Regulated Nonclinical
Studies, The University of Texas Medical Branch, Galveston, TX, USA (Temporary
Adviser)
Dr A.M.H.P. van den Besselaar, Department of Thrombosis and Haemostasis, University of
Leiden, Leiden, the Netherlands (Temporary Adviser)
Professor G.N. Vyas, Professor Laboratory Medicine, University of California, San Francisco,
CA, USA (Temporary Adviser)
Dr J. Wang, Deputy Director, National Institutes of Food and Drug Control, Beijing, China
(Temporary Adviser)
Dr J. Weir, Director, Division of Viral Products in the Office of Vaccines, Center for
Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD, USA
(Temporary Adviser)
Dr D. Wood, Coordinator, Quality, Safety and Standards, Essential Medicines and Health
Products, WHO, Geneva, Switzerland (Secretary)
Dr D. Xing, Principal Scientist, Bacteriology Division, National Institute for Biological
Standards and Control, Potters Bar, England (Temporary Adviser)
Dr N. Yasuda, International Planning Director, Ministry of Health, Labour and Welfare, c/o
Pharmaceutical Affairs Bureau, Tokyo, Japan (Temporary Adviser)
Dr S. Zhang, Director, Division of Biological Products, State Food and Drug Administration,
Beijing, China (Temporary Adviser)
Dr K. Zoon, Director, Division of Intramural Research, NIAID, National Institutes of Health,
Bethesda, MD, USA (Temporary Adviser)
x
Abbreviations
Ae Aedes
AE adverse event
AR adverse reaction
BCG bacille Calmette–Guérin vaccine
BRN Blood Regulators Network
CBER Center for Biologics Evaluation and Research
CCID50 cell culture infectious dose 50%
CDC Centers for Disease Control and Prevention
CFU colony-forming units
CMI cell-mediated immunity
DENV dengue virus
DFI dengue febrile illness
DTaP acellular pertussis component DTP vaccine
DTP diphtheria, tetanus and pertussis
E envelope
EDQM European Directorate for Quality of Medicines and HealthCare
ELISA enzyme-linked immunosorbent assay
ERA environmental risk assessment
FDA United States Food and Drug Administration
FSH follicle-stimulating hormone
GCP good clinical practice
GCV geometric coefficient of variation
GDP good distribution practice
GMO genetically modified organism
GMP good manufacturing practices
HBsAg hepatitis B surface antigen
HAV hepatitis A virus
HBV hepatitis B virus
xi
HCV hepatitis C virus

HEV hepatitis E virus
HPLC high-performance liquid chromatography
ICDRA International Conference of Drug Regulatory Authorities
IPV inactivated polio vaccine
ISBT International Society of Blood Transfusion
ISTH International Society on Thrombosis and Haemostasis
IU International Unit
LH luteinizing hormone
MHLW Ministry of Health, Labour and Welfare, Japan
NAT nucleic acid amplification technique
NCL national control laboratory
NIAID National Institute of Allergy and Infectious Diseases
NIBSC National Institute for Biological Standards and Control
NRA national regulatory authority
NS non-structural
OPV oral polio vaccine
PCR polymerase chain reaction
PDK primary dog kidney
PEI Paul-Ehrlich-Institute
prM premembrane
PRNT plaque-reduction neutralization test
RT-PCR reverse transcription-polymerase chain reaction
QMS quality management system
SOP standard operating procedure
SPC summary of product characteristics
SSC Scientific and Standardisation Subcommittee (ISTH)
TSE transmissible spongiform encephalopathy
TSH thyroid-stimulating hormone
xii
Abbreviations
UTR untranslated region

VE vaccine efficacy
VWF von Willebrand factor
VWF:Ag von Willebrand factor antigen
VWFpp von Willebrand factor propeptide
WHA World Health Assembly
YFV yellow fever virus
xiii
1. Introduction
The WHO Expert Committee on Biological Standardization met in Geneva
from 17 to 21 October 2011. The meeting was opened on behalf of the Director-
General by Dr Carissa Etienne, Assistant Director-General of the Health Systems
and Services cluster.
Dr Etienne outlined the major issues to be discussed during the meeting.
A major need in many developing countries is for guidance on the safety of blood
products, especially in relation to strengthening national and regional blood
regulatory systems. The Committee would also be requested to provide advice
on diagnostics for Chagas disease, and on hepatitis B viral genotype diagnostics.
Dr Etienne also emphasized the importance of WHO reference materials in
improving and ensuring access to quality biological products. She noted that a
record number of new and replacement vaccines would become available in the
coming years and would be presented for adoption during future meetings, with
guidelines on dengue vaccines and bacille Calmette–Guérin (BCG) vaccines,
for example, being put forward for consideration at the current meeting. She
reiterated that a major goal of WHO was to assure the availability of safe, effective
and affordable vaccines, diagnostics, and reagents, and that this included capacity-
building for regulatory authorities. Dr Etienne also told the Committee that the
“Decade of Vaccines” initiative, endorsed by the World Health Assembly (WHA)
in 2011, offered a unique opportunity for all involved sectors to work together to
realize the full potential of vaccines in enhancing public health worldwide.
Dr Etienne concluded by reminding the members of the Committee
that they served as individual experts and not as representatives of their parent
organizations or countries. She also reminded them that they should participate
fully in the discussions so that maximum use could be made of their expertise and
that the decisions reached should be based on sound scientific considerations.
Dr Elwyn Griffiths was elected Chairman of both the overall meeting and
vaccine track with Dr John Petricciani as Rapporteur, and Dr Harvey Klein was
elected Chair of the blood track with Dr Anthony Hubbard and Dr Micha Nübling
as Rapporteurs. The Committee then adopted the agenda (WHO/BS/2011.2161)
and the timetable proposed.
1
2. General
2.1 Current directions
2.1.1 Strategic directions in biological standardization
Dr David Wood informed the Committee that the International Conference
of Drug Regulatory Authorities (ICDRA) recommended collaboration and
cooperation with WHO in several areas, including: (a) continuing to assist
national regulatory authorities (NRAs) in garnering political support at national
or regional levels to build regulatory capacity; (b) providing a mechanism for
sharing ongoing regional harmonization activities; (c) collecting best practices
for collaboration and cooperation between NRAs including information
exchange, joint assessments and inspections and activities aimed at reducing
duplication; and (d) facilitating the twinning of less-developed agencies with
well-established agencies for capacity-building and training. NRA strengthening
was one of the top 10 priorities for WHO, and a range of initiatives had been or
were being developed.
The Committee was informed of the Decade of Vaccines initiative which
aimed to enhance coordination across the global community by creating a global
vaccine action plan (GVAP) with four working groups: (a) public and political
support; (b) delivery; (c) research and development; and (d) global access.
Groups b–d incorporated regulatory aspects. Priorities in the short-, middle- and
long-term were to be set and a regulatory research agenda was to be developed
by the end of 2011 with broad input from interested parties. The Committee
noted this development and supported proposals for WHO to lead a process of
regulator engagement as part of the Decade of Vaccines initiative, and to further
the development of a global regulatory research agenda. Consideration may be
given to the potential for extending this approach to other areas of biologicals.
In that regard, the Committee recommended that WHO obtain the views of
various groups and committees involved with biologicals.
The results of a survey undertaken by WHO showed that norms and

standards were valued by both developed and developing countries. However,
indicators were needed of the impact of WHO norms and standards in order to
quantify their value to stakeholders. Survey participants also indicated that the
process of priority-setting for new norms and standards should be made more
transparent and inclusive. Other issues arising from the survey findings included
the need for ongoing review of the continued fitness for purpose of Reference
Preparations provided by WHO, and for the Committee to continue to meet
at least once a year in order to provide up-to-date guidance to stakeholders. It
was recognized that implementation programmes for complex standards were
essential but were resource-intensive. WHO should therefore carefully evaluate
the needs of stakeholders and set priorities appropriately. The need for efforts
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to increase the implementation of the standards adopted by the Committee

should be more clearly highlighted as part of achieving improved international
harmonization.
Although there continued to be strong demand for the outcomes of
WHO standardization activities, reforms were now under way to adjust the work
of WHO in accordance with a new and constrained global economic reality. This
would require WHO to re-evaluate its ability to maintain some of its current
activities and to undertake new initiatives.
Dr Wood also informed the Committee that Sanofi Pasteur had donated
type-3 polio vaccine seed strain to WHO that would be held at the National
Institute for Biological Standards and Control (NIBSC), Potters Bar, Herts,
England.
2.1.2 Vaccines and biological therapeutics: recent and

planned activities in biological standardization
An important element of WHO biological standardization activities is to facilitate
the implementation of WHO guidance in the regulation and control of product
development and manufacturing. In this context, a joint meeting had been
held with the International Alliance for Biological Standardization (IABS) to
discuss the use of thermal stability data for vaccines. Additionally, steps were
being taken to strengthen collaboration between WHO collaborating centres
and NRAs. One aim was to ensure that such centres could assist countries in
their respective regions, while promoting the implementation of WHO written
standards and inter-laboratory collaboration. The adoption of a networking
approach by WHO collaborating centres in this and other areas was seen as a
potential aid to this process.
Proposals were then set out to the Committee to make draft WHO
guidelines and recommendations available for public comment in order to allow
revision as necessary before final presentation to the Committee for adoption.
This step was intended to help speed up adoption and to facilitate the inclusion of
up-to-date scientific knowledge and best practices.
WHO written standards for vaccines have evolved over the years to
include technical specifications to help define safe and efficacious vaccines.
These specifications are intended to be scientific and advisory in nature, and
can be used as a starting point for setting national requirements and for vaccine
prequalification. Proposed written standards for 2010–2013 were reviewed for
a range of specific products and activities, including oral polio vaccine (OPV);
combined vaccines based on diphtheria, tetanus and pertussis (DTP) vaccine;
malaria vaccine; Japanese encephalitis vaccine (live, attenuated); dengue vaccine;
bacille Calmette–Guérin (BCG) vaccine; acellular pertussis (aP) vaccine;
inactivated polio vaccine (IPV); risk assessment activities; nonclinical evaluation;
and biologicals derived by DNA technology.
3
Another important activity is the development of WHO guidance on

the regulatory evaluation of copies of biological medicines that have come off-
patent – also known as similar biotherapeutic products (SBPs) or “biosimilars”
– based on sound scientific principles but without inhibiting the development
and introduction of such products in a wider range of countries. A plan for the
implementation of written standards in 2012–2013 covered SBPs in China, and
vaccine lot releases in Latvia and India. The development of guidance on SBPs
in Latin America could also usefully be considered.
A consultation on the strategic direction for standardization of vaccines
and biotherapeutics was planned for 23–24 April 2012 and a first meeting of a
proposed network of WHO collaborating centres was planned immediately
afterwards to discuss issues such as the role of WHO and other standard-setting
bodies in developing standards, and establishing networks of NRAs and national
control laboratories (NCLs).
The Committee was also advised that feedback had been obtained from
stakeholders concerning the biological standardization web site (http://www.
who.int/biologicals) and improvements had been made as a result. Further
changes would be incorporated as appropriate.
The Committee was then reminded that WHO was in the process of
reform which would result in decreased resources at a time when there were
increasing expectations of assistance from countries. As a result, the Committee
recommended that a review of the work of the WHO Quality, Safety and
Standards unit be undertaken that should include the unit business plan, the
WHO constitutional mandates, country expectations, workload, impact on
global public health, priority-setting, and funding sources and opportunities. The
Committee noted that throughout the course of the meeting, recommendations
were made for such a review of the work of the unit which it strongly endorsed.
The Committee noted the report of activities in this area and indicated
its support for the proposal for WHO to provide technical support to countries
on selected recently adopted written standards, and to develop impact indicators
for this area of work. The Committee also recommended that WHO should
consider the establishment of a process for formally numbering the different
versions of written standards that were recommended by the Committee for
adoption and published on the WHO web site prior to finalized publication in
the WHO Technical Report Series. The Committee also recommended that in
future more specific guidance should be provided by the WHO Quality, Safety
and Standards unit regarding the degree of detail to be incorporated into the
report of the decisions made by the Committee. The Committee considered this
to be an important step in harmonizing the work of the current two meeting
tracks on vaccine-related and blood-related materials.
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2.1.3 Blood products and related in vitro diagnostics: recent

and planned activities in biological standardization
The Committee was reminded that World Health Assembly resolution WHA63.12
recognized that stringent regulatory control was vital in assuring the quality and
safety of blood products, and urged Member States:
to update their national regulations…in order to ensure that regulatory
control in the area of quality and safety of blood products across the entire
transfusion chain meets internationally recognized standards.
The Committee agreed that strengthening regulatory systems for blood
products and building the technical capacities of national and regional blood
regulatory authorities were recognized as fundamental requirements in ensuring
the global availability of safe blood products.
Traceability from donor to patient was also an important component as
it linked safety issues to individuals. Examples of contributions to the improved
regulation of blood products were provided, including the development of
several guidelines and reference materials for the control of blood products, and
the identification of essential control functions and their standard indicators.
The need for stringent blood product regulation arises from: (a) the
inherent risks of blood products that require an organized national or regional
blood system to deal with the complexities of providing adequate, timely and
equitable access to safe products; and (b) the need within such a system for a
competent blood regulatory authority to ensure that appropriate production and
safety monitoring standards are met.
ICDRA indicated that WHO should focus on capacity-building for the
implementation of quality assurance systems for blood and blood products
through the development of independent regulatory authorities. A strong focus
was required on strengthening regional regulatory networks and the provision of
education and training to ensure the best use of current guidelines and assessment
criteria. Resolution WHA63.12 requested the Director-General:
to ensure sustainable development and provision of International Biological
Reference Preparations (International Standards) for use in the quality
control and regulation of blood products and related in vitro diagnostic
devices.
In addition, WHO was requested to improve access by developing
countries to WHO biological Reference Preparations and to the scientific
information from collaborative studies conducted during their validation in
order to ensure their appropriate use. WHO was expected to report back to the
WHA by May 2014.
5
An often under-appreciated but essential contribution of NIBSC and

the International Society on Thrombosis and Haemostasis (ISTH) lies in the
procuring and supplying of the required reference materials. The Committee was
informed of future plans for WHO reference materials.
The Committee indicated its support for the proposal to encourage
the increased involvement of laboratories in developing countries in WHO
collaborative studies of candidate global reference materials. The Committee also
agreed that impact indicators should be developed for the standards it endorsed.
2.2 Reports
2.2.1 Report of the WHO Blood Regulators Network
Dr Jay Epstein, Chairman of the WHO Blood Regulators Network (BRN),
presented an overview of the activities of the network which was first convened
in 2006 and which addressed issues related to advancing technical expertise in
the areas of blood, blood products and associated drugs and medical devices
including in vitro diagnostics. The specific objectives of the network were to
identify issues in the blood field, share expertise and information, promote
a convergence of regulatory policy, and propose solutions to emerging public
health challenges. BRN members were national authorities with comprehensive
responsibility for blood regulation and the necessary expertise and capacity to
address emerging public health challenges.
Since 2006, network members had provided input to WHO in the drafting
of guidelines on the production, control and regulation of blood products. In
addition, the exchange of information on topical issues was an ongoing BRN
activity and a number of teleconferences and other events had been held. The
role of the BRN had also been outlined at a number of international meetings,
most recently in 2011 at the first meeting of the International Society of Blood
Transfusion (ISBT) Working Party for Global Blood Safety in Lisbon, and at
the 7th IABS International Symposium on Advances in Transfusion Safety in

Singapore. Network members had also taken steps to ensure preparedness for
the selection of blood donors in a pandemic situation, and were actively involved
in the resolution of problems in the area of potency assays for recombinant
products, most notably factor VIII. In November 2010, BRN assessment criteria
for national blood regulatory systems had been drafted and had now undergone
a process of review. An associated national system assessment tool was being
developed and adoption by WHO of the BRN criteria would now be considered
by the Committee. Membership of the network, for which WHO provided the
Secretariat, had recently expanded from six control and regulatory authorities
to seven with the inclusion of the regulatory agency of the Japanese Ministry of
Health, Labour and Welfare (MHLW).
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The Committee noted the developments in this area, and welcomed the
inclusion of MHLW as a new member of the BRN.
2.2.2 Reports from international laboratories and WHO

collaborating centres for biological standards
The Committee was informed of recent developments at the various international
laboratories and collaborating centres for biological standards.
National Institute for Biological Standards and Control (NIBSC), Potters Bar, England
The Committee was provided with an overview of current activities and
developments at NIBSC in relation to the WHO programme for biological
standardization. NIBSC had completed its studies of four replacement and
six new candidate standards for adoption by the Committee. In addition, 10
project proposals were presented for endorsement by the Committee. Two
stability-monitoring projects were ongoing. Other WHO-related activities
included promoting vaccine quality control, developing guidelines for product
development and quality control, workshop training in biological standardization
and medicines control, participation in a variety of consultation and advisory
committees, and the development of a variety of other standards including three
haematological product standards.
NIBSC had contributed to international efforts in the fields of influenza,
tuberculosis and blood products, and to collaborative projects with organizations
including the Paul-Ehrlich-Institute, the Center for Biologics Evaluation
and Research, and the European Directorate for the Quality of Medicines &
HealthCare (EDQM).
NIBSC had also been exploring new technological developments such as
non-destructive testing (O2 monitoring and moisture content) using infrared and
Raman spectroscopy. There were many potential advantages to this technique,
including no wasting of material.
During the past year, approximately 20 000 vials and ampoules of
reference materials had been shipped, with only a very small number of delays
and other problems occurring in transit. Capacity for filling infectious materials
was expected to be operational soon. However, the development and distribution
of reference materials was increasingly giving rise to intellectual property issues.
The Committee was also informed that NIBSC faced a number of
challenges due to the fact that the programme was already very large and there
was a continuing demand for replacement standards in addition to the demand
to expand activities in emerging areas such as cell standards. This was occurring
at a time of severe financial pressures and decreasing government support.
Although several options for identifying alternative or supplemental sources of
income were being explored, current activity levels would become unsustainable
in the face of decreasing financial resources.
7
Fourteen new and replacement reference materials from NIBSC were

on the agenda of the current meeting. The preparation of all such materials was
placing heavy demands on Institute resources, with increasing demands arising
for work on reference materials in new areas and for new purposes, such as the
provision of positive and negative control preparations and of genetic reference
materials. For example, the Institute was taking on activities at national level
in the field of clinical virology to improve the provision of reference materials,
and this work had international implications. Progress was also being made in
developing diagnostic testing for variant Creutzfeld–Jacob disease (vCJD).
A balance was thus required between replacements and new standards
in established fields, and the need to support innovative products and new
technologies such as viral diagnostics, genetic diagnosis and cell counting
standards. Currently unresolved issues included whether the programme should
expand into new areas, deciding on the most efficient and effective organizational
structure, and securing adequate financial resources to carry out the mission of
NIBSC. The prioritization of activities and decisions on whether to expand into
new areas were matters which the Committee should consider.
The Committee noted the issues raised and recommended that WHO
should consider the establishment of a review process to advise the Director-
General of WHO on optimal strategies and policies to ensure that the
international biological standardization endeavour remained fit for purpose and
adequately resourced.
WHO Collaborating Centre for Quality Assurance of Blood Products and in

vitro Diagnostic Devices, Paul-Ehrlich-Institute (PEI), Langen, Germany
The PEI was originally designated as a WHO collaborating centre in 2005 and
subsequently re-designated in 2009 as the WHO Collaborating Centre for Quality
Assurance of Blood Products and in vitro Diagnostic Devices. The PEI was also
an active BRN participant and acted as its first chair in the period 2006–2008.
In addition, the Institute also collaborated with WHO in other areas, including
vaccines and advanced therapy medicinal products (cell and gene therapy, and
xenotransplantation), tissue repair and stem cells. The PEI had also been involved
in several specific WHO projects, including work on hepatitis E, transfusion-
relevant bacterial strains, a hepatitis B virus (HBV) genotype Reference
Preparation for HBV DNA assays and HBsAg tests, exploring a new factor VIII
potency assay, and development of a WHO International Standard for hepatitis C
virus (HCV) core antigen.
PEI training courses for assessors working in regulatory agencies were
conducted to support the key WHO objective of improving the regulation and
control of blood products. This was in line with resolution WHA58.13 and report
EB113/10, and with resolution WHA63.12. Because of its extensive experience in
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the regulation of in vitro devices and blood products, the PEI assessor training
programme was used by regulatory authorities and agencies worldwide.
Other collaborative activities with WHO had included work on the WHO
prequalification programme for in vitro diagnostics. Starting in 2009, recent
collaborations had focused on the prequalification of rapid assays for the detection
of HIV and malaria with PEI involvement in dossier reviews; site inspections;
development of guidance for reviews; provision of suggestions for fast tracking
product dossiers; and the preparation and organization of a workshop on HIV
nucleic acid amplification techniques (NATs).
Recent collaborative activities with other WHO collaborating centres
included the Third Meeting of WHO Collaborating Centres for Biological
Standards to support the development of WHO biological Reference Preparations
for blood products and in vitro diagnostic devices, and participation in
collaborative studies of WHO international blood product standards.
Center for Biologics Evaluation and Research (CBER), Bethesda, MD, USA
The Committee was provided with a brief overview of the regulatory responsibilities
of CBER – which did not include diagnostics or certain therapeutic biologicals
– and its attention was drawn to a number of recent organizational changes. In
its work, CBER interacted with a range of governmental and nongovernmental
organizations, and adopted a strategic approach to reference materials involving
the external accreditation of producers.
Recent CBER activities in the field of vaccines included the successful
negotiation of a CBER-WHO Cooperative Agreement with substantial funding
provided by the United States Food and Drug Administration (FDA) to WHO
with the potential for further funding for up to five years. This FDA and
WHO collaboration aimed to support scientific collaboration and enhance the
regulatory capabilities of NRAs in order to advance global access to safe and
effective vaccines and other biologicals that meet international standards. In
its first year the programme would focus on enhancing regulatory capacity for
seasonal and pandemic influenza vaccines. Strengthening regulatory capacity
would bring wider benefits in terms of vaccine access and global supply.
Other recent activities included meetings and training on the regulation of
biologicals, and the introduction of a publicly available web-based programme
intended to provide information on the regulatory work carried out at CBER.
CBER had also collaborated closely with a number of organizations
particularly in the area of influenza preparedness, including continued
collaboration with WHO and other partners in strengthening vaccine virus strain
selection and reagent preparation for seasonal influenza vaccines, and in the
development of candidate vaccine strains for viruses with pandemic potential.
9
Other activities included work on developing a process (“animal rule”) to

allow for the licensing of vaccines when human efficacy studies were not ethical
or feasible. Although no vaccines had yet been licensed using the animal rule,
potential applications included the use of anthrax vaccines for post-exposure
prophylaxis and new-generation smallpox vaccines.
The Committee was also informed of CBER-sponsored workshops and
research activities conducted or scheduled for 2011–2012. These included one
workshop on the development and evaluation of next-generation smallpox
vaccines, and one on the development and evaluation of human cytomegalovirus
vaccines. Selected research projects were also initiated to develop and evaluate
novel methods that enhance the safety of vaccine-related products, and to
advance the use of the animal rule for efficacy evaluation of vaccines and facilitate
pre-clinical evaluation.
2.3 Issues
2.3.1 Scientific issues identified by users of WHO
biological Reference Preparations
The Committee was informed of the links that existed between the EDQM and
WHO. The EDQM was responsible for producing the European Pharmacopoeia
and, specifically in the biological field, for European reference materials which
were calibrated against WHO primary reference materials. The EDQM was also
responsible for a programme of method development and standardization overseen
by a Steering Group on which WHO sat. The EDQM was also the custodian of
the remaining WHO antibiotic reference materials.
The Committee was informed that 114 projects had been initiated or
concluded – 36 on method development and 78 on reference materials. Some
of the projects involved collaboration with WHO (for example, replacement
of the tetanus vaccine standard and of the endotoxin standard replacement,
both of which were ongoing). Other ongoing projects of potential interest

to WHO included: (a) alternative assays for testing for pertussis toxin, with
the goal of validation and introduction of the best method into the European
Pharmacopoeia; (b) pertussis vaccine (acellular) potency assay improvement;
(c) pertussis vaccine (whole cell) potency assay improvement; (d) veterinary use
of rabies vaccine involving the replacement of the mouse potency test that may
have implications for the human rabies vaccine assay; and (e) recombinant major
allergens calibrated in SI units.
As part of a European Union commitment to develop alternatives to
animal testing, WHO was invited to send a representative to participate in
ongoing efforts to refine, reduce and replace the use of animal testing methods
in the quality control of biologicals. Five projects on improving testing methods
10
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included three that were intended to replace in vivo with in vitro techniques,
and may have a significant impact on the published WHO recommendations for
quality control of the products involved.
The Committee was also informed that there were many potential new
areas of work in the biologicals field such as cell and tissue therapy. As a result,
it was difficult to determine which new areas should be pursued. One potential
mechanism for assisting WHO in this activity would be to establish an external
review of the WHO biological standardization programme that would include
an assessment of opportunities in new areas.
2.3.2 Issues shared with the WHO Expert Committee on

Specifications for Pharmaceutical Preparations
Request from the WHO Immunization Practices Advisory Committee (IPAC)
to establish harmonized standards for the labelling of vaccines
The Committee was informed about the role of IPAC which was established
in 2010 to advise WHO on issues related to immunization practices as part of
efforts to strengthen and improve the delivery of immunization programmes
at country level. In this capacity the attention of the Committee was brought
to a number of labelling legibility and harmonization issues identified through
an open process by the Vaccine Presentation and Packaging Advisory Group
(VPPAG), which was established in 2007 by the GAVI Alliance. These issues
included text legibility, minimum requirements, multiple language requirements,
package insert information, use of generic names on labels, ability to observe vial
content by allowing a minimal clear area and the date format. The Committee was
reminded that it had responsibility for good manufacturing practice (GMP) for
biologicals and standards for specific products such as vaccines. The Committee
was requested to review these issues and to consider possible solutions to the
issues raised.
The Committee noted the report and agreed with a proposal that the
WHO Secretariat should explore two options for the harmonization of labelling
standards for vaccines, namely (a) to prepare a revision of section 7 (labelling)
of the current WHO GMP for biologicals; or (b) to investigate the promotion of
a new standard through tender specifications for United Nations procurement.
Proposed Third WHO International Standard for bacterial endotoxin

There was agreement that the only reliable approach to maintaining harmonization
of the endotoxin unit for pyrogen testing was through the establishment of a
shared and harmonized reference material. Current procedures for endotoxin
testing were fully harmonized between the European, Japanese and United States
pharmacopoeias. However, the Committee was informed that the current Second
11
WHO International Standard for endotoxin established in 1996 now needed

to be replaced because it was nearly depleted (WHO/BS/2011.2182). The
production of 75 000 vials of a candidate Third WHO International Standard
was being completed. Following filling at NIBSC in November 2010, the United
States Pharmacopeial Convention (USP), NIBSC, and EDQM sponsored a
collaborative evaluation study involving 33 laboratories worldwide. The results
of the study would be submitted to the Committee for consideration in 2012 and
the replacement International Standard would be proposed.
The Committee endorsed the project as proposed.
Proposed global legally binding instrument on mercury:

implications for pharmaceuticals
The Committee was informed that there was growing concern about the human
health implications of mercury, especially in children who receive mercury in
vaccines. Thiomersal is widely used in vaccines in both developed and developing
countries. Data suggested that such vaccines were very safe. In addition, alternative
acceptable preservatives were not available, and the use of small-dose vials would
probably be cost prohibitive and have adverse environmental impacts. WHO
continued to recommend multi-dose vaccine vials for routine immunization
programmes in many countries because they are safe and effective, they limit the
required storage capacity and help reduce vaccine costs.
Other potential sources of mercury exposure include dental amalgam,
thermometers and blood pressure measuring devices. In all of these contexts there
had been a general trend to reduce and ultimately eliminate mercury exposure.
However, almost all countries still use dental amalgam and alternatives are
more expensive and have technical limitations. At a 2009 technical meeting
co-supported by the United Nations Environment Programme (UNEP), WHO
had recommended that a phase-down be pursued by promoting disease prevention
and alternatives to amalgam; conducting research and developing cost–effective
alternatives; educating dental professionals; and raising public awareness.

At the upcoming third session of the Intergovernmental Negotiating
Committee to prepare a global legally binding instrument on Mercury (INC3)
from 31 October to 4 November 2011, the use of mercury in pharmaceuticals
would be considered. WHO participates in the INC process with observer
status. As well as being present at the INC sessions, WHO provided submissions,
technical briefings, and contributions to Secretariat meeting documents. The aim
of WHO participation in the treaty discussions would be to exclude vaccines
containing mercury from being prohibited, by excluding them from the scope
of the treaty so as to allow their continued availability in multi-dose vials that
provide safe and effective prevention of serious diseases worldwide.
The Committee noted the report.
12
3. International Recommendations, Guidelines
and other matters related to the manufacture
and quality control of biologicals
All Recommendations and Guidelines established at the meeting are listed in
Annex 1, which provides an updated listing of all current WHO Recommendations,
Guidelines and other documents related to the manufacture and quality control
of biological substances used in medicine.
3.1 Vaccines and related substances

3.1.1 Guidelines on the quality, safety and efficacy of
dengue tetravalent vaccines (live, attenuated)
Dengue is a mosquito-borne disease that represents a major public health problem
in tropical regions of the world. The draft Guidelines (WHO/BS/2011.2159) that
were considered by the Committee covered dengue tetravalent vaccines (live,
attenuated) under clinical development, and were intended to provide guidance
to NRAs and vaccine manufacturers on their quality, safety and efficacy to
facilitate their international licensure and use.
Other types of dengue virus vaccines under development (such as subunit
or inactivated vaccines) were outside the scope of these Guidelines. However, some
of the guiding principles provided – for example, in Part C on clinical evaluation –
may be useful for the evaluation of these other vaccine types. In addition, in terms of
quality-control aspects, the guiding principles applicable to subunit, inactivated or
other vaccine types were available elsewhere if the product in development shared a
similar manufacturing process. For example, guidelines for human papillomavirus
and hepatitis B vaccines may also be useful for subunit dengue vaccines.
The draft Guidelines had been based on the experience gained from
developing candidate tetravalent dengue vaccines (live, attenuated), and would
need to be updated as new data became available from additional studies.
Following a brief overview of dengue disease and dengue vaccine development,
Part A sets out the guidelines for manufacture and quality control, with the
specific issues of nonclinical evaluation, clinical evaluation and environmental
risk assessment covered in Parts B, C and D respectively. Part E then provides
guidelines for NRAs.
After making suitable amendments, the Committee recommended that
the Guidelines be adopted and appended to its report (Annex 2).
3.1.2 Recommendations to assure the quality,

safety and efficacy of BCG vaccines
Tuberculosis was declared a global emergency by WHO in 1993, and
Mycobacterium tuberculosis (M. tuberculosis) was now considered to be responsible
13
for more adult deaths than any other pathogen. Vaccination with the BCG vaccine
remained the standard for tuberculosis prevention in most countries because
of its efficacy in preventing life-threatening forms of the disease in infants and
young children. It was inexpensive and usually required only one administration
in either neonates or adolescents. As there was currently no suitable alternative,
BCG would remain in use in the foreseeable future and may continue to be used
as a prime vaccine in a prime-boost immunization schedule in conjunction with
new tuberculosis vaccines.
The last revision of the Requirements for BCG vaccine for human
use was in 1985, with an amendment updating the section on the expiry date
published in 1998. Recent WHO consultations had addressed the issues of
improving BCG vaccine characterization and quality-control assays to reflect
current state-of-the-art technology. In addition, a proposal had been made
to replace the WHO International Reference Preparation for BCG vaccine by
substrain specific Reference Reagents evaluated by collaborative studies. The draft
Recommendations (WHO/BS/2011.2157) that were considered by the Committee
covered the production and control of BCG vaccines in Part A, with guidelines
for nonclinical evaluation provided in Part B. Guidelines on the content of the
clinical development programme applicable to BCG vaccines were provided in
Part C, and recommendations for NRAs in Part D.
The nonclinical evaluation guidelines applied to classical BCG vaccine
products still in need of such evaluation, including newly manufactured products
requiring clinical trial studies or in case of manufacturing changes.
the Recommendations be adopted and appended to its report (Annex 3).
3.1.3 Recommendations to assure the quality, safety

and efficacy of acellular pertussis vaccines
Pertussis immunization is an integral part of immunization programmes in all
regions of the world. It is recommended for all infants and children and in some
countries also for adults and adolescents. Whole-cell pertussis vaccines have
been used for more than 50 years, have been shown to provide protection and
are still the foundation of global pertussis control. However, there is increasing
interest in acellular pertussis vaccines, which have also been shown to be safe
and effective and which have been successfully introduced into many national
immunization programmes.
As a consequence of increasing demand for acellular pertussis vaccines,
new manufacturers are entering the field. The expansion in the number and use
of acellular pertussis vaccines, the development of new vaccines and advances in
the standardization of quality-control methods have prompted WHO to update
the current WHO Guidelines for the production and control of the acellular
pertussis component of monovalent or combined vaccines.
14
International Recommendations, Guidelines and other matters
The Committee was informed that the current Requirements for

acellular pertussis vaccine were adopted in 1996 with the recognition that further
improvements in their production and evaluation would follow. Since 1996,
further experience had been gained and it had become clear that a revision should
be undertaken. Acellular pertussis vaccines were almost exclusively administered
in combination with diphtheria and tetanus toxoid vaccines. Moreover, in recent
years, there had been increased interest in the use of more complex combination
vaccines, a trend that increased the challenges of clinical evaluation. Furthermore,
evaluating the clinical efficacy of new acellular pertussis vaccine formulations
had become increasingly difficult due to the decreased prevalence of pertussis
cases worldwide.
The draft revised Recommendations (WHO/BS2011.2158) that were
considered by the Committee highlighted advances in the development,
manufacturing and testing of acellular pertussis vaccines and aimed to provide
guidance on: (a) improving the quality control of existing vaccines based on
new information and experience; and (b) evaluating new products and new
combinations through manufacturing control and through both nonclinical
and clinical studies. The parameters measured during testing should include
immunogenicity and safety, and the candidate vaccine should be shown to be non-
inferior to the licensed comparator vaccine. After making suitable amendments,
the Committee recommended that the Recommendations be adopted and
appended to its report (Annex 4).
3.1.4 Assessing risk when a potential adventitious agent is found

The Committee was reminded that in 2010 it had recommended that WHO
lead the development of a risk assessment process subsequent to marketing
authorization. In addition, the ICDRA had recommended that WHO assist
countries in developing risk management strategies for responding to scientific
advances in detecting adventitious agents in biological products.
Following the discovery of porcine circovirus (PCV-1,2) in rotavirus
vaccines in 2010, NRAs and manufacturers assessed the emerging data on an
ongoing basis and made decisions accordingly, in line with previous discoveries
of an adventitious agent in licensed biological products. WHO was requested
to develop a document on assessing risk in such circumstances, and a first draft
version was considered by the Committee in 2010, who requested that it be
revised after broad consultation. Since then, several meetings had been held to
discuss the revised document which was now in the process of further revision
with an expectation that a final document would be submitted to the Committee
in 2012 for adoption.
A progress report was presented to the Committee who then provided
a number of suggestions to be considered during the revision process, while
15
emphasizing the importance of coordination and collaboration among WHO,

regulatory agencies and manufacturers whenever a potential adventitious agent
was identified.
3.1.5 Calibration of seasonal and pandemic influenza antigen

working reagents by WHO essential regulatory laboratories
The Committee was informed of the process used to develop a generic protocol
for the calibration of seasonal and pandemic influenza antigen working reagents
by WHO essential regulatory laboratories (ERLs) and a draft generic protocol was
provided for the Committee to review (WHO/BS/2011.2183). The Committee
expressed its appreciation for the work that had gone into the development of
both the process and the generic protocol.
Reagents for assessing the potency of influenza vaccines using the single
radial immunodiffusion (SRID) assay are prepared annually as the strains in
the vaccine are updated. The calibration process involves the preparation of a
primary liquid standard (PLS) and a large batch of freeze-dried antigen by one
of the ERLs. The PLS is then distributed to all the other ERLs for independent
calibration. Samples of the freeze-dried antigen are also distributed to the other
ERLs and calibrated against the PLS using the SRID assay.
Four ERLs are involved in this activity which had hitherto been outside
the Committee process because of the intense time constraints involved. To make
the process more transparent, the generic protocol provided described in detail
the process by which the four ERLs developed and calibrated the reagents. After
making suitable amendments, the Committee recommended that the generic
protocol be adopted, appended to its report (Annex 5) and published on the
WHO web site.
3.1.6 Proposed new projects for developing or updating written standards

The Committee was informed that the WHO Prequalification unit had
established a priority list (high, medium, and low) for vaccines to be used in
global immunization programmes. The Secretariat also had a list of new and
replacement regulatory written standards for vaccines and other biologicals. The
Committee was further informed that 12 new vaccines were in various stages of
development and were expected to be introduced by 2021. Of those 12, seven were
improvements on existing vaccines, and five were new vaccines. The Committee
was requested to assist the WHO Quality, Safety and Standards unit by providing
guidance on identifying the criteria that should be used in prioritizing those
biologicals for which written standards should be developed.
After a full discussion, the Committee recommended that the unit
consider the following factors: (a) public health emergencies are likely to arise
from time to time and will of necessity become a top priority; (b) the global public
16
health importance of a specific standard; (c) the vaccines assigned high priority
by the WHO Prequalification unit; (d) the establishing of high-, medium- and
low-priority categories within the unit based on input from stakeholders (such
as Member States through WHO regional offices, international organizations,
the Committee, regulatory agencies and industry organizations); (e) platform
technology documents; (f) the availability of guidance documents from other
organizations and whether a WHO document on the same topic added sufficient
value to warrant its production; (g) building on currently available documents
in new areas such as cell and gene therapy; (h) assessment of the availability of
funding for new document development; (i) the occasional inclusion of several
low-priority items as resources permit; and (j) the needs of countries and regions
for guidance on non-vaccine products such as SBPs and biotherapeutics. The
Committee suggested that the most efficient way to obtain input on priorities
from stakeholders would be to request comments on a draft based on the factors
listed above. The Committee also noted that a balance should be struck between
revisions of existing documents and the development of new documents. Another
factor that must be taken into consideration was the implementation of written
standards. All such activities were directly related to NRA strengthening and
would require financial resources.
The Committee welcomed the initiative for establishing a procedure for
priority-setting, and it was agreed that the vaccine track Secretariat would follow
up on the Committee’s suggestions, update the one-page outlines of proposed
projects and provide subsequent feedback.
3.2 Blood products and related substances

3.2.1 Guidelines for thromboplastins and plasma used to control
oral anticoagulant therapy with vitamin K antagonists
The draft Guidelines (WHO/BS/2011.2165) that were considered by the
Committee were a revision of the previous Guidelines introduced in 1999, and
described the establishment and maintenance of the International Normalized
Ratio (INR)/International Sensitivity Index (ISI) system used to control oral
anticoagulant therapy, and the development of the International Reference
Preparations for thromboplastins. The Guidelines covered the ISI calibration
of secondary standards (Procedure 1), individual batches of thromboplastin
reagents (Procedure 2) and local system calibration (Procedure 3).
It was suggested that there should be a better separation between the
sections relevant to manufacturers and those relevant to clinical laboratories.
However, it was considered extremely difficult to separate these aspects.
Manufacturers were expected to be aware of the whole document and to be
responsible for instructing clinical laboratories on the use of products. It was agreed
that a paragraph be added to the beginning which addressed how the document
should be used and its relevance to manufacturers and clinical laboratories.
17
The draft Guidelines incorporated references to the SSC/ISTH Guidelines

on the preparation, certification and use of certified plasmas for ISI calibration and
INR determination which were published in 2004. There was still a requirement
to use the manual technique with the current WHO International Standard
and some concern was expressed over the maintenance of expertise when most
laboratories used automated methods. However, the performance of more than
20 participating laboratories in the calibration of the WHO International Standard
rTF/09 indicated that the required expertise was still present. It was announced
that WHO would be willing to support workshops to maintain expertise in the
manual tilt tube technique.
The possibility of replacing the manual technique (used with the
International Standard thromboplastins in Procedure 1) with an automated
procedure which has been validated and found to correlate with the manual
technique was also discussed. Information on suitable automated procedures
could be obtained through a collaborative study and included later in an appendix.
It was agreed that the opinion of the SSC/ISTH Subcommittee on Control of
Anticoagulation would be sought to see if this alternative would be useful.
It was agreed that a new section addressing the criteria to apply in the
choice of thromboplastin reagents should also be added, possibly as an appendix.
The Committee recommended that the revised Guidelines be adopted
and appended to its report (Annex 6).
3.2.2 Assessment criteria for national blood regulatory systems

These assessment criteria for national blood regulatory systems (WHO/
BS/2011.2174; QAS/2011.431) were the result of a BRN initiative which aimed
to promote robust NRAs in both developed and developing countries.
The tool consisted of an assessment framework to highlight the strengths
of NRA programmes for the regulation of blood, blood components, plasma-
derived products, and associated substances and medical devices including in
vitro diagnostics. The assessment criteria were designed to identify gaps for
future development and thus strengthening of the regulatory oversight of NRAs.
They were structured into two essential elements (national regulatory system
and national regulatory authority) and 12 core functions. The main criteria
were broken down into indicators which were then divided into two categories
– required (R) or desirable/suggested (S) – in order to accord with international
best practices for national blood regulatory systems. This tool would support
NRA establishment and capacity-building and promote regulatory convergence.
Using previous versions of this tool, Health Canada, Swissmedic and two Latin
American countries had undertaken self-assessments. Based on the experience
gained, modifications had then been introduced into the document. Further
input had been obtained at the ICDRA workshop in Singapore in December
18
2010. A recent broad consultation process then generated comments from all
six WHO regions, involving 40 countries, which had been reviewed by the BRN.
It was expected that the tool would help to identify gaps and main
priorities based upon which capacity-building programmes could be developed
to support the introduction of regulations for blood products at global level, and
to sustain implementation of resolution WHA63.12 on the availability, quality
and safety of blood products.
The Committee highlighted a need for WHO to develop an implementation
plan, including a guidance document on the correct use of the tool, as part of the
capacity-building activities requested in the above resolution.
the assessment criteria be adopted and appended to its report (Annex 7).
19
4. International reference materials –
vaccines and related substances
All reference materials established at the meeting are listed in Annex 8.
4.1 WHO International Standards and Reference

Reagents – vaccines and related substances
4.1.1 First WHO International Standard for meningococcal
serogroup C polysaccharide
Meningococcal serogroup C (MenC) plain polysaccharide (PS) and conjugate
vaccines are primarily evaluated by physicochemical methods to ensure that
batches are consistently manufactured. As different assays are employed to
quantify the MenC PS content of final formulations and bulk intermediaries,
there was a need for an International MenC PS Standard to calibrate internal
references used in the different laboratories. Twelve laboratories from nine
different countries participated in a collaborative study (WHO/BS/2011.2169)
to assess the suitability and determine the MenC PS content of a candidate
International Standard MenC PS preparation (08/214).
The Committee recommended the adoption of preparation 08/214
as the First WHO International Standard for meningococcal serogroup C
polysaccharide, with a content of 1.192 mg MenC PS/ampoule.
4.1.2 First WHO International Standard for

anti‑pneumococcal antibodies in serum
To develop and evaluate the efficacy of pneumococcal vaccines it was important
to have an accurate method for measuring the concentration of human
antibodies directed against pneumococcal capsular polysaccharides (Pn PS). In
2000, representatives from academia, government and industry met at WHO and
selected an enzyme-linked immunosorbent assay protocol for the quantification

of human IgG antibodies specific for Streptococcus pneumoniae capsular
polysaccharides (Pn PS ELISA). In order to estimate the concentration of
antibodies, the reference serum 89SF was produced and antibody concentrations
established. As quantities of 89SF were now approaching depletion, a replacement
(007sp) had been produced in order to maintain the link to clinical efficacy.
Five laboratories from three countries had participated in a collaborative study
(WHO/BS/2011.2164) to assess the suitability and determine the pneumococcal
serotype specific antibody content of the candidate 007sp International Standard
serum preparation.
The Committee recommended the adoption of preparation 007sp as the
First WHO International Standard for anti-pneumococcal antibodies in serum.
20
International reference materials – vaccines and related substances
The Committee noted that μg/ml was used in order to maintain continuity with
89SF which was linked to clinical efficacy. The proposed WHO International
Standard was being held in two custodian laboratories: NIBSC (5000 vials) and
CBER (10 000 vials) and was being stored at –20 °C. Stability data during shipping
at room temperature were requested by the Committee.
4.1.3 Third WHO International Standard for

trivalent inactivated polio vaccine
The potency of inactivated polio vaccine (IPV) is measured in vitro using a
validated ELISA test with suitable Reference Preparations and is expressed in
D-Antigen units. The Second WHO International Standard for IPV (NIBSC
Code: 91/574) was established in 1994. It was shown to be suitable for determining
the antigenic content and immunogenicity of IPVs by in vitro and in vivo assays
respectively. In anticipation of 91/574 stock depletion, a collaborative study
had been conducted in 2009 to assess the suitability of candidate replacements.
However, for reasons which were not clear, this study had demonstrated excessive
variability in potency estimates for the candidate standards between laboratories.
Following the suggestion that this may have been due to a lack of homogeneity
between vials of the candidate standards, a further pilot study had been conducted
in 2010 (WHO/BS/2011.2162). A subset of six laboratories that had participated
in the original study was selected to be representative of the range of potency
estimates observed in the original study.
The aims of the 2010 study were to see if the variability observed
between laboratories in the original study could be confirmed, and to assess the
contribution of any vial-to-vial variability to the outcome.
The Committee was informed that the study had confirmed that variability
between laboratories was an issue. A new collaborative study was planned for
early 2012 using samples from various IPV manufacturers that would be filled at
facilities other than NIBSC.
4.2 Proposed new projects – vaccines and related substances

4.2.1 Replacement of the First WHO International Standard for
antibody to influenza A virus subtype H1N1pdm
To facilitate and improve transparency in the priority-setting process for new
projects, a simple tool had been developed that described their salient features.
The resulting documentation provided a means for the Committee and other
stakeholders to review and comment on proposed new projects that were under
consideration. Among the proposals made in document WHO/BS/11.2177
(Appendix 1) was the development of the Second WHO International Standard
for antibody to influenza A virus subtype H1N1pdm.
21
Influenza is considered to be of considerable public health importance

in most countries, and the H1N1pdm virus continues to circulate globally. The
variability of serology assays means that it is very difficult to compare different
vaccines, with resulting scientific and regulatory challenges. Nevertheless,
H1N1pdm virus antigen is a component of numerous influenza vaccines which
are licensed and in use.
Stock levels of the current International Standard were low and were
not expected to be sufficient for future levels of demand. Anticipated uses of a
replacement Reference Preparation include calibration of serology assays for
vaccine trials and serum surveys with users ranging from NCLs, independent
serology laboratories, clinical laboratories and vaccine manufacturers. Issues
raised by this proposal included the need to determine whether the new candidate
International Standard would reduce assay variability to a similar extent to the
current standard and whether the assigned unitage would be similar.
The Committee endorsed the proposal to develop the Second WHO
International Standard for antibody to influenza A virus subtype H1N1pdm, and
urged that efforts be made to match the characteristics of the source material
used for the current International Standard.
4.3 Ongoing stability monitoring – vaccines

and related substances
4.3.1 Inactivated hepatitis A vaccine
The First WHO International Standard for inactivated hepatitis A vaccine
was established in 1999 and has been widely used without problem. However,
a loss of potency (complete loss of antigenic activity) was reported in one
laboratory. This was investigated and attributed to the presence of a low-level
bacterial contaminant in a small percentage of samples. For this reason a post-
establishment stability evaluation was conducted (WHO/BS/2011.2160) of the
standard preparation (95/500).
In addition, although stability samples were laid down at the time of

production no evaluation of stability was performed for the establishment report.
At its meeting in 1999, the Committee had recommended ongoing stability
monitoring as the preparation was in liquid form and predictions of its long-term
stability were not available because of the inherent variability of the assays. The
current stability evaluation only included data on vaccine antigen.
Samples were tested for antigen content by comparison with frozen
baselines stored continuously at –150 °C. No evidence was found of significant
loss of stability over the 16 years of sample storage at –70 °C. Although extended
studies confirmed an appropriate level of stability for use, it was proposed that
this International Standard be replaced as soon as possible given the small
percentage of vials that were found to be contaminated. The Committee was
informed, however, that vials not contaminated were stable and fit for purpose.
22
International reference materials – vaccines and related substances
The Committee recommended that information on potential contamination

be included with future shipments of the current standard.
4.4 Progress reports – vaccines and related substances

4.4.1 Characteristics of an improved potency assay
for inactivated influenza vaccines
The Committee was informed of a draft document on the proposed characteristics
of an improved potency assay for inactivated influenza vaccines. The Committee
expressed appreciation for the efforts involved in developing the draft, and
encouraged further discussion with appropriate groups.
4.4.2 Hepatitis B vaccine

The Committee was informed of interest from laboratories around the world in
developing a standardized assay for assessing the potency of hepatitis B vaccines
based on a protocol developed by a manufacturer. A feasibility study had been
undertaken by three NCLs to evaluate the impact of using different reagents on
the proposed standard assay. Initial results had been encouraging and indicated
that the assay could be used with a variety of different reagents. Further discussion
was now needed on whether a larger collaborative study should be undertaken.
The Committee requested that it be kept informed of progress.
23
5. International reference materials – blood
products and related substances

Reagents – blood products and related substances
5.1.1 Assignment of a value for von Willebrand factor propeptide to the
Sixth WHO International Standard for factor VIII/VWF plasma
The ratio of von Willebrand factor propeptide to von Willebrand factor antigen
(VWFpp/VWF:Ag) is used to identify bleeding disorders associated with a
decreased half-life of VWF in the circulation. Since there was no internationally
agreed reference material or International Unitage (IU) for VWFpp, laboratories
have had to rely on locally calibrated reference materials, leading to increased
inter-laboratory variability. The objective of this project (WHO/BS/2011.2171)
was to assign a value for VWFpp to the Sixth WHO International Standard
for factor VIII/VWF plasma (07/316) in order to define the IU and provide
long-term continuity for the calibration of all secondary standards. As part of
a collaborative study, 13 laboratories had estimated VWFpp (and VWF:Ag) in
the International Standard relative to their local reference materials. The inter-
laboratory variability for VWFpp estimates was surprisingly low (GCV 8.1%)
and this provided an opportunity to assign a consensus mean value of 1.03 IU/
ampoule to the International Standard. When estimates of VWFpp in a second
lyophilized plasma were calculated using the International Standard as standard,
the inter-laboratory variability was further reduced to a GCV of 3.0%, indicating
that a VWFpp standard should lead to improved agreement between laboratories.
It was noted that estimates of VWF:Ag in the International Standard, relative
to local reference materials, were more variable (GCV 12.2%) than estimates
of VWFpp (GCV 8.1%) and this probably indicates inaccurate calibration of
local VWF:Ag Reference Preparations. It was proposed that the Sixth WHO
International Standard for factor VIII/VWF plasma (07/316) be assigned a value
of 1.03 IU/ampoule for VWFpp.
The Committee recommended the adoption of this assigned value.
5.1.2 Third WHO International Standard for fibrinogen (plasma)

The International Standard for fibrinogen (plasma) was introduced to improve
agreement between laboratories measuring plasma fibrinogen levels in connection
with the diagnosis of deficiency or in relation to the risk of cardiovascular disease.
Declining stocks of the Second WHO International Standard for fibrinogen
24
International reference materials – blood products and related substances
(plasma) necessitates the preparation of a replacement standard. A collaborative

study (WHO/BS/2011.68) involving 21 laboratories from 11 countries was
undertaken to assign a value for clottable protein to the proposed Third WHO
International Standard for fibrinogen (plasma) (09/264) by assay relative to the
current International Standard. Value assignment was based on a combination of
results from Clauss assays (22 estimates) and clot-removal methods (two estimates)
which provided a consensus mean value of 2.67 mg/ampoule (n=24) with inter-
laboratory variability (expressed as GCV) of 3.3%. Results from immunological
methods were not included in the calculated overall mean value. Stability had
been assessed by an accelerated degradation study which had predicted a loss of
0.004% per year for ampoules stored at 20 °C.
The Committee recommended the adoption of 09/264 as the Third WHO
International Standard for fibrinogen (plasma) with an assigned value of 2.7 mg
per ampoule.
5.1.3 First WHO International Reference Reagent for

anti‑human neutrophil antigen‑1a antibody
Transfusion-related acute lung injury (TRALI) is a recognized cause of mortality
and morbidity following blood transfusion. TRALI can be caused by antibodies
in the donor plasma directed against human leukocyte antigen (HLA) or
human neutrophil antigen (HNA) in the recipient. Many laboratories now test
for neutrophil antibodies but there are no reference reagents to determine
detection sensitivity. A pilot study of a candidate lyophilized reference plasma
preparation (09/284), containing a clinically relevant anti-human neutrophil
antigen (anti-HNA-1a) antibody confirmed the absence of other HNA and HLA
antibodies and indicated a suitable titre range. Furthermore, the study indicated
the unsuitability of using one monoclonal antibody (3G8) as capture antibody
for anti-HNA-1a detection in the MAIPA assay. A subsequent collaborative
study (WHO/BS/2011.2167) involving 24 laboratories returned titres (highest
dilution giving a positive result) ranging from 1 in 2 to 1 in 256. This wide
range indicated the need for improved sensitivity in some laboratories. There
was overall agreement that the minimum sensitivity required should be set at
a dilution of 1 in 4. It was proposed that the preparation 09/284 be established
as the First WHO International Reference Reagent for anti-human neutrophil
antigen-1a (minimum potency). It was noted that antibodies to other human
neutrophil antigens were also of clinical significance and could be addressed in
future Reference Preparations.
The Committee recommended the adoption of 09/284 as the First WHO
International Reference Reagent for anti-human neutrophil antigen-1a antibody.
25
5.1.4 First WHO International Reference Reagents

for blood group genotyping
Blood group genotyping is increasingly being used as it has a number of
advantages over conventional blood group serology. However, ISBT workshops
have demonstrated a need for DNA reference materials to improve diagnostic
accuracy. Four lyophilized genomic DNA preparations from genotyped and
phenotyped donors were evaluated for their suitability to serve as International
Reference Reagents for common Caucasian (samples RBC1, RBC4 and RBC5)
and black African (sample RBC12) alleles in an international collaborative study
involving 29 laboratories from 19 countries (WHO/BS/2011.2166). Although the
study demonstrated an overall high level of accuracy in blood group genotyping
for common alleles, the identification of errors and inconsistencies, and the
limited genotyping capabilities of many laboratories, highlighted a need for
validated reference materials to control test procedures and ensure that they
are sufficiently sensitive. The finding that the majority of laboratories reported
genotypes in accordance with those determined by the study organizers and with
the serological phenotypes validated the use of RBC1, RBC4, RBC5 and RBC12 as
Reference Reagents for blood group genotyping. Accelerated degradation studies
indicated that the preparations would be stable at –20 °C for many years. These
Reference Reagents were intended for qualitative use only – i.e. to give positive
or negative results for a particular allele from which red blood cell phenotype
could be inferred; the quantification of DNA was not required. It was proposed
that RBC1 (10/232), RBC4 (10/236), RBC5 (10/238) and RBC12 (10/234) be
collectively established as the First WHO International Reference Reagents
for blood group genotyping to control test procedures for common alleles in
Caucasian and black African populations. It was anticipated that up to four
more preparations may be developed in the future to cover all major clinically
important blood group genotypes.
The Committee recommended the adoption of the four preparations
RBC1 (10/232), RBC4 (10/236), RBC5 (10/238) and RBC12 (10/234) as the First
WHO International Reference Reagents for blood group genotyping.
5.1.5 Revision of the instructions for use of the First WHO International
Reference Repository for platelet transfusion relevant bacterial strains
Both bacterial-screening and pathogen-reduction systems have been developed
to address the bacterial contamination of platelet concentrates which remains a
significant problem in transfusion medicine. A four-member panel consisting
of deep-frozen bacterial strains of different bacterial species (Staphylococcus
epidermidis, Klebsiella pneumoniae, Streptococcus pyogenes and Escherichia coli)
able to grow in platelet concentrates after low-count spiking was assessed in a
worldwide validation study.
26
In 2010, this panel was adopted by the Committee as the First WHO
International Reference Repository for platelet transfusion relevant bacterial
strains. The proposed future expansion of the repository was also endorsed.
Enlargement studies using a revised protocol were in progress and
would be presented to the Committee in due course. These studies involved
representative laboratories from additional WHO regions and would incorporate
the validation of bacterial detection after the direct inoculation of platelets.
The instructions for use for the current repository bacterial strains were
presented and questions raised by the Committee and by customer laboratories
were discussed. It was agreed that on the basis of current data a number of
modifications to the instructions for use should be made including: (a) a restriction
on “Intended Use” for bacterial detection methods until suitability for pathogen-
inactivation methods had been demonstrated; (b) the inclusion of a handling
and dilution protocol; (c) the definition of precautionary measures to be followed
by users; (d) potential offer of training in panel use; and (e) the removal or
revision of inadequacies in phrasing and comprehensibility. In the meantime,
modifications to the instructions for use had been submitted by Dr Thomas
Montag-Lessing (PEI).
The Committee accepted the revised instructions for use, subject to
final edits.
5.1.6 Apolipoprotein B
There is increasing evidence that elevated serum apolipoprotein B concentration
is an important risk factor for coronary heart disease, and many groups
recommend the measurement of this analyte over low-density lipoprotein
(LDL) cholesterol. The standardization of apolipoprotein B assays has been
achieved through use of the First WHO International Reference Reagent for
Apolipoprotein B (SP3-07) which is a frozen pooled human serum preparation
stored at the Centers for Disease Control and Prevention (CDC), Atlanta. As
stocks of SP3-07 were approaching depletion, a replacement (SP3-08) had been
prepared which also consisted of a frozen pooled human serum preparation.
Value assignment of SP3-08 was performed relative to SP3-07 in one laboratory
using an immunonephelometric method. A mean value of 1.18 g/l from 186
determinations with a CV of 2.4% was assigned. Evaluation studies had indicated
that SP3-08 was suitable for use in terms of stability, linearity of dilutions and the
calibration of manufacturers’ in-house standards.
The establishment of SP3-08 was deferred pending receipt of
documentation in conformance with established WHO procedures. The
Committee proposed that the endorsement of this project be conducted as a
joint International Federation of Clinical Chemistry (IFCC) and WHO initiative
to replace SP3-07.
27
5.2 Proposed new projects – blood products

and related substances
5.2.1 Replacement of the Second WHO International
Reference Preparation for serum IgE
To facilitate and improve transparency in the priority-setting process for new
projects, a simple tool had been developed that described their salient features.
The resulting documentation provided a means for the Committee and other
stakeholders to review and comment on proposed new projects that were under
consideration. Among the proposals made in document WHO/BS/11.2179 was
the development of a replacement for the Second WHO International Reference
Preparation for serum IgE.
Allergic disorders are common and their incidence is increasing. Serum
IgE measurements are essential for the diagnosis and management of allergic
disease. There is frequent demand for the current Reference Preparation which
is used by assay manufacturers and clinical laboratories to standardize in vitro
diagnostic tests for allergen-specific IgE in serum. As stocks of the Second WHO
International Reference Preparation for serum IgE were nearing exhaustion, the
preparation of a replacement consisting of pooled sera from patients with allergic
disease was proposed, calibrated against the current Reference Preparation in an
international collaborative study.
The Committee endorsed this proposal.
5.2.2 Replacement of the Third WHO International Standard for plasmin

Plasmin is the key enzyme responsible for the digestion of fibrin clots, and
there is renewed interest in its use as a thrombolytic drug. The Third WHO
International Standard for plasmin is currently used to estimate the direct relative
potency of plasmin, and can also be applied to the wider standardization of
fibrinolysis assays.
As stocks of the Third WHO International Standard for plasmin were

now low, a replacement preparation was needed. The candidate replacement
would consist of therapeutic-grade plasmin manufactured from human plasma.
A truncated form of plasmin and full-length recombinant plasmin may also be
included in the proposed collaborative study (WHO/BS/2011.2179) to provide
valuable information on the utility of the replacement standard for their potency
estimation. Value assignment of the replacement standard in IU would involve
conventional chromogenic and fibrin clot-based assays relative to the current
International Standard. However, the determination of molar concentration by
active-site titration would also be investigated. The objective was to establish a
replacement for the current International Standard in 2013.
The Committee endorsed this proposal.
28
5.2.3 Haemoglobin A2
The quantification of haemoglobin A2 (HbA2) is essential for the routine
diagnosis of the beta thalassaemia trait, and hence for the identification of
couples at risk of having a child with beta thalassaemia major. The First WHO
International Reference Reagent for haemoglobin A2 (89/666) is currently the
only international reference material available for the control of test procedures.
A number of issues exist in relation to the current situation. First, only
one HbA2 level is covered by the reference material even though the calibration
and control of routine methods would benefit from the use of at least two levels.
More information on the methods used to establish the preparation should
be made available. In addition, there exists a potential commutability issue,
with an observed discrepancy reported between two high-performance liquid
chromatography (HPLC) methods relating to a patient sample. There is also a
lack of stability data on the current reference material. Finally, there may be a
need to remind manufacturers that the current preparation is for the control of
test procedures and not for calibration.
The IFCC had now established a working group on the standardization
of HbA2 with the objective of defining an international reference system,
including reference procedures and primary and secondary reference materials,
in collaboration with the Institute of Reference Materials and Measurements
(IRMM). It was suggested by the IFCC/IRMM that the First WHO International
Reference Reagent for haemoglobin A2 (89/666) might be withdrawn when a
new reference material had been developed with the IRMM.
The issue of commutability of the current International Reference Reagent
was considered to be unresolved due to insufficient information. It was agreed
that further information be obtained from diagnostics manufacturers on the
use of this reagent, and on any related issues. It was also suggested that the
information provided in the instructions for use for the current International
Reference Reagent should be updated.
The Committee recommended that WHO communicate its procedures
to the IFCC/IRMM for the establishment of international reference materials
and standards. The Committee further advised that the instructions for use for
the current International Reference Reagent be critically reviewed.
29
in vitro diagnostic device reagents
Reagents – in vitro diagnostic device reagents
6.1.1 Third WHO International Standard for HIV‑1 for NAT‑based assays
Currently the Second WHO International Standard for HIV-1 for assays based on
nucleic acid amplification techniques (NATs) is in place but stocks are running
low due to high demand for this material. Therefore, a replacement standard
(10/152) comprising heat-inactivated (1 hour at 60 °C) HIV-1 subtype B virus
diluted in citrated human plasma had been prepared. Heat inactivation had been
shown to have no negative effect on the detectability of HIV RNA by NAT-based
assays. After lyophilization, the candidate standard was characterized in an
international collaborative study (WHO/BS/2011.2178) together with the current
International Standard, and the candidate represented in coded duplicates.
Fifteen laboratories from 11 countries returned 17 data sets from 10 different
assay systems (six quantitative and four qualitative). The results showed excellent
correlation between the different assays used. Based on the results of this study,
it was proposed that candidate material 10/152 was suitable as a replacement
standard and should be established as the Third WHO International Standard
for HIV1-RNA for NAT-based assays and be assigned a unitage of 185 000 IU/ml
(5.27 log10 IU/ml).
The Committee recommended the adoption of candidate material 10/152
as the Third WHO International Standard for HIV1-RNA for NAT-based assays,
with an assigned unitage of 185 000 IU/ml.
6.1.2 Third WHO International Standard for hepatitis B

virus for NAT‑based assays
This International Standard is used by in vitro diagnostic device manufacturers,

blood transfusion centres, control authorities and clinical laboratories to
calibrate secondary reference materials and in the validation of hepatitis B virus
NAT-based assays. As stocks of the Second WHO International Standard for
hepatitis B virus for NAT-based assays are diminishing a replacement is required.
Two candidate materials were therefore prepared from the same source
(Eurohep R1 preparation; genotype A2) as that used for the current International
Standard with a targeted concentration of 10 6 IU/ml. Both bulk materials were
lyophilized as 0.5 ml aliquots, with post-fill testing including assessments of
residual moisture and oxygen content by non-invasive near-infrared (NIR)
spectroscopy. A collaborative study (WHO/BS/2011.2170) involving 16 laboratories
from nine countries was conducted to evaluate the suitability and potency of
30
International reference materials – in vitro diagnostic device reagents
the two candidate freeze-dried preparations (NIBSC codes 10/264 and 10/266) in
parallel with the Second WHO International Standard for hepatitis B virus for
NAT-based assays using a range of such assays. When the results were expressed
in IU/ml directly relative to the concurrently tested International Standard, the
overall mean estimates from the quantitative assays were 5.93 (10/264) and 5.98
(10/266) log10 IU/ml. The results of qualitative assays were characterized by
higher levels of variation.
It was proposed that the candidate standard 10/264 be established as the
Third WHO International Standard for hepatitis B virus DNA for NAT-based
assays, with an assigned potency of 850 000 IU/ml (~5.93 log10 IU/ml). It was
noted that the second candidate (10/266) would also be a suitable potential
replacement in due course, depending upon ongoing stability assessments.
The Committee recommended the adoption of preparation 10/264 as the
Third WHO International Standard for hepatitis B virus DNA for NAT-based
assays, with an assigned potency of 850 000 IU/ml.
6.1.3 First WHO International Reference Panel for hepatitis B

virus genotype for HBsAg assays
Infection with the hepatitis B virus (HBV) is a major global health problem and
the most common cause of liver cirrhosis and hepatocellular cancer. About two
billion people worldwide have been infected with the virus and about 350 million
live with chronic infection. An estimated 600 000 people die each year due to
the chronic consequences of hepatitis B. The HBV is preferentially transmitted
through contact with blood or other body fluid from an infected person. Sensitive
screening and accurate diagnostic assays play a crucial role in the prevention and
management of the disease.
The current International Standards for hepatitis B surface antigen
(HBsAg) and for HBV DNA are both based on HBV-genotype A2 material.
Concerns have been raised over the extent to which such preparations are
representative of other HBV-genotypes (A–H) which are widely distributed
globally, with clear regional variations observed in their distribution. In order
to address this issue, two HBV-genotype panels were designed for use with HBV
NAT-based assays or HBsAg assays. The first HBV-genotype panel, designed
for use with HBV NAT-based assays, was established by the 2009 Committee.
The HBV-genotype panel for use with HBsAg assays consists of 15 members
representing the HBV genotypes A(3), B(2), C(3), D(3), E, F(2) and H. The
panel members were derived from plasma samples collected worldwide with
the majority of infectious HBV particles removed by ultracentrifugation.
The HBsAg content of the source materials was determined by different
methods, including chemiluminescent immunoassay, quantitative immune
electrophoresis and antigen purification. The resulting HBsAg content was
31
expressed in IU/ml, PEI u/ml and ng/ml. Following dilution of the source
materials, aliquoting and lyophilization, the panel members were characterized
against the current First WHO International Standard for hepatitis B surface
antigen. A resulting collaborative study (WHO/BS/2011.2180) resulted in 28
data sets from 15 laboratories, covering 20 different HBsAg assays. The results
obtained demonstrated the consistent detection of HBV genotypes A–F and H
by the majority of the test kits investigated, with some assays showing genotype-
dependent effects on detection efficiency. This panel would be a very useful tool
for regulatory authorities in assessing the relative HBsAg-detection efficiency in
regard to HBV genotypes prevalent in their respective regions.
The Committee adopted the First WHO International Reference Panel
for hepatitis B virus genotypes for use with HBsAg assays on the understanding
that the instructions for use would be revised in compliance with the format used
for the HBV genotype panel for use with HBV NAT-based assays.
6.1.4 Fourth WHO International Standard for hepatitis C

Infection with the hepatitis C virus (HCV) remains a major public health
problem worldwide. Approximately 130–170 million people (2.2–3% of the
world’s population) are infected with HCV. Chronic infection can lead to liver
cirrhosis, liver failure and hepatocellular carcinoma. Although, the introduction
of routine screening for HCV antibody from the late 1980s greatly reduced the
risk of transmission via infected blood and blood products, there remained a
major risk of transfusion-transmitted infection due to window-period donations.
As a result, NAT-based assays were introduced to detect HCV RNA in human
plasma as part of the safety testing of blood and blood products, and are now
mandated in many parts of the world. HCV NAT-based assays are also widely
used in the clinical management of HCV, particularly for diagnosing infection
and monitoring response to antiviral therapies. A range of both commercial and
laboratory-developed NAT-based assays are currently in use.

It is proposed that the current Third WHO International Standard for
hepatitis C virus for NAT-based assays now be replaced by the freeze-dried
preparation 06/102. This proposed replacement was prepared in 2006 from
the same bulk as the current International Standard (06/100) but was filled
and freeze-dried in a separate processing run. Both preparations containing
hepatitis C virus (HCV) genotype 1a from anti-HCV-negative window phase
donations were characterized in a worldwide collaborative study in 2007, and
exhibited HCV-RNA concentrations of 5.19 (06/100) and 5.41 log10 IU/ml
(06/102). In a more recent study (WHO/BS/2011.2173) seven laboratories from
five countries participated in evaluating the potency and real-time stability of
06/102 by comparing it with both the current (06/100) and previous (96/798)
32
Reference Preparations. Surprisingly the mean potency estimates were 5.24,

5.08 and 5.05 log10 IU/ml respectively – for the samples 06/102 and 06/100 there
was a drop in potency of approximately 0.2 log10 when compared to the results
obtained in the original 2007 collaborative study, while sample 96/798 appeared
to be still stable. Subsequent investigations provided evidence of the degradation
of HCV RNA in 06/102 and 06/100 under elevated temperature during shipping,
rather than a deterioration of the product during storage at –20 °C. Consequently,
the candidate preparation is considered as a suitable replacement Fourth
WHO International Standard for hepatitis C virus for NAT-based assays when
maintained at or below –20 °C. Furthermore, it was agreed that the concentration
determined in the 2007 collaborative study should be applied.
It was therefore proposed that the candidate sample 06/102 be established
as the Fourth WHO International Standard for hepatitis C virus RNA for
NAT-based assays with an assigned potency of 260 000 IU/ml (~5.41 log10 IU/ml)
when reconstituted in 0.5 ml of nuclease-free water. As shipment with dry ice
and storage of the material at –20 °C would be a vital requirement, it was
further suggested that priority should be given to its future replacement with a
temperature-stable preparation. The problem of air entry into crimp-sealed vials
during storage was also discussed, and it was agreed that efforts should be made
to investigate the stability of other International Standards stored in such vials.
The Committee recommended the adoption of preparation 06/102 as the
Fourth WHO International Standard for hepatitis C virus RNA for NAT-based
assays, with an assigned potency of 260 000 IU/ml.
6.1.5 First WHO International Standard for hepatitis E

Hepatitis E virus (HEV) is a major cause of acute hepatitis and raises major
public health concerns in endemic areas. HEV is also an emerging (or more-
recognized) infection in developed countries. Certain patient populations (for
example, individuals with liver disease, pregnant women and immune-suppressed
patients) suffer from increased mortality rates associated with HEV infections.
Chronic HEV infections are also being increasingly recognized. Viral load testing
is important in the evaluation of antiviral therapy regimes, while NAT-based
assays are also used as sensitive assays for the detection of recent infection. In
humans four main HEV genotypes (1–4) have been described, with two (3 and 4)
also associated with animal infections.
An initial collaborative study involving 20 laboratories from 10 countries
was conducted to assess the consistency of results obtained by HEV NAT-based
assays when testing dilution series of four HEV-positive human plasma materials.
Wide variations in assay sensitivity (typically ranging from 100–1000-fold
differences) were observed between different NAT-based assays, with significant
33
variations in reporting of results. A candidate International Standard preparation

(6329/10) was selected from this study and alongside another preparation
designed as national standard for Japan (HEV genotype 3b) was chosen for
detailed characterization in a second collaborative study involving 24 laboratories
from 10 countries (WHO/BS/2011.2175). This study confirmed the feasibility of
standardizing HEV NAT-based assays using the candidate preparation 6329/10.
Commutability would be addressed in a further study where the candidate
would be tested in selected assays alongside a set of HEV-positive patient
samples. The current report would also be supplemented with a consideration
of the stability of the preparation (preliminary data are already present), more
detailed lyophilization conditions and a summary of the potency data in tables.
It was proposed that 6329/10 be established as the First WHO International
Standard for hepatitis E virus RNA for NAT-based assays with an assigned
unitage of 250 000 IU/ml.
The Committee recommended the adoption of preparation 6329/10 as
the First WHO International Standard for hepatitis E virus for NAT-based assays,
with an assigned unitage of 250 000 IU/ml and with the proviso that the report
would be updated as described.
6.1.6 First WHO International Reference Standard for

anti‑Trypanosoma cruzi antibodies
Chagas disease (human American trypanosomiasis) is caused by infection with
the protozoan parasite Trypanosoma cruzi which is endemic in Latin America
and is transmitted by triatomine bugs. It is estimated that around 10 million
people are currently infected by T. cruzi, and more than 10 000 people die from its
chronic clinical manifestations every year. Large-scale insect-control programmes
in Central and South American countries, together with the mandatory screening
of blood donations for anti-T. cruzi antibodies, have reduced the incidence and
prevalence of the disease. Two groups of T. cruzi are differentiated, with T. cruzi I
being mainly prevalent north of the Amazon basin (for example in Mexico)
whereas T. cruzi II is more prevalent in southern Latin American countries
(for example, Brazil and Chile). However, these two groups have still not been
characterized for genetic and antigenic differences. A small number of anti-Chagas
seropositive plasma samples obtained respectively from autochthonous blood
donors from either Mexico or from Brazil and/or Chile were pooled to result
in presumably anti-T. cruzi I-positive and anti-T. cruzi II-positive preparations
respectively. The two preparations were aliquoted, lyophilized and provided as
coded materials for a global collaborative study involving 24 laboratories from
16 countries (WHO/BS/2011.2181) with the inclusion of different serological
assays used for anti-Chagas screening, serodiagnosis or supplemental information
provision. The preparations were found suitable for assessing the analytical
sensitivities of the assays.
34
It was proposed to assign distinct unitages of 1 IU/ml (undiluted, after

reconstitution) to each of the materials and to establish them as the First WHO
International Reference Standard for anti-Trypanosoma cruzi antibodies to be
used concomitantly. It was understood that neither the anti-T. cruzi II unitage
of the anti-T. cruzi I reference standard nor the anti-T. cruzi I unitage of the
anti-T. cruzi II reference standard had been determined.
Preparation 09/186 is defined as the anti-T. cruzi antibody standard
representative of the region where T. cruzi II is predominant, while preparation
09/188 is defined as the anti-T. cruzi antibody standard representative of the
region where T. cruzi I is predominant.
The Committee recommended the concomitant adoption of preparations
09/186 and 09/188 as the First WHO International Reference Standard for anti-
Trypanosoma cruzi antibodies, each with an assigned unitage of 1 IU/ml and
subject to final editing of the instructions for use.
6.1.7 First WHO International Standard for

Epstein–Barr virus for NAT‑based assays
Both proficiency-testing programmes and feedback from the clinical diagnostic
community have indicated a need for the standardization of Epstein–Barr virus
(EBV) viral load assays. Viral load measurements are important in the prevention,
diagnosis and monitoring of EBV infections. With non-standardized NAT-based
assays currently in use it is not easily possible to compare clinical practice at
different sites and to standardize patient management. Whole B95-8 virus was
chosen as a candidate strain in order to allow for the standardization of the entire
assay, including extraction. The candidate preparation (09/260; ~1×107 copies/ml)
was formulated in universal buffer for subsequent dilution in the appropriate
sample matrices used in the routine testing of plasma, serum and whole blood.
The candidate was evaluated both as lyophilized and frozen liquid preparations
alongside EBV-positive Raji and Namalwa cells.
A collaborative study was conducted involving 28 laboratories from
16 countries (WHO/BS/2011.2172) selected for experience in EBV NAT-based
assays and for geographical representativeness, and including in vitro diagnostic
device manufacturers and clinical, reference and research laboratories. In total,
38 datasets (36 quantitative assays and two qualitative assays) were returned
and used for the statistical evaluation. The analysis confirmed the wide range of
results reported for the same preparation by different assays (as copies, genome
equivalents etc.) independent of the nature of the specimen (virus or cells). When
lyophilized B95-8 EBV preparation was used as a common standard material,
the results for the frozen liquid preparation became quite consistent between
the different assays. However, for the cell preparations (Raji and Namalwa)
this standardization effect appeared to be less pronounced despite a clear
35
improvement in the overall consistency of result reporting for the different assays.
This clear difference in the consistency of reporting plasma or cell-associated
loads of EBV by different NAT-based assays will be investigated further by
NIBSC. At a Standardization of Genome Amplification Techniques (SoGAT)
Clinical Diagnostics meeting, potential users proposed the establishment of
this preparation as the WHO standard despite the standardization limitations
observed in the analysis of cell-associated EBV. It was therefore proposed that
the candidate standard 09/260 be established as the First WHO International
Standard for Epstein–Barr virus for NAT-based assays with an assigned unitage
of 5×10 6 IU when reconstituted in 1 ml of nuclease-free water. The potential need
to develop an EBV type-2 standard was noted.
It was also suggested that the final edited instructions for use should
address: (a) the limited ability of the standard to control for efficiency of
extraction of cell-associated EBV DNA; (b) the possibility that methylation of
cell-associated virus DNA could cause artefactually reduced detection, compared
with NAT-based assays; and (c) the fact that the current standard is a type-1 EBV.
The Committee recommended the adoption of preparation 09/260 as
the First WHO International Standard for Epstein–Barr virus for NAT-based
assays, with an assigned unitage of 5×10 6 IU when reconstituted in 1 ml of
nuclease-free water.
6.2 Proposed new projects – in vitro diagnostic device reagents

6.2.1 Third Meeting of WHO Collaborating Centres for Biological Standards
The Third Meeting of WHO Collaborating Centres for Biological Standards
was hosted by NIBSC on 7–8 March 2011 and was attended by representatives
from FDA, NIBSC, PEI and WHO. The aim of the meeting was to strengthen
cooperation between the collaborating centres, and to share knowledge and
resources in order to advance the field of standardization of in vitro diagnostic
devices.
The commutability of Reference Preparations for use in the calibration
of diagnostic tests was reported to be an important area of scientific debate.
The collaborating centres proposed that WHO convene a meeting to review
the principles of commutability and their applicability to WHO International
Standards. Meeting participants would ideally include experts in the field
of metrology, with the overall goal of the meeting being the development of
guidelines on evaluating commutability during the validation of international
reference materials for in vitro diagnostic devices (including clinical diagnostics
and infectious-disease tests). Updates were then provided of ongoing collaborative
studies to evaluate new or replacement standards.
The Committee endorsed the proposal to convene a meeting on the
commutability of Reference Preparations for in vitro diagnostic devices.
36
6.2.2 Replacement of the First WHO International Standard

for hepatitis A virus for NAT‑based assays
The hepatitis A virus (HAV) is responsible for approximately half of all cases of
hepatitis diagnosed worldwide. It is also recognized as one of the most important
human foodborne pathogens.
The current International Standard (00/560) was prepared from a wild-
type isolate (representing HAV genotype 1a) derived from human plasma, and
was filled and freeze-dried in 2001. A potential replacement candidate (00/562)
was also prepared from the same bulk. At the current rate of depletion, it is
estimated that the stock of 00/560 will need to be replaced in ~2–3 years. However,
following its storage at +4 °C for five years and 10 months, a stability assessment
of samples of 00/562 suggested that there was some loss of potency (~1 log10 ).
Another more recent stability assessment of accelerated thermal degradation of
samples of 00/560 and 00/562 stored at +4 °C for nine years and 10 months also
suggested that 00/562 might not be the optimal replacement candidate.
NIBSC was therefore in the process of sourcing HAV RNA-positive
(genotype 1a) human plasma sample(s) negative for other viral markers (such
as HBV, HCV, HIV and B19V) for the design and manufacturing of a suitable
replacement standard. Once a suitable material had been identified, a collaborative
study involving approximately 20 laboratories would be conducted.
The Committee endorsed the proposed project (WHO/BS/2011.2179) to
develop a replacement for the First WHO International Standard for hepatitis A
virus for NAT-based assays.
6.2.3 Replacement WHO International Standard for hepatitis B e‑antigen

HBV infection is a major health problem worldwide with an estimated 350 million
chronic carriers. It is estimated that around one third of the human population has
encountered the virus. Hepatitis B e-antigen (HBeAg) is a diagnostic marker for
HBV infectivity that circulates in the blood when the virus is actively replicating,
and is thus indicative of both viral replication and potential infectivity. The
marker is used to determine the status of HBV infection and to monitor patients.
European legislation (Common Technical Specifications 2009/886/EC) requires
traceability of in vitro diagnostic test results to a higher-order standard. Since
1982 the PEI has provided an HBeAg standard for the determination of the
analytical sensitivity of HBeAg tests used by manufacturers worldwide. There
is a continuous need for the standard, with the material historically having been
assigned a PEI unit (u/ml). The PEI material now had to be replaced, ideally by
an International Standard expressed in IU/ml.
develop a replacement WHO International Standard for HBeAg.
37
6.2.4 Replacement WHO International Standard for hepatitis B e‑antibodies

HBV infection is spread worldwide with around two billion people having been
infected with the virus and about 350 million people living with chronic infection.
The diagnosis of HBV requires a combination of various tests including the
detection of antibodies to hepatitis B e-antigen (anti-HBeAg). The anti-HBeAg
test is particularly meaningful in association with the HBeAg test for monitoring
the course of HBV infection.
Anti-HBeAg preparations are used for the validation, standardization,
quality control and comparison of anti-HBe tests. The PEI anti-HBeAg reference
serum established in 1982 is used by in vitro diagnostic device manufacturers
worldwide. As with HBeAg there is a continuous need for an anti-HBeAg
standard with the material historically having been assigned a PEI unit (u/ml).
The PEI material now had to be replaced, ideally by an International Standard
expressed in IU/ml.
develop a replacement WHO International Standard for anti-HBeAg.
6.2.5 First WHO International Reference Panel for hepatitis E

virus genotypes for NAT‑based assays
Hepatitis E virus (HEV) is represented by a single serotype but can be classified
into at least four main genotypes (1–4). While HEV genotypes 1 and 2 are
exclusively detected in humans, genotypes 3 and 4 are found in both humans and
in a range of other animals (for example, pigs, wild boar, deer and rodents). The
geographical distribution of HEV genotypes is complex and there is approximately
74% nucleotide identity between genotypes. Once the First WHO International
Standard for hepatitis E virus for NAT-based assays has been established, a bias
towards the sensitive detection of HEV genotype 3a (as included in the WHO
candidate preparation) cannot be ruled out.
A tool to assess genotype specificity and the relative genotype sensitivity

of HEV NAT-based assays is therefore needed, especially for assays used in
regions with a high prevalence and incidence of one or more genotypes other
than genotype 3a. Laboratories in such regions should be included in the study.
The HEV genotype panel project aims to incorporate different HEV
genotypes and sub-genotypes into a reference panel which may be very helpful
for assessing assay features. Currently, different materials representing three
genotypes are already available at PEI, and efforts are being undertaken to obtain
source material representing the missing genotype 2.
The Committee endorsed the proposed project (WHO/BS/2011.2179)
to develop a First WHO International Reference Panel for hepatitis E virus
genotypes for NAT-based assays.
38
6.2.6 Replacement of the First WHO International

Reference Panel for HIV‑1 subtypes
The First WHO International Reference Panel for HIV-1 subtypes (01/466)
was established by the Committee in 2003. This original panel was a liquid
preparation stored at –80 °C and was composed of different subtypes of HIV-1
(A, B, C, D, AE, F, G and AGH) as well as representatives of group N and O.
The replacement panel would be of the same composition and from the same
isolates but would undergo heat inactivation (1 hour at 60 °C) and lyophilization.
Full-length sequences had been published previously for these isolates and the
sequences would be reconfirmed by NIBSC. A full collaborative study would also
be performed.
to develop a replacement for the First WHO International Reference Panel for
HIV-1 subtypes.
6.2.7 First WHO International Reference Panel for HIV‑1

circulating recombinant forms
The diversity of HIV was well documented with increasing epidemiological
evidence of inter-subtype recombinant forms of HIV-1 strains circulating in
different parts of the world. It was now widely appreciated that the recombinant
nature of HIV had allowed for the evolution of circulating recombinant forms
(CRFs) of HIV in which the virus may exhibit different genotypes across genomic
regions. There was therefore a need to establish respective panels to be able to
perform state-of-the-art assessments of HIV-1 using NAT-based assays. It was
intended that high-titre stocks (grown in cell culture) of HIV-1 subtypes G, H, J,
K , group O and a range of CRFs would be used as panel members. These would
be diluted in negative human plasma and heat inactivated prior to lyophilization.
The panel would be evaluated in a full international collaboration involving
countries known to have a variety of genotypes in circulation.
to develop a First WHO International Reference Panel for HIV-1 circulating
recombinant forms.
6.3 Ongoing stability monitoring

6.3.1 Prostate‑specific antigen
The principle application of International Standards in this area is the calibration
of diagnostic assays for prostate-specific antigen (PSA). As part of their
original establishment, PSA standards were recommended for ongoing stability
monitoring. In the absence of frozen baselines for such standards, a second
accelerated degradation study was performed (WHO/BS/2011.2161). At 12 years
39
post-fill, ampouled materials (stored at elevated temperatures for 13 months)

were compared with ampoules stored at –20 °C. A lack of degradation at +4 °C
and +20 °C precluded a prediction of degradation rate using the Arrhenius
equation but supported the predictions on stability made at establishment. For
samples stored at –20 °C the predicted degradation rates were 0.042% per year
(PSA free) and 0.027% per year (PSA 90:10).
As the remaining stocks of these standards were low and were predicted
to last no more than 4–5 years, no further stability assessment was proposed.
The Committee noted that these stability studies had confirmed the
predictions made at the time of standard establishment. The Committee asked
that this information be included in shipments of this material.
40
biotherapeutics other than blood products
7.1 WHO International Standards and Reference Reagents –

biotherapeutics other than blood products
7.1.1 First WHO International Standard for transforming growth factor beta‑3
Transforming growth factor beta-3 (TGF-β3) is a member of the TGF-β
superfamily (30 proteins) which includes activins, bone morphogenetic proteins
and growth and differentiation factors. Three closely related isoforms exist in
mammals – TGF-β1, -β2 and -β3 – each is derived from a distinct gene but
exhibits greater than 70% sequence homology and similar functional responses
in vitro. Produced by several immune and non-immune cell types such as
fibroblasts, endothelium, smooth muscle cells, TGF-β acts in an autocrine
and paracrine manner to regulate multiple cellular processes, such as immune
function, proliferation, differentiation, extracellular matrix production, migration
and survival.
The therapeutic potential of TGF-β3 in the treatment of oral mucositis
and in the prevention and reduction of scarring following surgical procedures and
wound repair has been shown in various phase I/II clinical trials. Additionally,
TGF-β3 appears to be a potentially useful growth factor in engineered
organogenesis and in the context of regenerative medicine.
Based on its activities (pro-inflammatory, immunosuppressive), TGF-β
has also been implicated in many human diseases, including certain cancers,
onset of several autoimmune diseases including joint destruction in arthritis
and in pathogenesis of fibrotic diseases associated with skin, lung, liver and
eye (cornea).
A reference standard for TGF-β3 could facilitate measurement of the
potency and stability of therapeutic preparations or for preparations in use in
regenerative medicine, its antagonists and in addition, measurement of TGF-β3
levels for research purposes. Currently, a Reference Reagent for TGF-β3 (98/608)
is available from NIBSC with an arbitrary unitage of 10 000 IU/ampoule. The
objective of the current study (WHO/BS/2011.2163) was therefore to characterize
a candidate International Standard for the bioassay of human TGF-β3 and assign
a unitage for its activity.
The results clearly indicated that the candidate preparation 09/234 was
suitable for use as an International Standard for TGF-β3. It was proposed that
the new international unit should preserve continuity with the existing NIBSC
Reference Reagent (98/608) with a proposed value of 19 000 IU/ampoule to
be assigned.
41
The Committee endorsed the proposal to establish the preparation 09/234

as the First WHO International Standard for transforming growth factor beta-3,
with an assigned unitage of 19 000 IU/ampoule. The Committee requested that
future studies be conducted with mammalian cell expressed material, when it
became available, to assess the performance of the standard.
7.2 Proposed new projects – biotherapeutics

other than blood products
7.2.1 Replacement of the First WHO International Standard for interleukin‑2
The current First WHO International Standard for interleukin-2 (86/504; 100 IU/
ampoule) is used for the potency labelling of interleukin-2 products approved
for the treatment of metastatic renal cell carcinoma and metastatic melanoma
patients. Additionally, based on its ability to induce proliferation of CD4+ cells, it
has been clinically tested in HIV-positive patients either alone or as combination
therapy with antiviral agents. Based on promising results from preliminary studies
using combination therapy, further clinical trials were ongoing in HIV-positive
patients. As stocks of the standard preparation would be almost exhausted in
2014 a replacement was required.
A proposed multi-centre international study (WHO/BS/2011.2177)
would include the evaluation of two candidate preparations from the previous
collaborative study, predominantly using bioassays. Unitage would be assigned
relative to the current International Standard. One of the candidate rDNA-derived
human interleukin-2 (86/564) preparations from the previous collaborative study
was currently being distributed with no reported issues. Limited stability studies
after 22 years of lyophilization had indicated no loss of biological activity.
The Committee endorsed the proposal to evaluate the suitability of a
new preparation of interleukin-2 as the Second WHO International Standard for
interleukin-2.
7.2.2 Replacement of the Second WHO International Standard

for recombinant human erythropoietin
Erythropoietin (EPO) is used globally in the treatment of anaemia. Defining
a standard IU value for EPO activity enables manufacturers to correctly label
the potency of therapeutic EPO products used to treat anaemia in end-stage
renal disease and anaemia associated with cancer and other diseases. As stocks
of the current Second WHO International Standard for recombinant human
erythropoietin (rhuEPO) were running low there was an urgent requirement for
a new International Standard.
Participants in the proposed collaborative study (WHO/BS/2011.2177)
would be asked to calibrate the candidate standard in terms of the current standard
42
International reference materials – biotherapeutics other than blood products
using polycythaemic or normocythaemic mouse bioassay. Confirmatory data

would also be requested from physicochemical analyses.
The Committee endorsed the proposal to evaluate the suitability of a new
preparation of rhuEPO as the Third WHO International Standard for recombinant
human erythropoietin.
7.2.3 Replacement of the Fourth WHO International Standard for urinary

follicle‑stimulating hormone and urinary luteinizing hormone
Urinary follicle-stimulating hormone (FSH) and luteinizing hormone (LH)
products are manufactured and marketed worldwide. The current International
Standard defines the IU value which is essential for the correct potency labelling
of the therapeutic preparations of urinary FSH/LH used to treat anovulation in
women and hypo- or normo-gonadotrophic hypogonadism in men. It is also
used for ovarian hyperstimulation as part of assisted reproductive technologies.
Strong demand for low-cost preparations, particularly for assisted reproductive
technologies in countries where it is not funded by government or healthcare
plans, necessitates effective standardization.
As stocks of the current Fourth WHO International Standard for urinary
follicle-stimulating hormone and urinary luteinizing hormone (98/704) were
almost exhausted there was an urgent requirement for a new International
Standard.
Participants in the proposed collaborative study (WHO/BS/2011.2177)
would be asked to calibrate the standard in terms of the current standard using
rat ovarian weight gain (for FSH) and immature rat seminal vesicle weight
gain (for LH) bioassays. Confirmatory data would also be requested from in
vitro bioassays.
The Committee endorsed the proposal to evaluate the suitability of a new
preparation of FSH/LH as the Fifth WHO International Standard for urinary
follicle-stimulating hormone and urinary luteinizing hormone.
7.2.4 Replacement of the Second WHO International Standard

for luteinizing hormone (human, pituitary)
Licensed LH immunoassays are an established component of the battery of
clinical endocrinology tests used in the diagnosis of a range of diseases. For
example, measurements of LH are used in the diagnosis of hypothalamic,
pituitary or gonadal dysfunction. In addition, LH levels are used to determine
menopause, pinpoint ovulation and monitor endocrine therapy. As stocks of the
current Second WHO International Standard for luteinizing hormone (human,
pituitary) are running low there is a requirement for a replacement International
Standard for the calibration of LH immunoassays.
43
As part of the proposed collaborative study (WHO/BS/2011.2177) the

candidate preparation would be value-assigned against the current standard
using commercial immunoassay methods.
new preparation of LH as the Third WHO International Standard for luteinizing
hormone, human, pituitary, and noted that this would be the last natural LH
standard.
44
8. International reference materials – antibiotics
8.1 WHO International Standards and

Reference Reagents – antibiotics
8.1.1 Third WHO International Standard for dihydrostreptomycin
Dihydrostreptomycin is an antibiotic consisting of a hydrogenated form of
streptomycin. It is used in the prophylaxis of tuberculosis and tularaemia, and for
the treatment of infections caused by Gram-negative bacteria. By preventing the
initiation of protein synthesis through binding to the 30S subunit of the bacterial
ribosome it results in the death of microbial cells. Humans and other mammals
have structurally different ribosomes from bacteria thus explaining the selective
action of this antibiotic.
The Second WHO International Standard for dihydrostreptomycin was
established in 1964 on the basis of an international collaborative study. A potency
of 820 IU/mg was assigned, with each ampoule containing approximately 200 mg.
As stocks of the current standard are becoming exhausted, the EDQM – which
acts as the WHO custodian laboratory for antibiotics – was requested by the
Committee to undertake the appropriate steps for its replacement.
Using the current standard as a reference, participating laboratories in
an international collaborative study (WHO/BS/2011.2176) were requested to
estimate the potency and suitability of a candidate standard (EDQM code number
ISA_42688). It was concluded that the candidate material should be proposed as
the Third WHO International Standard for dihydrostreptomycin and assigned
an activity value of 19 425 IU per vial.
The Committee recommended that ISA_42688 be established as the
Third WHO International Standard for dihydrostreptomycin, with an assigned
activity value of 19 425 IU/vial.
8.2 Proposed new projects – antibiotics

8.2.1 Replacement of the First WHO International Standard for neomycin B
Neomycin B is a globally marketed antibiotic which requires the use of an
International Standard to enable quality-control assessments to be undertaken
by manufacturers and NCLs. It was envisaged that stocks of the First WHO
International Standard for neomycin B would be exhausted by 2012–2013.
Manufacturers and public control laboratories from all over the world
would be invited to participate in a collaborative study (WHO/BS/2011.2177) to
establish a replacement standard.
candidate Second WHO International Standard for neomycin B.
45
8.2.2 Replacement of the Second WHO International Standard for neomycin

Neomycin is another globally marketed antibiotic which requires the use of an
International Standard to enable quality-control assessments to be undertaken
by manufacturers and NCLs. It was envisaged that stocks of the Second WHO
International Standard for neomycin would be exhausted by 2012–2013.
Manufacturers and public control laboratories from all over the world
would be invited to participate in a collaborative study (WHO/BS/2011.2177) to
establish a replacement standard.
candidate Third WHO International Standard for neomycin.
46
Annex 1
WHO Recommendations, Guidelines and other documents
related to the manufacture and quality control of
biological substances used in medicine
The Recommendations (previously called Requirements) and Guidelines published
by WHO are scientific and advisory in nature but may be adopted by an NRA as
national requirements or used as the basis of such requirements.
These international Recommendations are intended to provide guidance
to those responsible for the production of biologicals as well as to others who
may have to decide upon appropriate methods of assay and control to ensure that
these products are safe, reliable and potent.
Recommendations concerned with biological substances used in medicine
are formulated by international groups of experts and are published in the WHO
Technical Report Series1 as listed below. A historical list of Requirements and
other sets of Recommendations is available on request from the World Health
Organization, 20 avenue Appia, 1211 Geneva 27, Switzerland.
Reports of the Expert Committee on Biological Standardization published
in the WHO Technical Report Series can be purchased from:
WHO Press
World Health Organization
20 avenue Appia
1211 Geneva 27
Switzerland
Telephone: +41 22 791 3246
Fax: +41 22 791 4857
E-mail: [email protected]
Web site: http://www.who.int/bookorders
Individual Recommendations and Guidelines may be obtained free of
charge as offprints by writing to:
Department of Essential Medicines and Health Products
20 avenue Appia
1211 Geneva 27
Switzerland
1
Abbreviated in the following pages to “TRS”.
47
Recommendations, Guidelines and other Reference

documents
Animal cells, use of, as in vitro substrates for the Revised 2010, TRS 978 (2013)
production of biologicals
BCG vaccine, dried Revised 2011, TRS 979 (2013)
Biological products: good manufacturing Adopted 1991, TRS 822 (1992)
practices
Biological products prepared by recombinant Adopted 1990, TRS 814 (1991)
DNA technology
Biological standardization and control: a scientific Unpublished document WHO/
review commissioned by the UK National BLG/97.1
Biological Standards Board (1997)
Biological substances: International Standards Revised 2004, TRS 932 (2006)

and Reference Reagents, guidelines for the
preparation, characterization and establishment
Biotherapeutic products, similar Adopted 2009, TRS 977 (2013)
Blood, blood components and plasma Revised 1992, TRS 840 (1994)
derivatives: collection, processing and quality
control
Blood establishments: good manufacturing Adopted 2010, TRS 961 (2011)
practices
Blood plasma, human, for fractionation Adopted 2005, TRS 941 (2007)
Blood plasma products, human viral inactivation Adopted 2001, TRS 924 (2004)
and removal procedures
WHO Technical Report Series No. 979, 2013
Blood regulatory systems, assessment criteria for Adopted 2011, TRS 979 (2013)
national
Cholera vaccine (inactivated, oral) Adopted 2001, TRS 924 (2004)
Dengue tetravalent vaccine (live, attenuated) Revised 2011, TRS 979 (2013)
Diphtheria, tetanus, pertussis (whole cell), and Revised 1989, TRS 800 (1990);
combined (DTwP) vaccines Addendum 2003, TRS 927 (2005);
Addendum 2005, TRS 941 (2007)
DNA vaccines: assuring quality and nonclinical Revised 2005, TRS 941 (2007)
safety
Haemophilus influenzae type b conjugate Revised 1998, TRS 897 (2000)
vaccines
48
Annex 1

documents
Haemorrhagic fever with renal syndrome (HFRS) Adopted 1993, TRS 848 (1994)
vaccine (inactivated)
Hepatitis A vaccine (inactivated) Adopted 1994, TRS 858 (1995)
Hepatitis B vaccine prepared from plasma Revised 1987, TRS 771 (1988)
Hepatitis B vaccines made by recombinant DNA Revised 2010, TRS 978 (2013)
techniques
Human interferons prepared from Adopted 1988, TRS 786 (1989)
lymphoblastoid cells
Influenza, biosafety risk assessment and safe Adopted 2005, TRS 941 (2007)
production and control for (human) pandemic
vaccines
Influenza vaccine (inactivated) Revised 2003, TRS 927 (2005)
Influenza vaccine (live) Revised 2009, TRS 977 (2013)
Influenza vaccines, human, pandemic, regulatory Adopted 2007, TRS 963 (2011)
preparedness
Japanese encephalitis vaccine (inactivated) for Revised 2007, TRS 963 (2011)
human use
Japanese encephalitis vaccine (live) for Adopted 2000, TRS 910 (2002)
human use
Louse-borne human typhus vaccine (live) Adopted 1982, TRS 687 (1983)
Measles, mumps and rubella vaccines and Adopted 1992, TRS 848 (1994);
combined vaccine (live) Note TRS 848 (1994)
Meningococcal A conjugate vaccines Adopted 2006, TRS 962 (2011)
Meningococcal C conjugate vaccines Adopted 2001, TRS 924 (2004);
Addendum (revised) 2007,
TRS 963 (2011)
Meningococcal polysaccharide vaccine Adopted 1975, TRS 594 (1976);
Addendum 1980, TRS 658 (1981);
Amendment 1999, TRS 904 (2002)
Monoclonal antibodies Adopted 1991, TRS 822 (1992)
Papillomavirus vaccine, human Adopted 2006, TRS 962 (2011)
Pertussis vaccine (acellular) Revised 2011, TRS 979 (2013)
Pertussis vaccine (whole-cell) Revised 2005, TRS 941 (2007)
49

documents
Pharmaceutical products, storage and transport Adopted 2010, TRS 961 (2011)
of time- and temperature-sensitive
Pneumococcal conjugate vaccines Revised 2009, TRS 977 (2013)
Poliomyelitis vaccine (inactivated) Revised 2000, TRS 910 (2002);
Poliomyelitis vaccine (inactivated): guidelines Adopted 2003, TRS 926 (2004)
for the safe production and quality control of
inactivated poliovirus manufactured from wild
polioviruses
Poliomyelitis vaccine (oral) Revised 1999, TRS 904 (2002);
Addendum 2000, TRS 910 (2002)
Quality assurance for biological products, Adopted 1991, TRS 822 (1992)
guidelines for national authorities
Rabies vaccine for human use (inactivated) Revised 2005, TRS 941 (2007)
produced in cell substrates and embryonated
eggs
Regulation and licensing of biological products Adopted 1994, TRS 858 (1995)
in countries with newly developing regulatory
authorities
Rotavirus vaccine (live, attenuated), oral Adopted 2005, TRS 941 (2007)
Smallpox vaccine Revised 2003, TRS 926 (2004)
Snake antivenom immunoglobulins Adopted 2008, TRS 964 (2012)
Sterility of biological substances Revised 1973, TRS 530 (1973);

Synthetic peptide vaccines Adopted 1997, TRS 889 (1999)
Thiomersal for vaccines: regulatory expectations Adopted 2003, TRS 926 (2004)
for elimination, reduction or removal
Thromboplastins and plasma used to control oral Revised 2011, TRS 979 (2013)
anticoagulant therapy
Tick-borne encephalitis vaccine (inactivated) Adopted 1997, TRS 889 (1999)
Transmissible spongiform encephalopathies Revised 2005, WHO (2006)
in relation to biological and pharmaceutical http://www.who.int/biologicals/
products, guidelines publications/en/whotse2003.pdf
50
Annex 1

documents
Transmissible spongiform encephalopathies, Revised 2010, WHO (2010)

WHO tables on tissue infectivity distribution http://www.who.int/bloodproducts/
tablestissueinfectivity.pdf
Tuberculins Revised 1985, TRS 745 (1987)
Typhoid vaccine Adopted 1966, TRS 361 (1967)
Typhoid vaccine, Vi polysaccharide Adopted 1992, TRS 840 (1994)
Vaccines, clinical evaluation: regulatory Adopted 2001, TRS 924 (2004)
expectations
Vaccines, lot release Adopted 2010, TRS 978 (2013)
Vaccines, nonclinical evaluation Adopted 2003, TRS 926 (2004)
Vaccines, prequalification procedure Adopted 2010, TRS 978 (2013)
Vaccines, stability evaluation Adopted 2006, TRS 962 (2011)
Varicella vaccine (live) Revised 1993, TRS 848 (1994)
Yellow fever vaccine Revised 2010, TRS 978 (2013)
Yellow fever vaccine, laboratories approved by Revised 1995, TRS 872 (1998)
WHO for the production of
Yellow fever virus, production and testing of Adopted 1985, TRS 745 (1987)
WHO primary seed lot 213-77 and reference
batch 168-736
51
Annex 2
Guidelines on the quality, safety and efficacy of dengue
tetravalent vaccines (live, attenuated)
Replacement of Annex 1 of WHO Technical Report Series, No. 932
Abbreviations 55
Introduction 55
General considerations 56
Part A. Guidelines on manufacturing and control of dengue
tetravalent vaccines (live, attenuated) 62
A.1 Definitions 62
A.2 General manufacturing requirements 65
A.3 Control of source materials 65
A.4 Control of vaccine production 71
A.5 Filling and containers 76
A.6 Control tests on final lot 77
A.7 Records 79
A.8 Samples 79
A.9 Labelling 79
A.10 Distribution and shipping 80
A.11 Stability, storage and expiry date 80
Part B. Nonclinical evaluation of dengue tetravalent vaccines
B.1 General remarks 81
B.2 Product development and characterization 83
B.3 Nonclinical immunogenicity and protective activity 83
B.4 Nonclinical toxicity and safety 84
B.5 Environmental risk 87
Part C. Clinical evaluation of dengue tetravalent vaccines
C.1 General considerations for clinical studies 87
C.2 Immunogenicity 89
C.3 Clinical studies 91
C.4 Post-licensure investigations 102
Part D. Environmental risk assessment of dengue tetravalent vaccines
(live, attenuated) derived by recombinant DNA technology 103
D.1 Introduction 103
53
D.2 Procedure for environmental risk assessment 105

D.3 Special considerations for live recombinant dengue vaccines 106
Part E. Guidelines for NRAs 109
E.1 General 109
E.2 Release and certification 109
Authors 110
Acknowledgements 113
References 114
Appendix 1
Summary protocol for manufacturing and control of dengue tetravalent vaccine
Appendix 2
Model certificate for the release of dengue tetravalent vaccine (live, attenuated)
by NRAs 134
Guidelines published by WHO are intended to be scientific

and advisory in nature. It is recommended that modifications
be made only on condition that the modifications ensure that
the vaccine is at least as safe and efficacious as that prepared
in accordance with the Guidelines set out below. To facilitate
the international distribution of vaccine made in accordance
with these Guidelines, a summary protocol for the recording of
results of the tests is given in Appendix 1.
54
Annex 2
Abbreviations
Ae Aedes
CCID50 cell culture infectious dose 50%
CDC Centers for Disease Control and Prevention
CMI cell-mediated immunity
DENVs dengue viruses
DFI dengue febrile illness
E envelope
ELISA enzyme-linked immunosorbent assay
ERA environmental risk assessment
GMO genetically modified organism
IU International Unit
NIAID National Institute of Allergy and Infectious Diseases
NS non-structural
PDK primary dog kidney
prM premembrane
PRNT plaque-reduction neutralization test
RT-PCR reverse transcription-polymerase chain reaction
TRS Technical Report Series
TSE transmissible spongiform encephalopathy
UTR untranslated region
VE vaccine efficacy
YFV yellow fever virus
Introduction
These Guidelines are intended to provide national regulatory authorities (NRAs)
and vaccine manufacturers with guidance on the quality, safety and efficacy of
live tetravalent dengue vaccines currently under clinical development to facilitate
their international licensure and use.
55
These Guidelines update the Guidelines for the production and quality
control of candidate tetravalent dengue virus vaccines (live) (1). They should be
read in conjunction with other WHO guidelines that are referred to in each part.
The Guidelines cover dengue tetravalent vaccines (live, attenuated). Other types
of dengue virus vaccines under development (e.g. subunit or inactivated vaccines)
are outside the scope of these Guidelines. However, some guiding principles
provided in these Guidelines (e.g. Part C on clinical evaluation) may be useful
for the evaluation of other types of dengue vaccine. For quality control, guiding
principles applicable to other types of vaccines – such as inactivated or subunit
vaccines – are available elsewhere if the product in development shares similar
manufacturing processes. For example, guidelines for human papillomavirus and
hepatitis B vaccines may also be useful for subunit vaccines for dengue.
These Guidelines are based on experience gained from candidate dengue
tetravalent vaccines (live, attenuated) that have been developed as described below,
and will need to be updated as new data become available from additional studies.
Part A sets out guidelines for manufacture and quality control. Guidelines specific
to the nonclinical and clinical evaluation and environmental risk assessment are
provided in parts B, C and D, respectively. Part E provides guidelines for NRAs.
In the following section, brief overviews of dengue disease and dengue vaccine
development at the time of preparing this document are provided as a scientific
basis for each part.
General considerations
Dengue viruses
Dengue is a mosquito-borne disease and represents a major public health
problem throughout the tropical world. The causative dengue viruses (DENVs)
are members of the genus Flavivirus, within the family Flaviviridae. There are
four serotypes (termed DENV-1 to DENV-4) and at least three genetic groups
(genotypes) within each serotype.

All flaviviruses are lipid-enveloped, positive-sense, single-stranded RNA
viruses, approximately 55 nm in diameter. The genome is capped at the 5ʹ
terminus but does not have a poly A tract at the 3ʹ terminus, and is approximately
11 000 nucleotides in length. The virion RNA encodes a single open reading
frame that is flanked by a 5ʹ untranslated region (UTR) and a 3ʹ UTR. The open
reading frame is translated into a polyprotein that is co- and post-translationally
cleaved to yield at least 10 proteins. Three structural proteins are derived by
cleavages of the amino-terminal one third of the polyprotein: the capsid or
core protein forms a “nucleocapsid” complex with virion RNA that lies within
the lipid envelope. The premembrane (prM) and envelope (E) proteins are
embedded in the lipid envelope via carboxy-terminal transmembrane domains
56
Annex 2
and are displayed on the surface of virions. Cleavage of the carboxy-terminal

two thirds of the polyprotein yields seven non-structural (NS) proteins: NS1,
NS2A, NS2B, NS3, NS4A, NS4B and NS5. NS3 encodes a serine protease in the
N-terminal 180 amino acids and helicase, nucleotide triphosphatase, and RNA
5ʹ-triphosphatase activities in the C-terminal region. NS5 encodes two functions:
the first one third encodes a methyltransferase that sequentially methylates the
N7 and 2ʹ-O positions of the viral RNA cap using S-adenosyl-l-methionine as a
methyl donor, and the remainder a RNA-dependent RNA polymerase. NS1 plays
various roles in the virus replication cycle while NS2A, NS2B, NS4A and NS4B
are all small hydrophobic proteins with the central region of NS2B required for
the functioning of the NS3 protease.
Host range and transmission

DENVs are most commonly transmitted to humans by the bite of infected
Aedes aegypti mosquitoes, which are highly domesticated and the primary
mosquito vector. However, Aedes albopictus can also sustain human-to-human
transmission. The drastic increase in the incidence of DENV infection in the
Americas during the past 30 years is primarily due to the geographical spread of
Ae. aegypti following the decline in vector-control efforts. The DENV that infects
and causes disease in humans is maintained in a human-to-mosquito-to-human
cycle and does not require a sylvatic cycle in nonhuman primates. Certain strains
of DENV are known to be transmitted to nonhuman primates in western Africa
and Malaysia. However, transmission to humans via mosquitoes from nonhuman
primates is believed to be very limited.
Clinical and pathological manifestation in humans

Following infection resulting from the bite of an infected mosquito, the virus is
thought to replicate in local dendritic cells. Subsequent infection of macrophages
and lymphocytes is followed by entry into the bloodstream. Haematogenous spread
is the likely mechanism for seeding of peripheral organs and the occasional reports
of central nervous system infections, which can lead to symptomatic illness.
Most DENV infections are either asymptomatic or only mildly
symptomatic. The incubation period of dengue can range from 3 to 14 days,
but is generally 4–7 days. Most symptomatic DENV infections present with a
sudden onset of fever accompanied by headache, pain behind the eyes, generalized
myalgia and arthralgia, flushing of the face, anorexia, abdominal pain and nausea.
Rash is common in dengue and can be macular, maculopapular, morbilliform,
scarlatiniform or petechial in character. Rash is most often seen on the trunk, on
the insides of the arms and thighs, and on plantar and palmar surfaces. Laboratory
abnormalities that can be observed in dengue infection include leukopenia
and thrombocytopenia.
57
Dengue illness is classified as (i) dengue with or without warning signs

and (ii) severe dengue. A presumptive diagnosis of dengue can be made in a
patient living in or travelling from a dengue-endemic area who has fever and
at least two of the following clinical signs or symptoms: anorexia and nausea,
rash, body aches and pains, warning signs, leukopenia, and a positive tourniquet
test. “Warning signs” include abdominal pain or tenderness, persistent vomiting,
clinical fluid accumulation, mucosal bleeding, lethargy or restlessness, liver
enlargement of > 2 cm, or an increase in haematocrit concurrent with a rapid
decrease in platelet count. Dengue illness should be classified as severe in a patient
with presumptive dengue and any of the following: severe plasma leakage leading
to shock or respiratory compromise, clinically significant bleeding, or evidence
of severe organ involvement. Detailed case classification of dengue is provided in
a separate WHO document (or its subsequent update) (2).
Nonhuman primate dengue virus infection

Natural hosts for DENV infection are humans and mosquitoes. Serological
evidence from nonhuman primate studies indicates the existence of a sylvatic
DENV cycle involving several species of mosquitoes and several monkey species.
Although monkeys develop a viraemia and a neutralizing antibody response to
DENV infection, they do not develop the haematological abnormalities seen in
humans. However, in nonhuman primates (and AG129 mouse, see item below)
primary DENV infections cause a leukopenia. Thrombocytopenia has been
observed after a secondary infection (3–6).
In the rhesus macaque, viraemia typically begins 2–6 days after infection
and lasts for 3–6 days (3, 5, 7). Virus spreads to regional lymph nodes and can
be isolated from the skin, distant lymph nodes, and rarely from spleen, thymus
and other body organs. The nonhuman primate model for DENV is useful for
measuring the protection from viraemia conferred by vaccination or passively
acquired antibody. Disadvantages of the nonhuman primate model include the
lack of overt clinical signs of disease (4–6, 8).
Mouse dengue virus infection

Clinical isolates of DENV do not replicate well in genetically normal mice.
However, mouse-brain-adapted DENVs can induce fatal encephalitis after
intracranial inoculation of suckling mice. It has been demonstrated that adaptation
of a DENV-2 isolate to neurovirulence in suckling mice correlated positively with
attenuation of virulence in humans (9). Because of this ambiguity, the suckling
mouse/encephalitis model is probably not useful for studying the safety or
efficacy of candidate dengue vaccines. Nevertheless, it could be used to assess lot
consistency (see sections A.3.2.5.5.2 and A.4.2.4.7). In recent years, both chimeric
mice that are transplanted with human cells and severely immunocompromised
58
Annex 2
strains of mice have been used to elucidate the immune response to dengue
infection and to study pathogenesis (4, 10, 11). Interferon receptor-deficient
AG129 mice support replication of selected DENV strains which infect relevant
cell and tissue types comparable to human infection (10, 12). AG129 mice have
been used to investigate antibody-mediated protection. A strain of DENV-2 that
has been adapted to AG129 mice by serial passage between mice and mosquito cells
has a viscerotropic phenotype, causing thrombocytopenia and vascular leakage
in the infected animals. The phenomenon of antibody-dependent enhancement
of virus infection was observed in AG129 mice following passive transfer of
anti-DENV-1 antibodies and challenge with the adapted strain of DENV-2 (10,
12, 13). The relevance of such an immunocompromised mouse model may,
however, be limited with regard to vaccine evaluation (see section B.4.3).
Mosquito dengue virus infection

Vector competence refers to the efficiency with which the vector transfers
infection between hosts. Typically, this is a product of vector susceptibility to
infection, replication efficiency of the pathogen in the vector, and the sensitivity
of the host to infection transmitted by vector contact. Ae. aegypti mosquitoes
exhibit global variation in vector competence for flaviviruses. For example, in
sub-Saharan Africa, a black “sylvan” subspecies (Ae. formosus) predominates.
This mosquito has a low vector competence for flaviviruses due primarily to a
midgut infection barrier (14). Once ingested in an infectious blood meal, DENVs
should replicate in the midgut and disseminate to the salivary glands to facilitate
transmission to a new host during feeding. In this process, the virus should
overcome any midgut barrier that would limit replication and prevent spread of
the virus to other tissues in the mosquito (6, 15, 16).
None of the live dengue vaccine preparations currently in the clinical
trial phase of development is effectively transmitted by mosquito vectors (16, 17),
because vaccine viruses replicate poorly in mosquito midgut epithelium and/or
do not disseminate efficiently to the salivary glands, thereby effectively precluding
transmission to humans (6, 18, 19). In addition, the low peak titre and very short
duration of viraemia induced by these candidates in humans has been shown to
render vaccinees relatively non-infectious for feeding mosquitoes. The net effect
of these two phenomena is a drastic reduction in vector competence.
Populations at risk and global health importance

Dengue is the most rapidly spreading mosquito-borne viral disease in the world.
Since 1955, the incidence of dengue and severe dengue reported to WHO has
increased approximately 30-fold with increasing geographical expansion to new
countries and from urban to rural settings. Approximately 3.5 billion people live
in dengue-endemic countries which are located in the tropical and subtropical
59
regions of the world. An estimated 50 million dengue infections occur annually

and the number of cases reported annually to WHO ranged from 0.4 million to
1.3 million in the decade 1996–2005 (2).
Justification for vaccine development

Prevention of dengue by vector control has proven to be very difficult and costly.
While vector-control efforts should be sustained, vaccination holds substantial
potential in the control of the disease. Hence, there is an urgent need to develop
dengue vaccines, especially to protect people from disease in endemic countries.
Development of candidate dengue vaccines

Efforts to develop a vaccine against dengue have focused primarily on live,
attenuated viruses, derived by serial passage of virulent viruses in tissue culture
or via recombinant DNA technology that permits site-directed mutagenesis of
the genome of a virulent parent strain or chimerization between flaviviruses.
Success in the development, licensure and clinical use of live, attenuated flavivirus
vaccines, such as yellow fever 17D vaccines and Japanese encephalitis SA14-14-2,
suggests that a suitably attenuated live dengue vaccine could be highly efficacious.
Other vaccine candidates, based on inactivated whole virus, subunits that include
E protein, virus-like particles composed of prM and E proteins, and DNA
vaccines that induce expression of DENV prM/E proteins, are in the nonclinical
or early clinical stages of development.
There are no animal models that completely mimic the protean
manifestations of dengue. The lack of a suitable animal model makes it difficult
to assess the efficacy of vaccine candidates and to identify or establish possible
correlates of protection in vivo. Therefore, the protective capacity of any vaccine
candidate will be finally defined by its ability to protect humans from dengue
febrile illness (DFI). Results of nonclinical studies using monkeys and susceptible
mouse strains suggest, however, that protection from dengue is best correlated
with the presence of virus-neutralizing antibodies (3, 20–26). Studies in which

vaccinated volunteers were challenged with dengue viruses have been conducted
in the past but are not a required part of currently recommended clinical
development programmes.
There is general agreement that DENV vaccines should ideally induce
protective neutralizing antibodies to each of the four serotypes simultaneously.
In theory, a tetravalent immune response would protect against all DFI and would
also reduce or eliminate the risk of a phenomenon termed antibody-dependent
enhancement of disease, which is thought to be one of the mechanisms that
predispose to severe forms of dengue.
Several strategies have been employed to derive candidate live, attenuated
vaccines. There are four candidates in clinical development at the present time.
The Walter Reed Army Institute of Research developed attenuated DENV strains
60
Annex 2
by empirical serial passage in primary dog kidney (PDK) cells and produced
vaccine candidates in fetal rhesus lung cells. Tetravalent formulations of these
attenuated vaccine candidates have been evaluated in Phase 1 and Phase 2
clinical trials conducted by the Walter Reed Army Institute of Research and
GlaxoSmithKline (8, 27–29).
The other three candidates were developed using recombinant DNA
technology which involves first the generation of a full-length DNA copy of the
DENV genome. Site-specific mutations expected to affect virulence are then
introduced into the DNA, and mutant full-length DNAs can then be copied in
vitro to produce infectious RNA transcripts that can be used to generate mutant
DENVs in tissue culture. The United States National Institute of Allergy and
Infectious Diseases (NIAID) has thus derived a total of five candidate dengue
vaccine viruses that have been tested in clinical trials. Two were generated by
introduction of a 30-nucleotide deletion (termed Δ30) into the 3ʹ UTR of the
DENV-4 and DENV-1 genomes (8, 27–29). These DENV-1 and DENV-4 vaccine
candidates were shown to be attenuated and immunogenic in nonhuman
primates. A DENV-2 candidate vaccine was developed by replacing the gene
segments encoding the prM and E proteins of the DEN4Δ30 candidate vaccine
with those of DENV-2. An additional DENV-3 candidate vaccine was developed
by replacing the 3ʹ UTR of a DENV-3 wild-type virus with that of the DEN4Δ30
UTR. A third DENV-3 candidate vaccine was developed by introducing a
30 nucleotide deletion into the 3ʹ UTR homologous to that of the DEN4Δ30
vaccine virus and a second non-contiguous 31 nucleotide deletion, also in the 3ʹ
UTR (30). Phase 1 trials have been conducted with “Δ30” monovalent vaccines
(31), and Phase 1 trials with the tetravalent formulation were initiated in 2010.
Thai scientists at Mahidol University developed a candidate DENV-2
vaccine empirically by 53 serial passages of the virus in PDK cells, designated
DENV-2 strain PDK53, which was found to be highly attenuated and immunogenic
in Phase 1 and 2 clinical trials. The United States CDC determined that the
attenuation mutations of DENV-2 PDK53 virus were not located in the prM or
E proteins, and in collaboration with Inviragen used this genetic background
to derive chimeric DENV-1, DENV-3 and DENV-4 vaccines expressing the
respective prM/E genes in the context of the DENV-2 PDK53 genome “backbone”
(8, 27). A tetravalent formulation is in Phase 1 clinical trials.
Finally, a candidate live vaccine was developed by Acambis/Sanofi Pasteur
using the live, attenuated yellow fever virus (YFV) vaccine, 17D, as the backbone
for chimeric DENV vaccine candidate. In these viral genomes, the prM and E
genes from each of the four DENV serotypes, respectively, are substituted for
those of YFV in the context of the genetic background of the 17D vaccine. A
tetravalent chimeric YFV-DENV vaccine has been evaluated in Phase 1 and 2
clinical trials for safety and immunogenicity. Phase 2b trials to investigate
protective efficacy in children began late in 2009 (8, 27) and Phase 3 trials have
been in progress since late 2010.
61
Part A. Guidelines on manufacturing and control of

dengue tetravalent vaccines (live, attenuated)
A.1 Definitions
A.1.1 International name and proper name
Although there is no licensed dengue vaccine, the provision of a suggested
international name at this early stage of development will aid harmonization of
nomenclature if licensure is obtained. The international name should be “dengue
tetravalent vaccine (live, attenuated)”. The proper name should be the equivalent
to the international name in the language of the country of origin. The use of the
international name should be limited to vaccines that satisfy the specifications
formulated below.
A.1.2 Descriptive definition

A tetravalent dengue virus vaccine (live, attenuated), as defined in section A.1.1,
should contain live, attenuated dengue viruses representing each of the four
serotypes, or replication-competent viral vectors that express the major structural
antigen genes of each of the four dengue serotypes, that have been separately
prepared in cell culture. It may be presented as a sterile, aqueous suspension
or as freeze-dried material. The preparation should satisfy all the specifications
given below.
A.1.3 International reference materials

As the prospective vaccines are very different in type, no international reference
material for a candidate live dengue vaccine is available. However, an international
reference panel of human antisera against all four dengue serotypes is available
from the National Institute of Biological Standards and Control, Potters Bar,
England. The panel is intended to help calibrate the response to vaccines.
A.1.4 Expression of dose related to vaccine potency

Potency of a live vaccine is usually expressed in terms of the number of infectious
units of virus contained in a human dose, using a specified tissue culture
substrate and based on results of Phase 1 and Phase 2 clinical trials. In the case
of a tetravalent dengue vaccine, potency will have to be assessed in terms of the
individual titres of each of the four serotypes of vaccine virus contained in a
human dose. When international reference standards for the vaccine type under
production become available, the dose related to vaccine potency should be
calculated against the International Standard and expressed in International Units
(IU) to reduce variation between laboratories. Until then, the use of plaque-
forming unit, immunofocus-forming unit or cell culture infectious dose 50%
62
Annex 2
(CCID50) to express the potency and doses of vaccine can be an alternative. The
dose should also serve as a basis for establishing parameters for stability and for
the expiry date.
A.1.5 Terminology
The definitions given below apply to the terms as used in these Guidelines. They
may have different meanings in other contexts.
Adventitious agents: contaminating microorganisms of the cell culture
or source materials including bacteria, fungi, mycoplasmas/spiroplasmas,
mycobacteria, rickettsia, protozoa, parasites, transmissible spongiform encephalopathy
(TSE) agents and viruses that have been unintentionally introduced into the
manufacturing process of a biological product.
Cell bank: a collection of appropriate containers whose contents are of
uniform composition stored under defined conditions. Each container represents
an aliquot of a single pool of cells.
Cell culture infectious dose 50%: the amount of a virus sufficient to
cause a cytopathic effect in 50% of inoculated replicate cell cultures, as determined
in an end-point dilution assay in monolayer cell cultures.
Cell seed: a quantity of vials containing well-characterized cells derived
from a single tissue or cell of human or animal origin stored frozen in liquid
nitrogen in aliquots of uniform composition, one or more of which would be
used for the production of a master cell bank.
Cell substrates: cells used for the production of a vaccine.
Dengue febrile illness (DFI): the virological confirmation of dengue
virus infection in patients with two days of fever irrespective of the severity
of illness.
Final lot: a collection of sealed final containers of finished vaccine
that is homogeneous with respect to the risk of contamination during filling
and freeze-drying. All the final containers should, therefore, have been filled
from one vessel of final tetravalent bulk and freeze-dried under standardized
conditions in a common chamber in one working session.
Final tetravalent bulk: the finished tetravalent vaccine prepared from
virus harvest pools in the vessel from which the final containers are filled.
Genetically modified organism: an organism in which the genetic
material has been altered in a way that does not occur naturally by mating and/
or natural recombination.
Immunofocus-forming unit: the amount of a virus required to generate
one focus of infected cells that can be detected by dengue-specific antisera and a
counter-stain in monolayer cell cultures.
Master cell bank: a quantity of well-characterized cells of animal or other
origin, derived from a cell seed at a specific population doubling level or passage
level, dispensed into multiple containers, cryopreserved, and stored frozen under
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defined conditions, such as the vapour or liquid phase of liquid nitrogen in

aliquots of uniform composition. The master cell bank is prepared from a single
homogeneously mixed pool of cells. It is considered best practice for the master
cell bank to be used to derive working cell banks.
Virus master seed: a suspension of vaccine virus that has been aliquoted
into identical vials and stored at a temperature and under conditions deemed to
stabilize the virus in each container. The virus master seed is used as a source of
infectious virus for the generation of each virus working seed lot.
Neurovirulence: the capacity of a microorganism to cause disease of
the nervous system, leading to paralysis or dysfunction of the nervous system.
In animal experimental settings, clinical and pathological evaluations are often
carried out after intracranial inoculation of a microorganism.
Neurotropism: the affinity of a microorganism for, or for localizing
selectively in, nerve tissue.
Monovalent virus pool: a suspension of single serotype of dengue virus
that may be the result of one or more single harvests or multiple parallel harvests
of the same virus serotype collected into a single vessel before clarification.
Multiple parallel harvest: a pool of harvests coming from multiple
cultures that are initiated in parallel from the same ampoule of the same working
cell bank infected together by the same virus suspension of the same virus
working seed lot.
Plaque-forming unit: the amount of a virus sufficient to cause a single
visible focus of infection due to cytopathic effect in a cell culture monolayer after
proper staining of cells.
Production cell culture: a collection of cell cultures used for biological
production that have been prepared together from one or more containers from
the working cell bank or, in the case of primary cell cultures, from the tissues of
one or more animals.
Single harvest: a quantity of virus suspension harvested from production
cell cultures inoculated with the same virus working seed and processed together
in a single production run.
Working cell bank: a quantity of well-characterized cells of animal or
other origin, derived from the master cell bank at a specific population doubling
level or passage level, dispensed into multiple containers, cryopreserved and
stored frozen under defined conditions, such as in the vapour or liquid phase
of liquid nitrogen in aliquots of uniform composition. The working cell bank is
prepared from a single homogeneously mixed pool of cells. One or more of the
working cell bank containers is used for each production culture.
Virus working seed: a quantity of virus of uniform composition, well
characterized and derived from a virus master seed lot (see above) in a production
cell line. The working seed lot is used for the production of a single harvest.
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A.2 General manufacturing requirements

The general requirements for manufacturing establishments contained in
WHO’s Good manufacturing practices for biological products (32) should be
applied by establishments manufacturing dengue tetravalent vaccine. Separate
manufacturing areas for each of the four dengue serotypes and for tetravalent
vaccine formulation may be used. Alternatively, manufacturing areas may be
used on a campaign basis with adequate cleaning between campaigns to ensure
that cross-contamination does not occur.
Production steps and quality-control operations involving manipulations
of live virus should be conducted under the appropriate biosafety level, as agreed
with the NRA and in accordance with country biosafety laws.
A.3 Control of source materials

A.3.1 Cell cultures for virus production
A.3.1.1 Conformity with WHO recommendations
Dengue viruses used in producing tetravalent dengue vaccine should be propagated
in cell substrates which meet the Recommendations for the evaluation of animal
cell cultures as substrates for the manufacture of biological medicinal products
and for the characterization of cell banks (33) and should be approved by the
NRA. All information on the source and method of preparation of the cell culture
system used should be made available to the NRA.
A.3.1.2 Types of cell culture

Dengue vaccine candidates have been produced in fetal rhesus lung diploid cells
and in continuous cell lines. For fetal rhesus lung diploid cells and continuous
cells, sections A.3.1.3 and A.3.1.4 apply.
A.3.1.3 Cell banks

The use of a cell line such as fetal rhesus lung diploid cells or Vero cells for the
manufacture of dengue vaccines should be based on the cell bank system. The
cell seed should be approved by the NRA. The maximum number of passages
or population doubling allowable between the cell seed, the working cell bank
and the production passage levels should be established by the manufacturer
and approved by the NRA. Additional tests may include, but are not limited
to: propagation of the master cell bank or working cell bank cells to or beyond
the maximum in vitro age for production, and examination for the presence of
retroviruses and tumorigenicity in an animal test system (33).
WHO has established a bank of Vero cells, designated as WHO Vero
reference cell bank 10-87 that has been characterized in accordance with the
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WHO Requirements for continuous cell lines used for biologicals production
(34). The cell bank is available to manufacturers, as is well-characterized starting
material for manufacturers to prepare their own master and working cell banks
on application to the Coordinator, Quality, Safety and Standards, WHO, Geneva,
Switzerland (33).
In normal practice, a master cell bank is expanded by serial subculture
up to a passage number (or population doubling, as appropriate) selected by the
manufacturer and approved by the NRA, at which point the cells are combined
to give a single pool distributed into ampoules and preserved cryogenically
to form the working cell bank. The manufacturer’s working cell bank is used
for the preparation of production cell culture, and thus for the production of
vaccine batches.
A.3.1.4 Characterization of cell banks

The cell seed (if applicable), master and working cell banks and end-of-
production cells or extended cell bank should be characterized according to the
WHO Recommendations for the evaluation of animal cell cultures as substrates
for the manufacture of biological medicinal products and for the characterization
of cell banks (33).
A.3.1.5 Cell culture medium

Serum used for propagating cells should be tested to demonstrate freedom from
bacteria, fungi and mycoplasmas, according to the requirements given in Part A,
sections 5.2 and 5.3 of the General requirements for the sterility of biological
substances (Requirements for biological substances, no. 6) (35, 36), as well as
from infectious viruses.
Detailed guidelines for detecting bovine viruses in serum for establishing
a master cell bank and working cell bank are given in Appendix 1 of the WHO
Recommendations for the evaluation of animal cell cultures as substrates for the
manufacture of biological medicinal products and for the characterization of cell
banks (33). The principles outlined in the cell substrate recommendations should
be applied as appropriate, and the guidelines for detecting bovine viruses in serum
for establishing the cell banks may be applicable to production cell cultures as
well. In particular, validated molecular tests for bovine viruses might replace the
cell culture tests of bovine sera if agreed by the NRA. As an additional monitor
of quality, sera may be examined for freedom from phage and endotoxin. Gamma
irradiation may be used to inactivate potential contaminant viruses.
The sources of animal components used in culture medium should be
approved by the NRA. These components should comply with current guidelines
in relation to animal TSE (37, 38).
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Human serum should not be used. If human albumin is used, it should

meet the revised Requirements for the collection, processing and quality control
of blood, blood components and plasma derivatives (Requirements for biological
substances no. 27) (39), as well as current guidelines in relation to human TSE
(37, 38).
The use of human albumin as a component of a cell culture medium
requires careful consideration due to potential difficulties with the validity period
of albumin (which is based on the length of time for which it is suitable for use
in clinical practice) in relation to the potential long-term storage of monovalent
bulks of each dengue serotype. In addition, if human albumin is used, it should
be tested according to the WHO Recommendations for the evaluation of animal
cell cultures as substrates for the manufacture of biological medicinal products
and for the characterization of cell banks (33).
Penicillin and other beta-lactams should not be used at any stage of the
manufacture. Other antibiotics may be used at any stage in the manufacture
provided that the quantity present in the final product is acceptable to the NRA.
Nontoxic pH indicators may be added (e.g. phenol red at a concentration of
0.002%). Only substances that have been approved by the NRA may be added.
If porcine or bovine trypsin is used for preparing cell cultures, it should
be prepared, tested and found free of cultivable bacteria, fungi, mycoplasmas and
infectious viruses, as described in the WHO Recommendations for the evaluation
of animal cell cultures as substrates for the manufacture of biological medicinal
products and for the characterization of cell banks (33). The methods used to
ensure this should be approved by the NRA. The source(s) of trypsin of bovine
origin, if used, should be approved by the NRA and should comply with current
guidelines in relation to animal TSE (37, 38).
A.3.2 Virus seeds

A.3.2.1 Vaccine virus strains
The strains of DENV 1–4 viruses attenuated either by serial passage in cell
cultures or by recombinant DNA technology used in the production of candidate
tetravalent dengue vaccine should be thoroughly characterized. This will include
historical records (such as information on the origin of the strain, cell culture
passage history, method of attenuation, results of preclinical and clinical studies
demonstrating attenuation, and whether the strains have been biologically
or molecularly cloned prior to generation of the master seed), their genome
sequence, the passage level at which clinical trials were performed, and the results
of clinical studies. Only strains approved by the NRA should be used.
Strains of dengue recombinant viruses used for master and working seeds
to produce vaccine candidates should comply with the additional specifications
given in section A.3.2.2.
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A.3.2.2 Strains derived by molecular methods

If vaccine seeds derived by recombinant DNA technology are used, and because
this is a live, attenuated vaccine, the candidate vaccine is considered a genetically
modified organism (GMO) in several countries and should comply with the
regulations of the producing and recipient countries regarding GMOs. An
environmental risk assessment should be undertaken according to Part D of
these Guidelines.
The nucleotide sequence of any cDNA clone used to generate vaccine
virus stocks should be determined some time prior to any further nonclinical
or clinical trial. The cell substrate used for transfection to generate the virus
should be appropriate for human vaccine production and should be approved
by the NRA.
Pre-seed lot virus stocks derived from passaging of the primary virus
stock should also be sequenced as part of nonclinical evaluation.
Viral vaccine seeds that are directly re-derived from RNA extracted
from virus in order to reduce the risk of previous contamination by TSEs or
other adventitious agents are considered as new vaccine seeds, and they should
be appropriately characterized to demonstrate comparability with the starting
virus seed.
A.3.2.3 Virus seed lot system

The production of vaccine should be based on the master and working seed lot
system to minimize the number of tissue culture passages needed for vaccine
production. Seed lots should be prepared in the same type of cells using the same
conditions for virus growth (other than scale) as those used for production of
final vaccine.
The virus working seed should have a well-defined relationship to the
virus master seed with respect to passage level and method of preparation, such
that the virus working seed retains all of the in vitro and in vivo phenotypes and
the genetic character of the virus master seed. Nonclinical and clinical data are
needed to support this relationship. Once the passage level of the virus working
seed with respect to the virus master seed is established, it may not be changed
without approval from the NRA.
Virus seed lots should be stored in a dedicated temperature-monitored
freezer at a temperature that ensures stability upon storage. It is recommended
that a large virus working seed lot should be set aside as the basic material for
use by the manufacturer for the preparation of each batch of vaccine.
Full in vitro and in vivo testing for detecting adventitious agents should
be conducted on either master or working seed lots.
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A.3.2.4 Control cell cultures for virus seeds

In agreement with NRAs, tests on control cell cultures may be required and
performed as described in section A.4.1.
A.3.2.5 Tests on virus master and working seed lots

A.3.2.5.1 Identity
The serotype of all dengue virus master seeds and working seeds should be
confirmed by immunological assay or by molecular methods.
A.3.2.5.2 Genetic/phenotypic characterization

Different live dengue vaccine viruses may have significantly different properties.
Such differences may influence the tests to be used to examine their genetic and
phenotypic stability relevant to consistency of production. The applicable tests
will be identified in the course of the nonclinical evaluation of the strains. Each
seed should be characterized by full-length nucleotide sequence determination
and by other relevant laboratory and animal tests, which will provide information
on the consistency of each virus seed.
Mutations introduced during derivation of each vaccine strain should be
maintained in the consensus sequence, unless spontaneous mutations induced
during tissue culture passage were shown to be innocuous in nonclinical and
small-scale clinical trials. Some variations in the nucleotide sequence of the virus
population on passage are to be expected, but what is acceptable should be based
on experience in production and clinical use.
For any new virus master seeds and working seeds, it is recommended
that the first three consecutive consistency bulk vaccine lots be analysed for
consensus sequence changes from virus master seed. The nucleotide sequence
results should be used to demonstrate the consistency of the production process.
Routine nucleotide sequence analysis of bulk vaccine is not recommended.
A.3.2.5.3 Tests for bacteria, fungi, mycoplasmas and mycobacteria

Each virus master and working seed lot should be shown to be free from
bacterial, fungal and mycoplasmal contamination by appropriate tests as specified
in Part A, sections 5.2 and 5.3, of General requirements for the sterility of
biological substances (Requirements for biological substances, no. 6) (35, 36).
Nucleic acid amplification techniques alone or in combination with cell culture,
with an appropriate detection method, may be used as an alternative to one or
both of the compendial mycoplasma detection methods after suitable validation
and agreement from the NRA (33).
Seed lots should be shown to be free from mycobacteria by a method
approved by the NRA. Nucleic acid amplification techniques may be used as an
alternative to the microbiological culture method for mycobacteria and/or to the
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in vivo guinea-pig test for the detection of mycobacteria after suitable validation
and agreement from the NRA (33).
A.3.2.5.4 Tests for adventitious agents

Each virus working seed lot and/or master seed lot should be tested in cell
cultures for adventitious viruses relevant to the passage history of the seed virus.
Where antisera are used to neutralize dengue virus or the recombinant dengue
virus, the antigen used to generate the antisera should be produced in cell culture
from a species different from that used for the production of the vaccine and
free from extraneous agents. Monkey and human cell cultures inoculated with
the virus antibody mixture should be observed microscopically for cytopathic
changes. For virus grown in monkey or human cells, the neutralized virus is
tested on a separate culture of these cells. If other cell systems are used, cells
of that species, but from a separate batch, are also inoculated. At the end of the
observation period, the cells should be tested for haemadsorbing viruses.
Each virus master or working seed lot should also be tested in animals
that include guinea-pigs, adult mice, and suckling mice. For test details, refer
to section B.11 of WHO’s Recommendations for the evaluation of animal cell
cultures as substrates for the manufacture of biological medicinal products and
for the characterization of cell banks (33). Additional testing for adventitious
viruses may be performed using validated nucleic acid amplification techniques.
New molecular methods with broad detection capabilities are being
developed for adventitious agent detection. These methods include: (i) degenerate
nucleic acid amplification techniques for whole virus families with analysis of
the amplicons by hybridization, sequencing or mass spectrometry; (ii) nucleic
acid amplification techniques with random primers followed by analysis of the
amplicons on large oligonucleotide micro-arrays of conserved viral sequencing or
digital subtraction of expressed sequences; and (iii) high throughput sequencing.
These methods may be used in the future to supplement existing methods or as
alternative methods to both in vivo and in vitro tests after appropriate validation
and agreement from the NRA.
A.3.2.5.5 Tests in experimental animals

Tests in nonhuman primates: all vaccine candidates should be evaluated, at least
once during nonclinical development, for neurovirulence in nonhuman primates,
as detailed in Part B (nonclinical evaluation). Candidate vaccines that are
homogeneously “dengue” in terms of genetics are not expected to be neurotropic in
such a test but, where attenuation has been achieved by recombination of dengue
genes with those of a different virus species that itself displays neurovirulence
(e.g. dengue/yellow fever recombinants), the master seed should be tested in
nonhuman primates. If these tests were not performed at the master seed level,
they should be performed at working seed level.
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NRAs may decide that such testing does not need to be repeated each
time a novel working seed lot is derived, if results of a well-conducted monkey
neurovirulence assay on the master seed lot are negative. Recent data suggest
that certain small animal models for neurovirulence may serve as a surrogate
for nonhuman primates, at least where viruses expressing yellow fever strain
17D genes are concerned (40). NRAs may eventually wish to consider accepting
results of such studies as a surrogate for studies using nonhuman primates to
evaluate neurovirulence of novel dengue vaccines.
Test for neurovirulence: to provide assurance that a candidate vaccine virus
is not unexpectedly neurovirulent, each vaccine strain of each serotype, or a
tetravalent formulation if agreed by the NRA, should be tested for neurovirulence
in monkeys by inoculation of Macaca mulatta (rhesus), Macaca fascicularis
(cynomolgus) or other susceptible species of monkey, in the course of preclinical
evaluation.
Prior to testing for neurovirulence, the neutralizing antibody test should
be used to assess the immune status of nonhuman primates to both dengue and
yellow fever viruses. For further details on the test for neurovirulence see Part B.
Tests in suckling mice: the virulence of different vaccine candidates in mice will
depend on the strains of virus and mouse. Novel vaccines that reach the clinical
phase of development in many cases were tested for neurovirulence in suckling
and adult mice during the preclinical phase of development.
While mice are not considered a good model for dengue, suckling and
adult mice have been used to assess the neurovirulence of dengue/yellow fever
recombinant vaccines (21, 41). A mouse test might be considered in order to
demonstrate consistency of characteristics of dengue/yellow fever recombinant
viruses during production (see section A.4.2.4.7).
A.3.2.5.6 Virus titration for infectivity

Each virus master and working seed lot should be assayed for infectivity in a
sensitive assay in cell culture. Depending on the results obtained in preclinical
studies, plaque assays, CCID50 assays, immunofocus-forming unit assays or
CCID50 with a molecular readout such as quantitative polymerase chain reaction
may be used. All assays should be validated.
A.4 Control of vaccine production

A.4.1 Control of production cell cultures
Where the NRA requires the use of control cells, the following procedures should
be followed. From the cells used to prepare cultures for production of vaccine,
a fraction equivalent to at least 5% of the total or 500 ml of cell suspension, or
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100 million cells, should be used to prepare uninfected control cell cultures.
These control cultures should be observed microscopically for cytopathic and
morphological changes attributable to the presence of adventitious agents for at
least 14 days at a temperature of 35–37 °C after the day of inoculation of the
production cultures, or until the time of final virus harvest, whichever comes
last. At the end of the observation period, supernatant fluids collected from
the control culture should be tested for the presence of adventitious agents as
described below. Samples that are not tested immediately should be stored at
–60 °C or lower, until such tests can be conducted.
If adventitious agent testing of control cultures yields a positive result,
the harvest of virus from the parallel vaccine virus-infected cultures should not
be used for production.
For the test to be valid, not more than 20% of the control culture flasks
should have been discarded, for any reason, by the end of the test period.
A.4.1.1 Test for haemadsorbing viruses

At the end of the observation period, a fraction of control cells comprising not
less than 25% of the total should be tested for the presence of haemadsorbing
viruses, using guinea-pig red blood cells. If the guinea-pig red blood cells have
been stored prior to use in the haemadsorption assay, the duration of storage
should not have exceeded seven days, and the storage temperature should have
been in the range of 2–8 °C.
In some countries, the NRA requires that additional tests for
haemadsorbing viruses should be performed using red blood cells from other
species, including those from humans (blood group O), monkeys, and chickens
(or other avian species). For all tests, readings should be taken after incubation
for 30 minutes at 0–4 °C, and again after a further incubation for 30 minutes
at 20–25 °C. The test with monkey red cells should be read once more after
additional incubation for 30 minutes at 34–37 °C.
For the tests to be valid, not more than 20% of the culture vessels should
have been discarded for any reason by the end of the test period.
A.4.1.2 Tests for cytopathic, adventitious agents in control cell fluids

Supernatant culture fluids from each of the control cell culture flasks or bottles
collected at the time of harvest should be tested for adventitious agents. A 10 ml
sample of the pool should be tested in the same cell substrate, but not the same
cell batch, as that used for vaccine production, and an additional 10 ml sample of
each pool should be tested in both human and continuous simian (monkey) cells.
Each sample should be inoculated into cell cultures in such a way that
the dilution of the pooled fluid in the nutrient medium does not exceed 1:4. The
surface area of the flask should be at least 3 cm2 per ml of pooled fluid. At least
one flask of the cells should remain uninoculated, as a control.
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Annex 2
The inoculated cultures should be incubated at a temperature of 35–37 °C

and should be examined at intervals for cytopathic effects over a period of at least
14 days.
Some NRAs require that, at the end of this observation period, a subculture
is made in the same culture system and observed for at least an additional seven
days. Furthermore, some NRAs require that these cells should be tested for the
presence of haemadsorbing viruses.
For the tests to be valid, not more than 20% of the culture vessels should
have been discarded for any reason by the end of the test period.
A.4.1.3 Identity test

At the production level, the cells should be identified by means of tests approved
by the NRA. Suitable methods are (but are not limited to) biochemical tests (e.g.
isoenzyme analyses), immunological tests (e.g. major histocompatibility complex
assays), cytogenetic tests (e.g. for chromosomal markers), and tests for genetic
markers (e.g. DNA fingerprinting).
A.4.2 Production and harvest of monovalent virus

A.4.2.1 Cells used for vaccine production
On the day of inoculation with the working seed virus, each production cell
culture flask (or bottle etc.) and/or cell culture control flask should be examined
for cytopathic effect potentially caused by infectious agents. If the examination
shows evidence of the presence in any flask of an adventitious agent, all cell
cultures should be discarded.
If animal serum is used in growth medium, the medium should be
removed from the cell culture either before or after inoculation of the virus
working seed. Prior to beginning virus harvests, the cell cultures should be rinsed
and the growth medium should be replaced with serum-free maintenance medium.
Penicillin and other beta-lactam antibiotics should not be used at any
stage of manufacture. Minimal concentrations of other suitable antibiotics may
be used if approved by the NRA.
A.4.2.2 Virus inoculation

Cell cultures are inoculated with dengue virus working seed at an optimal and
defined multiplicity of infection. After viral adsorption, cell cultures are fed with
maintenance medium and are incubated at a temperature within a defined range
and for a defined period.
The multiplicity of infection, temperature range and duration of
incubation will depend on the vaccine strain and production method, and
specifications should be defined by each manufacturer.
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A.4.2.3 Monovalent virus harvest pools

Vaccine virus is harvested within a defined period post-inoculation. A
monovalent harvest may be the result of one or more single harvests or multiple
parallel harvests. Samples of monovalent virus harvest pools should be taken for
testing and should be stored at a temperature of –60 °C or below. The sponsor
should submit data to support the conditions chosen for these procedures.
The monovalent virus harvest pool may be clarified or filtered to remove
cell debris and stored at a temperature that ensures stability before being used to
prepare the tetravalent final bulk for filling. The sponsor should provide data to
support the stability of the bulk throughout the duration of the chosen storage
conditions, as well as to support the choice of storage temperature.
Harvests derived from continuous cell lines should be subjected to further
purification to minimize the amount of cellular DNA, and/or to treatment with
DNase to reduce the size of the DNA.
A.4.2.4 Tests on monovalent virus harvest pools

A.4.2.4.1 Identity
Each monovalent virus harvest pool should be identified as the appropriate
dengue virus serotype by immunological assay on cell cultures using specific
antibodies, or by molecular methods (see section A.6.1) approved by the NRA.
A.4.2.4.2 Tests for bacteria, fungi, mycoplasmas and mycobacteria

Each monovalent virus harvest pool should be shown by appropriate tests to
be free from bacterial, fungal, mycoplasmal and mycobacterial contamination.
Sterility tests are specified in Part A, sections 5.2 (bacteria and fungi) and 5.3
(mycoplasmas), of the General requirements for the sterility of biological
substances (Requirements for biological substances, no. 6) (35, 36).
Nucleic acid amplification techniques alone or in combination with cell
culture, with an appropriate detection method, might be used as an alternative to
one or both of the pharmacopoeial mycoplasma detection methods after suitable

validation and the agreement of the NRA (33).
The method for testing mycobacteria should be approved by the NRA.
Nucleic acid amplification techniques might be used as an alternative to the
microbiological culture method for mycobacteria after validation and agreement
by the NRA (33).
A.4.2.4.3 Tests for adventitious agents

Each monovalent virus harvest pool should be tested in cell culture for adventitious
viruses by inoculation into continuous simian kidney cells, cell lines of human
origin, and the cell line used for production, but from another batch. Where
antisera are used to neutralize dengue virus or the recombinant virus, the antigen
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Annex 2
used to generate the antisera should be produced in cell culture from a species
that is different from that used for the production of the vaccine and that is free
from extraneous agents. The cells inoculated should be observed microscopically
for cytopathic changes. At the end of the observation period, the cells should be
tested for haemadsorbing viruses. Additional testing for adventitious viruses
may be performed using validated nucleic acid amplification techniques.
A.4.2.4.4 Virus titration for infectivity

The titre for each monovalent virus harvest should be determined in a sensitive
assay in cell culture. Depending on the results obtained in preclinical studies,
plaque assays, CCID50 assays, immunofocus formation assays or CCID50 with a
molecular readout such as quantitative polymerase chain reaction may be used.
A.4.2.4.5 Tests for host cell proteins

The host cell protein profile should be examined as part of characterization
studies (33).
A.4.2.4.6 Tests for residual cellular DNA

For viruses grown in continuous cell line cells, the monovalent harvest pool
should be tested for the amount of residual cellular DNA, and the total amount
of cell DNA per dose of vaccine should be not more than the upper limit agreed
by the NRA. If this is technically feasible, the size distribution of the DNA should
be examined as a characterization test, taking into account the amount of DNA
detectable using state-of-the-art methods (33), as approved by the NRA.
A.4.2.4.7 Test for consistency of virus characteristics

The dengue virus in the monovalent harvest pool should be tested to compare
it with virus working seed, or another suitable comparator, to ensure that the
vaccine virus has not undergone critical changes during its multiplication in the
production culture system.
Relevant assays should be identified in nonclinical studies and may
include, for example, virus yield in tissue culture, plaque phenotype, or temperature
sensitivity. Other identifying characteristics may also be applicable.
Assays for the attenuation of dengue/yellow fever recombinants and
other vaccine viruses, if appropriate, include tests in suckling mice. Intracerebral
inoculation of suckling mice with serial dilutions of vaccine and yellow fever
17D is followed by the determination of the mortality ratio and survival time.
The results obtained with the vaccine are compared to the yellow fever 17D
control results.
The test for consistency may be omitted as a routine test once the
consistency of the production process has been demonstrated on a significant
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number of batches in agreement with the NRA. Where there is a significant

change in the manufacturing process, the test should be reintroduced.
A.4.2.5 Storage
Monovalent virus harvest pools should be stored at a temperature that ensures
stability.
A.4.3 Final tetravalent bulk lot

A.4.3.1 Preparation of final tetravalent bulk lot
The final tetravalent bulk lot should be prepared from monovalent virus pools
of the four dengue virus subtypes using a defined virus concentration of each
component. The operations necessary for preparing the final bulk lot should be
conducted in a manner that avoids contamination of the product.
In preparing the final bulk, any excipients (such as diluents or stabilizer)
that are added to the product should have been shown to the satisfaction of the
NRA not to impair the safety and efficacy of the vaccine in the concentration used.
A.4.3.2 Tests on the final tetravalent bulk lot

A.4.3.2.1 Residual animal serum protein
If appropriate, a sample of the final bulk should be tested to verify that the level
of serum is less than 50 ng per human dose.
A.4.3.2.2 Sterility
Except where it is subject to in-line sterile filtration as part of the filling process,
each final bulk suspension should be tested for bacterial and fungal sterility
according to Part A, section 5.2 of the General requirements for the sterility of
biological substances (Requirements for biological substances, no. 6) (35), or by
a method approved by the NRA.
A.4.3.3 Storage
Prior to filling, the final bulk suspension should be stored under conditions
shown by the manufacturer to retain the desired viral potency.
A.5 Filling and containers

The requirements concerning Good manufacturing practices for biological
products (32) appropriate to a vaccine should apply.
Care should be taken to ensure that the materials from which the container
and, if applicable, the closure are made do not adversely affect the infectivity
(potency) of the vaccine under the recommended conditions of storage.
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A final filtration could be included during the filling operations to

assure sterility.
The manufacturer should provide the NRA with adequate data to prove
that the product is stable under appropriate conditions of storage and shipping.
A.6 Control tests on final lot

The following tests should be carried out on the final lot.
A.6.1 Vaccine
A.6.1.1 Inspection of final containers
Each container in each final lot should be inspected visually, and those showing
abnormalities should be discarded.
A.6.1.1.1 Appearance
The appearance of the freeze-dried or liquid vaccine should be described
with respect to form and colour. In the case of freeze-dried vaccines, a visual
inspection should be performed of the freeze-dried vaccine, the diluent, and the
reconstituted vaccine.
A.6.1.2 pH
The pH of the final lot should be tested in a pool of final containers and an
appropriate limit should be set to guarantee virus stability. In the case of freeze-
dried vaccines, pH should be measured after reconstitution of the vaccine with
the diluent.
A.6.1.3 Identity
Each monovalent component of a tetravalent dengue vaccine lot should
be identified as dengue or recombinant virus type DENV-1, -2, -3 or -4 by
immunological assay using specific antibodies or by molecular methods. The
methods used for the potency assay (section A.6.1.5) may serve as the identity test.
A.6.1.4 Sterility
Vaccine should be tested for bacterial and fungal sterility according to the
requirements of Part A, section 5.2 of the General requirements for the sterility
of biological substances (Requirements for biological substances, no. 6) (35), or
by methods approved by the NRA.
A.6.1.5 Potency
At least three containers of each tetravalent vaccine lot should be assayed for
infectivity in a validated assay in appropriate cell culture. The assay should include
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a working reference preparation to control the accuracy and reproducibility of

the testing system. The titre of each serotype of dengue virus in the final tetravalent
mixture should be determined.
A.6.1.6 Thermal stability

The purpose of the thermal stability test is to demonstrate consistency of
production. Additional guidance on evaluation of vaccine stability is provided
in WHO’s Guidelines for stability evaluation of vaccines (42). At least three
containers of tetravalent vaccine should be incubated at the appropriate elevated
temperature for the appropriate time (e.g. 37 °C for seven days) depending on
the products. The geometric mean titre of infectious virus in the containers for
each individual virus serotype that has been exposed should not have decreased
during the period of exposure by more than a specified amount (e.g. 1 log) that is
justified by production data and approved by the NRA. Titration of non-exposed
and exposed containers should be carried out in parallel. A validity control
reagent of each of the four virus components should be included in each assay to
validate the assay.
A.6.1.7 General safety

Each filling lot should be tested for unexpected toxicity (sometimes called
abnormal toxicity) using a general safety test approved by the NRA. This test
may be omitted for routine lot release once consistency of production has
been established to the satisfaction of the NRA and when good manufacturing
practices are in place. Each lot, if tested, should pass a general safety test.
A.6.1.8 Residual moisture (if appropriate)

The residual moisture in each freeze-dried lot should be conducive to the stability
of the product, and the upper limit of the moisture content should be approved
by the NRA on the basis of the results of stability testing.
A.6.1.9 Residual antibiotics (if applicable)

If any antibiotics are added during the vaccine production, the content of the
residual antibiotics should be determined and should be within limits approved
by the NRA.
A.6.2 Diluent
The recommendations given in WHO’s Good manufacturing practices for
pharmaceutical products: main principles (43) should apply to the manufacturing
and control of diluents used to reconstitute live, attenuated dengue vaccines. An
expiry date should be established for the diluent on the basis of stability data.
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For lot release of the diluent, tests should be done for identity, appearance, pH,
volume, sterility, and the content of key components.
A.7 Records
The recommendations of Good manufacturing practices for biological products
(32) should apply, as appropriate to the level of development of the candidate
vaccine.
A.8 Samples
A sufficient number of samples should be retained for future studies and needs.
Vaccine lots that are to be used for clinical trials may serve as reference materials
in the future, and a sufficient number of vials should be reserved and stored
appropriately for that purpose.
A.9 Labelling
The recommendations of Good manufacturing practices for biological products
(32) should apply, as appropriate for a candidate vaccine, with the addition of
the following.
The label on the carton enclosing one or more final containers, or the
leaflet accompanying the container, should include:
■ a statement that the candidate vaccine fulfils Part A of these

Guidelines;
■ a statement of the nature of the preparation, specifying the
designation of the strains of dengue or recombinant viruses
contained in the live, attenuated tetravalent vaccine, the minimum
number of infective units per human dose, the nature of any cellular
systems used for the production of the vaccine, and whether the
vaccine strains were derived by molecular methods;
■ a statement of the nature and quantity, or upper limit, of any
antibiotic present in the vaccine;
■ an indication that contact with disinfectants is to be avoided;
■ a statement concerning the photosensitivity of the vaccine, cautioning
that both lyophilized and reconstituted vaccine should be protected
from light;
■ a statement indicating the volume and nature of diluent to be added
to reconstitute the vaccine, and specifying that the diluent to be used
is that supplied by the manufacturer;
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■ a statement that after it has been reconstituted, the vaccine should

be used without delay or, if not used immediately, stored at 2–8 °C
and protected from light for a maximum period defined by stability
studies.
A.10 Distribution and shipping

The recommendations given in Good manufacturing practices for biological
products (32) appropriate for a candidate vaccine should apply.
Shipments should be maintained within specified temperature ranges
and packages should contain cold-chain monitors (44).
A.11 Stability, storage and expiry date

products (32) and in Guidelines for stability evaluation of vaccines (42)
appropriate for a candidate vaccine should apply. The statements concerning
storage temperature and expiry date that appear on the primary or secondary
packaging should be based on experimental evidence and should be submitted
for approval to the NRA.
A.11.1 Stability testing

Stability testing should be performed at different stages of production – namely,
on single harvests, purified bulk, final bulk and final lot. Stability-indicating
parameters should be defined or selected as appropriate to the stage of production.
It is advisable to assign a shelf-life to all in-process materials during vaccine
production – in particular stored intermediates such as single harvests, purified
bulk and final bulk.
The stability of the vaccine in its final container and at the recommended
storage temperatures should be demonstrated to the satisfaction of the NRAs

on at least three lots of final product. Accelerated thermal stability tests may be
undertaken on each final lot to give additional information on the overall stability
of a vaccine (see section A.6.1.6).
The formulation of vaccine and adjuvant (if used) should be stable
throughout its shelf-life. Acceptable limits for stability should be agreed with NRAs.
A.11.2 Storage conditions

Before being distributed by the manufacturing establishment or before being
issued from a storage site, the vaccine should be stored at a temperature shown
by the manufacturer to be compatible with a minimal loss of titre. The maximum
duration of storage should be fixed with the approval of the NRA and should be
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such as to ensure that all quality specifications for final product, including the
minimum titre specified on the label of the container (or package), will still be
maintained until the end of the shelf-life.
A.11.3 Expiry date

The expiry date should be defined on the basis of shelf-life and should be
supported by the stability studies with the approval of the NRA. If the vaccine
is stored at a temperature lower than that used for stability studies and intended
for release without re-assay, the expiry date is calculated from the date of removal
from cold storage. The expiry dates for the vaccine and the diluent may differ.
A.11.4 Expiry of reconstituted vaccine

For single-dose containers, the reconstituted vaccine should be used immediately.
Multi-dose containers should be kept in the dark at 2–8 °C and the expiry time
for use of an opened container should be defined by stability studies approved
by the NRA, but should be not more than six hours.
Part B. Nonclinical evaluation of dengue

B.1 General remarks
Nonclinical evaluation of a live dengue vaccine includes in vitro and in vivo
testing that is required prior to initiation of the clinical phase of the vaccine
development programme. This testing should yield information suggesting
the safety and potential for efficacy of a dengue vaccine candidate. Testing
may continue in parallel with the clinical phase of product development. Tests
should include product characterization at each stage of manufacture (including
quantification of contaminants such as cellular proteins and DNA), proof of
concept/immunogenicity studies (including dose ranging in animals etc.),
toxicology if required by the NRA, establishment of a test for potency to be used
throughout, and safety testing in animals (see Table A2.1). These Guidelines,
which are specifically aimed at nonclinical evaluation of a live, attenuated dengue
vaccine, should be read in conjunction with the WHO Guidelines on nonclinical
evaluation of vaccines (45).
Although there is no animal model that precisely mimics dengue
disease in humans, animal models have been and are being used in studies on
immunogenicity, protective activity, toxicology and safety. Animal models were
briefly reviewed at the time of preparing these Guidelines to highlight the latest
developments and to provide a better understanding of their use in vaccine
development.
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Table A2.1
Nonclinical evaluation of dengue vaccines
Area of nonclinical Primary concern Scope of nonclinical evaluation

evaluation
In vitro nonclinical evaluation
Product Product risks are Mutations in the genome may impact
characterization appropriate for the infection efficiency and growth
anticipated use. capacity in different cell types,
including cells of a monocyte lineage.
Virus structural protein profiles;
serotype identity; consistency of
the manufacturing process; genetic
stability of vaccine candidates.
Process development, Process meets Sources of all media, cells and
quality control and all good seed viruses; purification and virus
quality assurance manufacturing concentration procedures; sources of
practice standards. all animal sera used to cultivate viruses
and cells; demonstrated efficiency of
purification processes; titration of virus
dose; safety of excipients; standardized
laboratory assays to measure
immunogenicity, etc.
In vivo nonclinical evaluation
Immunogenicity and Demonstrate that DENV live, attenuated vaccines
protective activity in the vaccine can are immunogenic in nonhuman
an animal model protect from some primates; candidate should induce
aspect of dengue limited viraemia and should protect
infection; estimate against viraemia following wild-
dose range for type DENV challenge in nonhuman
humans. primates. Interference between DENV

serotypes may be evaluated in mice
or nonhuman primates, but data may
not always correlate with data from
humans.
Toxicity and safety Product risks are Focus on unexpected consequences
appropriate for the of the effect of the vaccine dose and
anticipated use. direct effects due to vaccine virus
replication and tissue tropisms. The
evaluation includes scoring and
statistical analysis for histopathogical
lesions and clinical signs between
treatment and control groups.
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B.2 Product development and characterization

It is critical that vaccine production processes are standardized and controlled
to ensure consistency of manufacture in support of nonclinical data suggesting
potential safety and efficacy in humans. This is a prerequisite for entering the
clinical trial phase.
Each of the attenuated virus candidates in the tetravalent dengue vaccine
formulation should be characterized to define as far as is practical the critical
genetic markers of attenuation and phenotypic markers that suggest that the
genome of a vaccine virus has remained stable following tissue culture passage.
Each vaccine virus should also be evaluated to determine whether the genetic
basis of attenuation is stable enough to reduce the risk of reversion to virulence,
either during manufacture or during replication in a vaccinee, using available in
vivo and in vitro approaches. To this end, laboratory and animal studies should
define genetic changes in the virus genome.
Phenotypic markers may be useful for detecting reversion events and
to differentiate vaccine strains from wild-type virus strains in epidemiological
surveillance following human immunization.
Qualification of each attenuated vaccine strain should include obtaining
the consensus nucleotide sequence of the entire genome of the vaccine candidate,
using the consensus nucleotide sequence of the genome of the parent virus as
a comparator. This is essential for documenting the mutations in the vaccine
virus genome that may correlate with its attenuated phenotype. It is also good
practice to document any in vitro phenotypes of vaccine viruses that might serve
as indicators of the stability of the mutations that differentiate the vaccine virus
from its virulent parent. Such markers include, but are not limited to, plaque size,
replication efficiency in mosquito vectors, induction of viraemia in nonhuman
primates, suckling mouse neurovirulence, virulence in any other animal model,
and temperature sensitivity (4, 22, 46–48). Developers should bear in mind that
consensus genome sequencing is unsuitable for identifying minor or quasi-
species genomes in a vaccine seed or batch (6).
B.3 Nonclinical immunogenicity and protective activity

Assessment of innate and adaptive immune responses in animals provides
evidence that the dengue vaccine has replicated in the host, at the very least.
Animals, particularly mice, have also been valuable for assessing the various
elements of the immune response to DENV. Although there is no specific immune
correlate of protection, antibodies directed against the virus E protein neutralize
the virus and have been shown to protect animals when actively induced by
experimental vaccines or when passively administered prior to challenge. On the
basis of the accumulated data, it is generally accepted that protection in humans
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should require a DENV-specific neutralizing antibody response. However, a

correlation between the titre of neutralizing antibodies in serum, as determined
in an in vitro neutralizing antibody assay (e.g. PRNT50), and protection has not
been established for any of the four serotypes of virus.
While protective activity in an animal model does not necessarily predict
the protective effect in humans, it provides useful information regarding the
potency of the vaccine.
The immune response to or protective activity of each of the four
serotypes in a tetravalent DENV vaccine should be assessed, including the
quality of response and any potential virological/immunological interference
between types.
B.4 Nonclinical toxicity and safety

B.4.1 Considerations
General guidance on the nonclinical safety assessment and design of preclinical
studies that apply to dengue vaccines is provided in the WHO Guidelines on
nonclinical evaluation of vaccines (45). The term “toxicity” is generally associated
with the untoward consequences of the administration of a nonreplicating
medicine or biological that relate to its direct dose-dependent effect in the test
animal. Thus toxicity studies entail the careful analysis of all major organs, as well
as tissues near to and distal from the site of administration, to detect unanticipated
direct toxic effects typically of a drug or nonreplicating biological agent over a
wide range of doses, including doses sufficiently exceeding the intended clinically
relevant amount of dose. It is generally expected that, if a live, attenuated vaccine
does not replicate in the test animal, direct toxic effects are very unlikely to be
detected. For live vaccines the emphasis is on the demonstration of nonclinical
safety as a consequence of vaccine virus replication.
Nonclinical safety studies of live vaccines should be required for live,
attenuated vaccines in certain stages of development. Such studies are designed
with the primary purpose of demonstrating that the vaccine(s) is less “virulent”
in the animal host than comparable wild-type viruses, and that the vaccine does
not exhibit any unexpected harmful tissue tropism and damage or the capacity
to elicit a harmful immune response. There is no animal model that replicates
human dengue disease adequately (see sections B.2.1 and B.2.2). However,
nonhuman primates and mice may provide useful information for characterizing
the viruses (see sections B.4.2 and B.4.3). The design of preclinical safety studies
should reflect route and frequency of administration, as proposed in the protocol
to support clinical trials (45).
If the live, attenuated DENV vaccine is intended to be used to immunize
women of childbearing age, developmental/reproductive toxicity studies should
be performed according to WHO guidelines (45).
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B.4.2 Assessment in the nonhuman primate

B.4.2.1 Neurovirulence and neurotropism in nonhuman primates
The consensus of current opinion is that all live dengue vaccines should be
tested once for neurovirulence. If any vaccine virus strain is determined to be
neurovirulent to nonhuman primates on the basis of neurovirulence testing,
neurotropism in nonhuman primates via the clinical or peripheral inoculation
route should also be evaluated as part of the nonclinical safety study.
At this time, the most well-established model for vaccine neurovirulence
is the nonhuman primate, which has historically been used to evaluate new seeds
of yellow fever vaccines (17D substrains 17D204- or 17DD-derived) and live polio
vaccines. Novel rodent (hamster and mouse) models for yellow fever vaccine
virulence are currently under development. A rodent model could eventually be
considered in place of nonhuman primate testing (40) (see section A.3.2.5.5.1).
Involvement of the central nervous system in cases of dengue fever and
dengue haemorrhagic fever has usually been diagnosed as secondary to vasculitis
with resultant fluid extravasation. The rarity of reports of patients with dengue
encephalitis suggests that the virus does not typically cross the blood–brain
barrier and infect neuronal cells (49). However, since dengue vaccine viruses
are genetically altered compared to their wild-type parent viruses, it is advisable
to ensure that candidate vaccines have not acquired a neurotropic phenotype
as an unintended consequence of the attenuation process. This is a particular
concern with regard to dengue vaccine viruses that contain yellow fever 17D
chimeric genomes, and it would be of similar importance in the future if novel
dengue vaccines are derived from the genomes of any other known neuropathic
viruses. This evaluation could be done once at an early stage of development,
using a master seed or working seed lot of the vaccine. NRAs would need to
decide whether each component of the tetravalent formulation needs to be
tested separately for the property of neurovirulence or whether the tetravalent
formulation could be tested initially, in which case no further testing of the
individual vaccines would need to be done if results of the initial tests were
within predefined specifications.
Testing for neurovirulence in the nonhuman primate model via the
intracerebral inoculation route should follow the WHO recommendations for
neurovirulence testing of yellow fever vaccines (50, 51) as appropriate (see a brief
procedure below).
Groups of at least 10 monkeys, determined to be non-immune to
DENV and YFV prior to inoculation with the DENV master seed, should be
inoculated intracerebrally in the frontal lobe. A control group of 10 monkeys,
also demonstrated to be non-immune to DENV and YFV, should receive yellow
fever 17D. All monkeys should be observed for 30 days for signs of encephalitis,
prior to necropsy. If the number of monkeys, the observation period and/or
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time-point(s) for necropsy for histological examination are different from these
recommendations, they should be justified and agreed with the NRA. Clinical
scores, and the scores of histological lesions in the central nervous system, should
be recorded. An advanced histological scoring method such as automated image
analysis (52) may be implemented to provide quantitative assessment of virus-
induced histopathology in brain tissues if the method has been properly validated
and is acceptable to the NRA. The overall mean clinical and histological scores
of the test group should not exceed the scores of the yellow fever vaccine control
group. The significance level in statistical difference between test and control
groups should be agreed by the NRA.
B.4.2.2 Viraemia in nonhuman primates

Nonhuman primates, humans and mosquitoes are the only natural hosts of DENV
(4, 6, 8). Nonhuman primates have been widely used to evaluate replication and
immunogenicity of candidate dengue vaccines (3, 5, 10). Primary infection of
macaques with wild-type DENV results in moderate lymphadenopathy and a
robust immune response (4, 6). The nonhuman primate model has traditionally
been used as an important guide for selecting vaccine strains for further
development. In such studies, reduced peak titres and duration of viraemia
induced by a candidate vaccine, compared to those induced by the non-attenuated
parent virus, is often – but not always – a correlate of attenuation. Consequently,
if a dengue vaccine candidate causes viraemia in nonhuman primates comparable
to that caused by its wild-type parent virus, the vaccine developers may wish to
consider discontinuing further development.
B.4.3 Assessment in mouse models

DENV infection has been studied in many different mouse models (4, 10–13).
When appropriate, a mouse model may be selected to evaluate the potential of a
candidate vaccine to cause disease in comparison to its wild-type parent virus. In
such an experiment, the titres of virus in blood, spleen, liver, lymph nodes, lungs,
brain and other tissues at various post-infection time-points can be evaluated
(4). The AG129 interferon receptor-deficient mouse will support replication of
selected DENVs of all serotypes (22, 48). A DENV-2 strain adapted to replicate
in the AG129 mouse induces a physiologically relevant disease in that strain (10).
At present, the AG129 mouse seems suitable for safety studies, but NRAs should
be aware of the pitfalls of interpreting results since these animals do not possess
an intact innate immune response. For this same reason, as mentioned earlier, it
would not be advisable to use AG129 mice for classic toxicology studies. Other
inbred mouse strains with genes knocked out are under investigation as models
of DENV infection and disease. One or more of these may have applicability to
vaccine development in the future.
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B.4.4 DENV replication in vector mosquitoes

Transmission of DENV to arthropod vectors from humans is essential in
maintaining the virus in nature. As noted previously, none of the DENV live,
attenuated candidate vaccines studied to date induces a viraemia in vaccinees
that is sufficient in magnitude to infect feeding mosquitoes (6, 15, 19). Further,
if mosquitoes are infected with dengue vaccines, the viruses do not replicate
sufficiently to permit transmission of the virus. For these two reasons, Ae. aegypti
mosquitoes are not expected to transmit dengue vaccine viruses (6, 18, 19). As
a measure of attenuation and safety, future novel candidate vaccines should
be shown to have reduced ability to replicate and disseminate in Ae. aegypti
mosquitoes that have been infected in a controlled laboratory setting, using
parent strains as controls (6, 16, 18, 53).
B.5 Environmental risk

The primary environmental risks of live dengue vaccines relate to their capacity
to be spread from human to human by vector mosquitoes, and the risk that
prolonged or repeated cycles of replication in mosquitoes could permit reversion
to virulence. As previously noted, live vaccines currently under development have
been shown to replicate poorly both in vaccinees and in mosquitoes, such that
the risk for transmission by the mosquito vector is very low, if any risk exists at
all (14–16, 18, 19, 54). These factors should markedly reduce the chance that any
of these vaccines could revert in mosquitoes to a virulent phenotype when used
in a mass vaccination campaign in an endemic area. In addition, genetic stability
during multiple sequential passages in mosquitoes has also been demonstrated
for most existing live dengue vaccine candidates. For future candidate novel live
vaccines, similar studies would need to be done.
Some investigators have recently raised a concern regarding live dengue
vaccines, suggesting that vaccine viruses might revert to virulence in mosquitoes
via intragenic recombination with endogenous wild-type flaviviruses. Such a
phenomenon would seem to be highly unlikely due to the factors noted above
plus the controversial question of whether flaviviruses are able to undergo
recombination at all, even under ideal conditions in vitro.
Guidelines for live dengue vaccines derived by recombinant DNA
technology are described in Part D below.
Part C. Clinical evaluation of dengue tetravalent

vaccines (live, attenuated)
C.1 General considerations for clinical studies
The following should be read in conjunction with: WHO’s Guidelines on clinical
evaluation of vaccines: regulatory expectations (55); and Guidelines for the clinical
evaluation of dengue vaccines in endemic areas (56).
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C.1.1 Objectives of the clinical development programme

The clinical evaluation of a candidate live dengue tetravalent vaccine should
document:
■ the immune responses elicited by the vaccine against all four dengue
serotypes;
■ vaccine efficacy in the prevention of DFI of any severity caused by any
of the serotype 1, 2, 3 and 4 viruses over an appropriate minimum
period of observation;
■ the safety profile.
In addition, the programme should:
■ gather preliminary evidence that the immediate and longer-term
immune response to a candidate dengue vaccine does not predispose
vaccinated individuals to develop severe DFI (e.g. including
haemorrhagic manifestations and systemic shock) during natural
infections;
■ attempt to examine the association between neutralizing antibody
titres and protection against clinical disease (referred to as a
surrogate marker for efficacy in this document);
■ attempt to identify a neutralizing antibody titre that predicts (in the
short or longer term) protection against clinical disease (referred to
as an immunological correlate of protection in this document).
C.1.2 Outline of the clinical development programme

In the initial clinical studies (i.e. Phase 1 studies) it is expected that relatively
small numbers of healthy adults are vaccinated with investigational vaccine
formulations and that the primary focus is on assessing safety. These studies may
include exploration of immune responses to ascending doses of the four DENV

serotypes when administered alone and in combination.
The subsequent clinical studies (i.e. Phase 2 studies) should be designed to
select a dose of each DENV serotype for use in the tetravalent candidate vaccine
formulation and to identify an appropriate primary immunization schedule for
further study.
It is not currently possible to license candidate dengue vaccines only
on the basis of safety and immunogenicity data because there is no established
surrogate marker for protection and, hence, no immunological correlate of
protection has been identified.
Therefore, candidate tetravalent dengue vaccines should be evaluated for
protective efficacy against DFI.
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Sponsors may decide to conduct at least one preliminary study of protective

efficacy (sometimes referred to as a Phase 2b study) in order to identify a final
candidate vaccine and immunization schedule for further study. Alternatively,
depending on the data already accumulated (e.g. based on demonstration of a
robust neutralizing antibody response), sponsors may consider it appropriate to
omit such a study.
The selected candidate vaccine should be evaluated in at least one
adequately sized study of protective efficacy (i.e. Phase 3 study) that compares
numbers of cases of virologically confirmed DFI (see section C.3.3.5) between
groups of vaccinated and unvaccinated subjects. The total DFIs counted should
include those due to any of the four DENV serotypes and of any degree of clinical
severity that occur within a defined observation period.
Section C.3 gives more details of study designs and populations to be
enrolled in studies conducted at each phase of development.
C.2 Immunogenicity
C.2.1 Measurement of immune responses to vaccination
Current evidence suggests that neutralizing antibody against each DENV serotype
is likely to be the best surrogate marker for efficacy.
It is recommended that the methodology for determination of DENV
serotype-specific neutralizing antibody titres should follow WHO guidelines for
the plaque-reduction neutralization test (PRNT) (57). If alternative methods for
determining neutralizing antibody (e.g. high throughput microneutralization
assays) are developed, these should be validated against the PRNT.
Vero cells for dengue PRNT are available from the National Institute of
Biological Standards and Control, England. Serum neutralization titres should
be expressed in IUs calibrated against the reference panel of human antisera for
dengue (see section A.1.3). In-house virus strains may be used.
An assessment of neutralizing antibody titres against each of the four
serotypes of DENV is required. Additional testing against a range of strains of
those serotypes, including recent wild-type isolates, is encouraged. This would
be valuable information to obtain due to worldwide strain diversity and because
neutralizing antibody titres against specific isolates will be variable. Such
additional assays could be applied to subsets of sera collected from vaccinees
who have been selected randomly, or on the basis of a scientific justification (e.g.
to select sera known to cover a range of neutralizing antibody titres against the
reference or in-house strains).
The assay of DENV-specific antibody other than neutralizing antibody
(e.g. IgM and IgG ELISA) may be of interest but is not considered to be essential
for the assessment of potential vaccine efficacy.
89
It is considered unlikely that data on cell-mediated immunity will provide

an immunological correlate of protection. However, the exploration of cell-
mediated immunity is encouraged since specific cell-mediated immunity assays
may be useful for the assessment of immunological memory and durability
of protection. Assessments of cytokine responses may assist in the evaluation
of vaccine safety and may provide some indication of the potential risk that
vaccination could predispose subjects to develop severe DFI during subsequent
natural infection (58).
C.2.2 Investigation and interpretation of immune responses to vaccination

There is no established immunological correlate of protection against any DENV
serotype. In the initial clinical studies of safety and immunogenicity, including
the dose-finding and regimen-finding studies, it is essential to describe fully
the pre-vaccination and post-vaccination neutralizing antibody titres that are
observed against each of the four DENV serotypes (see also section C.3.1).
Adequate data should be generated to describe the kinetics of the neutralizing
antibody response in the short term. Longer-term antibody persistence data may
be collected in these and/or in later studies, as described below.
In a non-endemic population with no detectable pre-vaccination
neutralizing antibody in the majority of subjects, a comparison of percentages
with a detectable neutralization titre post-vaccination (which may be defined
as seroconversion in such a population) should be made against each DENV
serotype. The analyses should also look at proportions that seroconvert (in
accordance with an appropriate definition of seroconversion stated in the
protocol) to multiple serotypes (i.e. two, three or all four serotypes).
In an endemic population in which very high proportions of subjects
are already seropositive for neutralizing antibody with respect to at least one
dengue type, a comparison of pre-vaccination and post-vaccination geometric
mean titres with respect to those types will be informative, in addition to
analyses based on seroconversion rates and increments in antibody from pre- to

post-vaccination.
In endemic and non-endemic populations, detailed consideration of reverse
cumulative distribution curves is important. For example, it may be informative
to compare percentages achieving a predefined high titre of neutralizing antibody.
In protective efficacy studies, neutralizing antibody against DENV
serotypes should be determined and followed over time in predefined subsets
of the study population, including an assessment of antibody persistence after
the protocol-defined period for the primary evaluation of protective efficacy.
It is preferable that the subsets of subjects to be included in these detailed
immunogenicity evaluations should be identified at the time of randomization,
with stratification for age and any other factors that may have an important impact
on immune responses to vaccination. In any case, the data should be analysed
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according to predefined subsets. Immune responses should be determined for

vaccinated and unvaccinated subjects so that the effects of background exposure
to DENVs during the study period can be assessed.
Depending on the specific vaccine construct and taking into account any
pertinent results of nonclinical studies, sponsors may wish to undertake some
exploratory investigations of antibody against other antigens (e.g. those associated
with the attenuated yellow fever virus backbone in a chimeric vaccine).
Long-term storage of sera is encouraged since future developments in
the field, and/or emerging data on longer-term safety or efficacy, may point to
the need for additional investigations that cannot be predicted at the time of
conducting the study.
Subsets of subjects should also be identified for collection of peripheral
blood mononuclear cells, taking into account feasibility issues such as the blood
volumes required from different age groups to produce adequate cell numbers
for study and accessibility to adequate sample processing and storage facilities.
For the analysis of the relationship between neutralizing antibody titres
and protection against virologically confirmed DFI, sera should be collected at
timed intervals from a substantial cohort of subjects (and preferably from the
entire study population, if feasible). Once the protocol-defined double-blind
observation period has been completed, the initial analysis of the relationship
between immune response and protection against DFI should follow. The most
likely approach would be a cohort study in which one or more measures of the
immune response to vaccination are related to disease in all, or in a large subset
of, immunized subjects. Further analyses using longer-term follow-up data
should be planned.
The use of serology to help identify infections with dengue viruses
(whether or not clinically apparent) is a separate issue that is discussed in
section C.3.3.
C.3 Clinical studies

C.3.1 Phase 1 studies
The Phase 1 studies should be designed to provide an early indication of
whether severe local and/or systemic adverse events may occur commonly
after vaccination. These studies may also provide preliminary data on immune
responses to assist in the selection of DENVs (or constructs) and doses to be
included in candidate tetravalent vaccine formulations for further study.
Subjects enrolled in these initial studies should be healthy adults who
are naive to flaviviruses (based on medical and vaccine history and serological
studies). It is preferred that the subjects are resident in non-endemic areas so that
they are not at risk of natural infection with dengue or other flaviviruses. Eligible
subjects should not be in need of vaccination against other flaviviruses, at least
throughout the duration of the study.
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Sponsors may choose to commence studies with a monovalent vaccine

(i.e. containing a single live, attenuated DENV serotype) before progressing to
evaluate multivalent versions (which may include bivalent, trivalent and then
tetravalent formulations) of a candidate dengue vaccine.
If a candidate tetravalent vaccine formulation elicits a much lower
antibody titre to one (or more than one) DENV serotype than to others, it is
important that consideration is given to modification of the vaccine (e.g. by
modifying the infectious titres of serotypes), and/or the immunization schedule,
due to the potential implications for safety and efficacy.
If a likely candidate tetravalent vaccine is identified, it may be appropriate
for a preliminary exploration of safety and immunogenicity to be conducted in
healthy adult residents of an endemic area (i.e. including subjects with evidence
of some pre-existing immunity to dengue or other flaviviruses). Such a study
could provide further reassurance regarding the ability of the candidate vaccine
to elicit immune responses to all four DENV serotypes before progressing to
studies in larger numbers of subjects.

The Phase 2 studies should extend the information on safety and immunogenicity
of candidate vaccine formulations. They should include studies in residents of
endemic areas who are therefore at risk of natural infection with dengue and may
have some degree of pre-existing immunity to one or more DENV serotypes
and to other flaviviruses.
While the first data may be obtained in adults there should be a plan to
move down to younger age groups in a stepwise fashion. The age range should
reflect that proposed for the evaluation of protective efficacy of the tetravalent
vaccine candidate. Depending on the findings of the Phase 1 studies, the first
Phase 2 studies may further explore dose–response relationships. The data
generated on safety and immunogenicity should be sufficient to support the
selection of one or more candidate tetravalent vaccines and immunization

schedules (i.e. number of doses and dose intervals) for further evaluation.
If the sponsor chooses to undertake a preliminary (i.e. Phase 2b) study of
safety and efficacy, this should be of an appropriate design and of adequate size to
support a robust decision regarding the vaccine formulation and schedule to be
further evaluated (see section C.3.3). Even in a Phase 2b study, it is recommended
that subjects should be followed up for approximately 3–5 years from the time
of completion of vaccination to collect data on safety and to document antibody
to DENV serotypes in subsets of each treatment group.
The total number of subjects enrolled in Phase 2 studies should be
sufficient to describe at least common adverse reactions to vaccination with
some degree of confidence. Therefore it is expected that several hundred subjects
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should have been exposed to candidate tetravalent vaccines containing the final
or near-final doses of DENVs of each serotype. If any unusual, severe or serious
adverse reactions are documented, it may be appropriate for further studies to
include the assessment of safety as one of the primary objectives provided that
these reactions would not preclude further vaccine development.

Each tetravalent candidate vaccine should be evaluated in at least one study that
is of an appropriate design and adequate size to estimate vaccine efficacy. This
requirement may change in the future (see section C.3.3.7).
C.3.3.1 General issues for study design

Vaccine efficacy is estimated by comparing the total numbers of virologically
confirmed cases of DFI of any degree of severity, and due to any of the four
DENV serotypes, between the vaccinated and unvaccinated (control) groups.
The primary analysis of vaccine efficacy should be conducted at the conclusion
of a protocol-defined double-blind observation period. Each study should be of
sufficient size and duration to provide a robust estimate of vaccine efficacy and to
provide preliminary evidence that the vaccine does not predispose recipients to
develop one of the severe forms of DFI following natural infection.
Studies of protective efficacy should be performed in endemic areas
where a proportion of the population is likely to have some naturally acquired
immunity to one or more of the four DENV serotypes and/or other flaviviruses.
It is assumed that, in most – if not all – cases, each study will evaluate a single
tetravalent candidate dengue vaccine and immunization schedule. However,
the study design may be adapted as necessary if more than one possible active
vaccination group is to be included.
Studies that involve vaccination of a large proportion of subjects at any one
study locality carry the potential to interrupt DENV transmission significantly
during the observation period. The result could be a reduced likelihood of
demonstrating a difference in the numbers of virologically confirmed cases of
DFI between the vaccine and control groups. Consideration should be given to
this possibility when designing the study.
Randomization should be performed using a centralized system. When
using a 1:1 randomization ratio, the block size should be selected with the aim
of enrolling approximately equal numbers in test and control groups at each of
the study sites so that subjects in each group are at the same risk of developing
mild and severe DFI throughout the observation period. It is also possible to
consider the use of unbalanced randomization (e.g. vaccine:control = 3:2 or 2:1)
provided that care is taken to ensure that the desired ratio is applied at each study
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site (or geographically localized sites) and the sample size is calculated to provide
adequate power.
The decision to use unbalanced randomization should take the possible
advantages and disadvantages into consideration. Advantages include a larger
safety database and possibly easier enrolment due to the greater chance that any
one subject would receive the candidate dengue vaccine. Disadvantages include
the possibility that a larger proportion vaccinated against dengue could increase
the risk of achieving a reduction in DENV transmission sufficient to influence
the chance of obtaining a conclusive study result.
Whenever possible, subjects randomized to the control group should
receive an alternative active vaccine (i.e. not a dengue vaccine) that can be given
by the same route of administration as the candidate tetravalent dengue vaccine,
rather than injections of placebo. The active vaccine should be selected to provide
an anticipated benefit to study participants. However, such an appropriate
vaccine may not always be available and there may be no option to using placebo
injections to maintain the double-blind design. In addition, if the active control
vaccine cannot be given at the same schedule as the candidate dengue vaccine,
then placebo injections may need to be used within the schedule, as necessary,
to maintain a double-blind design.
If the active control vaccine has a different presentation or appearance
from those of the candidate dengue vaccine, study personnel who administer the
vaccinations should not have any other involvement in the conduct of the study.
Vaccine recipients should not be allowed to observe preparation of the vaccines
for injection (e.g. any reconstitution steps that may or may not be necessary) to
avoid the risk of their sharing this information and so identifying themselves
with one of the study groups.
If the use of a placebo control is necessary to achieve a double-blind
design, the protocol could plan to administer a suitable licensed vaccine to all
subjects in the study (i.e. those who do and who do not receive the candidate
dengue vaccine) at some time after completion of the assigned study treatments
and during the double-blind follow-up period. In this way, all study subjects
can derive some potential benefit from participation in the study without
compromising the study’s integrity.
It is expected that several different production lots of vaccine will be
used during protective efficacy studies. The decision whether a formal lot-to-
lot consistency study should be built into the protocol, with the specific aim of
comparing safety and immunogenicity between subjects who receive different lots
(usually three of the total used) according to predefined criteria, must be made
on a case-by-case basis. If such a formal comparison is to be made, additional
measures will be needed to ensure that adequately sized subsets of subjects are
randomized to receive each of the vaccine lots identified for this comparison.
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C.3.3.2 Study location and duration

The geographical areas selected for study should have background rates of
DFI that are sufficient to provide enough cases in the control group during the
observation period to facilitate the estimation of vaccine efficacy. In order to
assess background rates, efficacy studies should be preceded by the collection
of epidemiological information to document the expected incidences of DENV
serotype-specific DFI, and all DFI, preferably over several years. The data should
include information on seasonality of disease to identify periods of transmission
and case demographics (e.g. age and sex), so that the populations at highest risk
of DFI can be targeted for enrolment.
There should also be an assessment of the likely extent of exposure of
the population to other species of flaviviruses at potential study sites, because
such exposure may confound the interpretation of dengue-specific serological
data and may possibly affect the clinical course of DFI. This assessment should
take into account any available epidemiological data, serological studies, and
information on rates of vaccination against other flaviviruses.
Study sites should be endemic for dengue disease. Site selection should
be based on the information collected prior to study initiation regarding the
expected number of cases of dengue within the study population each season
during the observation period, which would probably range from one to three
years from the time of the first vaccination. Nevertheless, even if the study is
conducted over several seasons and at geographically dispersed study sites,
there may not be sufficient numbers of cases of DFI to support an estimation of
serotype-specific vaccine efficacy for some or all of the four serotypes. Additional
evidence for protective efficacy against individual DENV serotypes should be
sought from post-licensure (i.e. effectiveness) studies, as discussed in sections
C.3.3.7 and C.4.
There should be a plan for follow-up of subjects for safety and efficacy for
at least 3–5 years from the time of completion of primary vaccination. During
this period it is possible that an efficacious dengue vaccine may be offered to
subjects originally assigned to the control group with potential implications for
interpretation of the data that can be collected (see section C.3.3.7).
C.3.3.3 Study population

Since protective efficacy studies should be performed in endemic areas, there is a
need to consider that the ultimate target group for vaccination may range from a
subgroup (e.g. a specific age range) to the entire population. There are likely to be
concerns regarding the inclusion of infants in protective efficacy studies because
of the possible risk of DFI that has been reported in association with waning
maternal antibody against one or more DENV serotypes and the unknown effects
of vaccination in the presence of maternal antibody.
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Therefore, it is expected that protective efficacy studies would probably

exclude subjects aged under one year but should enrol children across a wide age
range subject to satisfactory results from the safety and immunogenicity studies.
Section C.3.3.7 considers bridging the observed vaccine efficacy to populations
that were not included in efficacy studies.
C.3.3.4 Objectives, end-points and analyses

The primary objective of an efficacy study is to estimate vaccine efficacy against
DFI. The primary analysis should seek to demonstrate superiority for the
vaccinated group versus the control group in terms of the total numbers of cases
of virologically confirmed DFI in subjects who have been fully vaccinated in
accordance with the protocol and have been followed up for the required time
with no major protocol deviations. In this analysis, counting of cases should
commence from a designated time-point after the last dose of protocol-assigned
doses has been administered.
Vaccine efficacy is estimated by comparing the total numbers of
virologically confirmed cases of DFI (i.e. summation of cases due to any DENV
serotype and of any degree of severity) that occur in vaccinated and unvaccinated
(control) groups during a protocol-defined double-blind observation period.
Vaccine efficacy should be calculated using the standard formula VE (%) = 100 ×
(1– r1/r0) (where VE = vaccine efficacy, r1 = incidence rate in the vaccine group,
and r0 = incidence rate in the control group).
The assessment of DENV serotype-specific vaccine efficacy should be
a major secondary objective and should be the subject of a planned secondary
analysis. It is not expected that the study would be powered to support a formal
statistical analysis of DENV serotype-specific efficacy.
The statistical analysis plan should explain how multiple episodes of DFI
in any one study participant will be handled in the analyses.
The following secondary analyses are suggested for inclusion in the
study protocol (although some of the data needed to complete these analyses
may not become available until some time after completion of the double-blind
observation period that precedes the primary analysis):
■ efficacy based on counting all DFI that occur after administration of
the first dose of protocol-assigned treatment;
■ efficacy in all vaccinated subjects regardless of protocol deviations
(including those with incomplete vaccination courses and missing
data);
■ efficacy according to pre-vaccination flavivirus serological status,
which might be determined in a randomized subset of enrolled
subjects who are followed serologically;
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Annex 2
■ efficacy according to severity of virologically confirmed DFI (with

adequate protocol definitions);
■ efficacy that includes prevention of “possible” or “probable” dengue
infection (e.g. applied to patients in whom serology is used as
the basis for dengue diagnosis without a virologically confirmed
diagnosis). The justification for this secondary analysis is based
on expectation that a dengue vaccine may reduce the viraemia, so
making it more difficult to detect in patients who may also have
abbreviated clinical signs and symptoms. Thus, serological secondary
end-points may help assess overall efficacy, assuming that serological
assays are equally sensitive and specific to DENV (but not to
individual serotypes) in detecting dengue infection in vaccine and
control groups; or
■ the effect of vaccination on the duration of hospitalization and/or
need for specific interventions to manage the clinical illness.
If more than one study of protective efficacy is performed with a single

candidate vaccine (e.g. perhaps covering different geographical regions) using
the same or a very similar study protocol, it may be appropriate to predefine a
pooled analysis of the data. This pooled analysis could provide additional insight
into serotype-specific vaccine efficacy and the risk of severe DFI in vaccine and
control groups.
Each study should have in place a data and safety monitoring board
consisting of persons with no involvement in study conduct and analysis and
including a statistician. The charter of the data and safety monitoring board should
enable it to unblind treatment assignments as necessary, and to recommend that
enrolment is halted or the study is terminated on the basis of predefined criteria
designed to protect subjects from harm. In addition, studies may include one or
more planned interim analyses with predefined stopping rules.
C.3.3.5 Case definitions

The case definitions for the primary and various secondary analyses, with details
of the criteria to be met, should be stated in the protocol and should be in
accordance with the latest WHO recommendations (2).
Clinical diagnosis: the most commonly diagnosed form of clinically apparent

dengue virus infection is characterized by the sudden onset of fever lasting at least
two, and up to seven, days. Fever is commonly accompanied by severe headache,
pain behind the eyes, gastrointestinal symptoms, muscle, joint and bone pain and
a rash. These cases are usually self-limiting and result in complete recovery.
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For the purposes of classification of cases it is important to characterize

the severity of each DFI. The criteria used to assess severity should be those
described by WHO that are current when the protocol is finalized (WHO/HTM/
NTD/DEN/2009.1 (2) at the time of preparation of these Guidelines). These
criteria should be used to determine the features of DFI that are captured in the
case report form.
Virological diagnosis: all methods used for the virological component of the
case definition should be fully validated. Virological confirmation of the clinical
diagnosis can be based on direct detection of dengue viraemia by isolation.
However, the use of alternative virological methods to confirm the diagnosis (e.g.
detection of NS1 to demonstrate the presence of DENV and the use of reverse
transcription-polymerase chain reaction (RT-PCR) to detect dengue viraemia and/
or determine the serotype) is acceptable. The standardization of viral diagnostic
methods is encouraged. Every effort should be made to conduct testing in one
or a small number of designated central laboratories with appropriate expertise.
Obtaining specimens to attempt virological confirmation of the diagnosis
should be triggered by a set of clinical features that are laid down in the study
protocol and that aim to identify all potential cases of DFI of any severity as
early as possible, taking into account the observation that virological diagnostic
methods (including virus isolation and PCR-based assays) are more sensitive
during the first five days of infection.
Serological diagnosis: commercial and/or in-house serological assays (e.g.
enzyme immunoassay, immunofluorescence and virus neutralization tests) may
be performed on paired acute and convalescent sera. The results may be used to
identify possible cases of DFI in which a virological diagnosis was not confirmed,
and the numbers may be compared between vaccinated and control groups in an
additional secondary analysis of vaccine efficacy.
Nevertheless, although an acute primary infection may be implied from
a rise in IgM levels during the first two weeks post-infection, such data need to
be interpreted with considerable caution. For example, the IgM response to acute
infection may be blunted in vaccinated subjects and in those infected previously
by wild-type DENV or by another flavivirus. Depending on the timing of the
illness, the results may also be confounded by the fact that IgM and IgG responses
may reflect recent dengue vaccination rather than acute infection with wild-type
dengue. In this regard, the ratio of IgM and IgG may assist in the differentiation
of primary and secondary infections.
The interpretation of serological data is also complicated by cross-
reacting antibody among flaviviruses. In those instances where cross-reaction
with other flaviviruses does not occur, a fourfold or greater rise in dengue
neutralizing antibodies makes it possible to attribute recent infection to a dengue
virus presumptively – but not definitively.
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Annex 2
C.3.3.6 Case detection and description

It is essential that there is adequate surveillance to detect any possible case of DFI
as early as possible in order to optimize the chances of virological confirmation
of the diagnosis. The surveillance mechanisms (e.g. including arrangements for
periodic home visits or telephone calls, and involvement of hospitals serving the
study catchment areas) should be tested before the study is initiated at each study
site. It is essential that study subjects are educated regarding the need to contact,
or directly present to, the designated study health-care facilities whenever they
develop signs or symptoms that may be indicative of DFI. A checklist of these
signs and symptoms should be provided to all study participants at the time
of enrolment.
In addition, measures should be in place to follow each possible case
of DFI for any change in disease course (e.g. progression from mild to severe
DFI, onset of complications) and to document the outcome, including the time
to recovery or death. In case of death before collection of specimens or in the
absence of virological confirmation of the diagnosis, permission should be
sought to perform a postmortem examination or, if this is refused, to at least
obtain a specimen for virological examination using needle puncture of the liver.
In some study sites that are otherwise considered suitable, it may not be
possible to identify a local health-care facility willing to participate in the study.
These sites should not be initiated unless there is at least agreement from local
health-care providers to notify study staff of possible cases of DFI within a time
frame that is sufficient to allow for specimens to be collected and transported for
virological diagnosis. Subjects should carry a study participant card, with contact
names and numbers, to ensure that study personnel are alerted and can arrange
for the collection of all the necessary clinical data and the transport of specimens
for virological diagnosis.
Despite taking the steps described, there will still be some cases of
possible DFI that are not confirmed virologically and for which serological
testing is inconclusive. In addition, some subjects may not comply with the study
requirement to present to a designated health-care facility when they have signs
and symptoms indicative of a possible DFI, or they may be so ill that they are
immediately admitted to a hospital not directly participating in the study and/
or may die without notification of study personnel in time to collect data and
specimens. It is important that as much information as possible is collected on
these cases whenever and however they come to light, and that they are taken
into account in a “worst-case scenario” analysis of vaccine efficacy that counts all
cases (proven and unproven and regardless of protocol deviations).
C.3.3.7 Need for additional studies of efficacy

Once the efficacy of at least one candidate dengue vaccine has been satisfactorily
demonstrated (and it is perhaps already licensed and introduced into the routine
99
vaccination programme in at least one country) there will be a need to reassess

the content of clinical development programmes for other candidate dengue
vaccines. For example, depending on the licensed dengue vaccine(s) available and
the data that have been generated during their development, it may or may not be
feasible or considered necessary to conduct studies that include an unvaccinated
control group with subsequent candidate tetravalent vaccines.
It is not currently possible to make a definitive recommendation regarding
what could or should be required in this scenario, since much will depend on the
findings reported from the first completed efficacy study of a candidate vaccine
or from ongoing studies with other candidate vaccines. Some pertinent issues are
discussed below.
Once one or more dengue vaccine(s) has been licensed and introduced
into the routine vaccination programme in one or more countries, the inclusion
of an unvaccinated group in subsequent studies in these (and possibly other)
countries may be considered unethical. However, studies that include an
unvaccinated group may be feasible in some regions after approval of the first
vaccine where there is appropriate justification and if approved by the NRA. In
such a case, standard care and protection of study subjects should be provided
as appropriate.
Efficacy studies using an unvaccinated control group and relative efficacy
studies (i.e. studies in which the test vaccine is compared to a licensed vaccine)
may not be feasible once a dengue vaccine has been introduced into the routine
vaccination programme in a country or region, because this may reduce the
incidence of DFI to levels that are too low to permit the estimation of vaccine
efficacy from further studies of feasible size and duration.
However, if there remains considerable uncertainty about vaccine
efficacy against one or more DENV serotype(s), efficacy studies that include an
unvaccinated control group might be possible in regions where such serotype(s)
are predicted to predominate.
It is very possible that immunological correlates of efficacy for each of

the four serotypes of dengue virus cannot be established by analysis of early
results of the first efficacy trials. Until surrogates and correlates of protection have
been established, it may not be possible to determine the efficacy of a novel live
vaccine by conducting a head-to-head comparison of its immunogenicity to that
of a licensed live vaccine (i.e. in a bridging study). It may also be problematic to
use bridging studies to support the extrapolation of efficacy observed with live
virus vaccines to other types of dengue vaccine (e.g. killed virus vaccines, DNA
vaccines, subunit vaccines). However, there may be no alternative to the use of
bridging studies because of the factors described above.
Before resorting to bridging studies, there should be a careful scientific
evaluation of the arguments for and against extrapolation of the efficacy observed
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for a particular vaccine to populations that differ in character from the population
in which the efficacy study was actually performed. Examples include populations
that differ in age, risk for severe dengue, ethnicity, and/or prior or concurrent
exposure to other flaviviruses.
C.3.3.8 Documentation of safety during pre-licensure studies

The routine monitoring of safety during all pre-licensure clinical studies should
follow the usual principles taking into account issues relevant to live, attenuated
vaccines. In addition to providing study-specific safety data, there should be
an analysis of safety data pooled across all study groups that received the final
selected vaccine formulation.
There is a particular need to assess whether DFI (which could be of any
degree of severity, including very mild illness) may be caused by vaccine strains.
In all cases of fever or other dengue-like signs or symptoms that occur following
vaccination during clinical studies, it is essential that laboratory investigations
are undertaken to determine whether or not the vaccine is responsible. Since the
results will not always allow for a clear judgement of relatedness to vaccination,
the protocol should provide criteria for ranking causality.
Low-grade and very transient fevers are to be expected and have
been routinely observed in a small fraction of vaccinees after exposure to live
dengue vaccines. NRAs will have to judge on a case-by-case basis whether the
incidence, duration and/or severity of febrile episodes that cannot be ascribed to
intercurrent illness that is observed during early studies in non-endemic areas
are unacceptable. In these studies the level of vaccine viraemia (determined
by RT-PCR and/or by direct culture methods) and level of vaccine virus NS1
antigenaemia should be determined in each vaccinee at one or more time-points
post-vaccination in order to establish an average profile for the novel live vaccine
under study. Levels higher than the predetermined average levels of vaccine
virus or NS1 protein in blood detected during a febrile episode could be taken as
evidence in favour of a direct causative role for the vaccine. During later studies
in endemic areas there is a need to distinguish vaccine-associated from naturally
occurring DFI, in addition to the other issues noted above.
There is a risk that vaccination could predispose recipients to developing
a severe form of DFI. The risk may increase with time elapsed since vaccination in
relation to waning titres of vaccine-induced antibodies in subjects who have not
been naturally boosted in the interim period. The monitoring and investigation
of all subjects who develop signs or symptoms potentially indicative of DFI
during pre-licensure studies in endemic regions should provide a preliminary
assessment of this risk. If no undue risk is identified and the vaccine is licensed,
it is essential that there is adequate follow-up of study subjects together with
further assessment of the risk in the post-licensure period (see section C.4).
101
The total safety database derived from all pre-licensure studies should
be sufficient to describe uncommon adverse reactions. It is desirable to rule out
events that occur at a frequency greater than 1:1000 vaccinees.
On the basis of the considerations outlined in section C.3.3.7, it may be that
vaccines that are developed subsequent to the approval of the first vaccine(s) will
not be evaluated in pre-licensure studies of protective efficacy, with implications
for the size of the safety database. NRAs will need to assess numbers that would
constitute an adequate safety database before initial licensure. In addition, NRAs
may make specific recommendations regarding the method of data collection,
classification and scoring of severity of adverse events that are captured, as well
as the post-vaccination duration of the safety data collection (i.e. after each dose
and following the last dose of a course).
C.4 Post‑licensure investigations

There is a need to ensure that adequate surveillance is in place and is maintained
to detect adverse reactions during the post-licensure period, in accordance with
requirements of the countries in which approval has been obtained.
The need for, and the design and extent of, specific studies of safety and/
or effectiveness following approval of a dengue vaccine should be given careful
consideration by sponsors and NRAs. There will be a clear need to try to collect
information on the following:
■ long-term evaluation of breakthrough cases of DFI, to detect any
waning of protective immunity against one or more DENV serotypes
and the possible need for booster doses (there may be considerable
practical difficulties in collecting reliable data in some settings;
therefore it is recommended that sponsors and NRAs discuss ways
in which such data could be obtained at least in some areas/regions);
■ persistence of the immune response to vaccination (e.g. based on
serial measurements of neutralizing antibody and detection of

sensitized B-cells);
■ responses to booster doses, which may be planned for predefined
subsets enrolled in studies or may be instituted when disease
surveillance indicates a possible need;
■ the possible increased risk of severe DFI in vaccine recipients
(e.g. in some areas/regions it may be possible to collect data on
hospitalization of vaccinees to capture severe dengue cases);
■ surveillance of dengue in areas/regions where routine vaccination is
introduced and, if possible, collection of sufficient data for a formal
estimate of vaccine effectiveness.
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There are several possible study designs and methods for estimating
vaccine effectiveness and it is essential that expert advice is sought. In addition, it
is likely that such studies would need to be performed in close liaison with public
health authorities.
There may be no information on vaccine co-administration at the time
of initial licensure. Sponsors and NRAs should consider the need to assess the
interaction of any novel dengue vaccine with other vaccines that are likely to
be co-administered. For instance, in countries where dengue vaccination will
become part of the routine childhood immunization programme, the interaction
with the other vaccines used in the programme needs to be studied. In addition,
if licensure is sought in non-endemic areas with the intention of protecting
travellers, it is advisable to study the possible interactions of a novel dengue
vaccine with other vaccines for travellers. Interaction studies should assess safety
and the immune response to all co-administered antigens.
Some or all post-licensure studies may be conducted as post-approval
commitments made to an individual NRA. In this regard, both sponsors and
NRAs that have approved a vaccine should communicate and cooperate to
ensure that studies are well-designed to answer the questions posed and to avoid
demands for numerous studies in individual countries that are likely to be too
small to provide reliable results. Provisional plans for appropriate post-licensure
studies should be submitted with the application dossier and these should be
refined during the assessment by the NRA and as necessary after initial approval.
Part D. Environmental risk assessment of dengue

derived by recombinant DNA technology
D.1 Introduction
D.1.1 Scope
Some countries have legislation covering environmental and other concerns
related to the use of live vaccines derived by recombinant DNA technology since
they may be considered as GMOs. However, similar concerns may be raised by
live vaccines derived by conventional methods.
This section of the Guidelines considers the environmental risk
assessment (ERA) that may be performed during DENV vaccine development.
The ERA assesses the risk to public health and the environment. It does not assess
the risk to the intended recipient of the vaccine which is assessed through clinical
studies of the vaccine. Nor does the ERA assess the risk to laboratory workers.
The environmental impact is not usually the responsibility of the NRA
but of other agencies. Nonetheless the NRA should receive a copy of the ERA
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and of any associated decisions taken, both for information and to ensure that
the appropriate procedures have been followed.
D.1.2 Principles and objectives

Live DENV vaccine in which the genome has been genetically modified by
recombinant DNA technology is considered a GMO. The manufacture, use
and transboundary shipping of such live recombinant vaccines for research
or commercial use should, when applicable, comply with relevant legislation
or regulations on GMOs in the producing and recipient countries. In some
regulatory regimes, in order to comply with environmental regulations, an ERA
should be undertaken if the live vaccine is being tested in a clinical trial or if
it is placed on the market. It should be noted that the following guidance on
the ERA of live recombinant DENV vaccines is not intended to replace existing
GMO legislation that is already in place in certain countries.
Generally, the objective of an ERA is to identify and evaluate, on a case-by-
case basis, the potential adverse effects (direct or indirect, immediate or delayed)
of a GMO on public health and the environment. This means that a separate ERA
should be performed for each different live recombinant dengue vaccine. “Direct
effects” are primary effects on human health or on the environment which result
from the GMO itself and which occur through a short causal chain of events.
“Indirect effects” are effects that occur through a more extended causal chain of
events, through mechanisms such as interactions with other organisms, transfer
of genetic material, or changes in use or management. “Immediate effects” are
observed during the period of the release of the GMO, whereas “delayed effects”
are those effects which may not be observed during the period of release of the
GMO but which become apparent as a direct or indirect effect either at a later
stage or after termination of the release.
The ERA should be performed in a scientifically sound and transparent
manner and should be based on available scientific and technical data. Important
aspects to be addressed in an ERA include the characteristics of: (i) the parental
organism, (ii) the recipient organism, (iii) viral vector characteristics, (iv) the
donor sequence, (v) genetic modification, (vi) the intended use and (vii) the
receiving environment. The data needed to evaluate the ERA do not have to
derive solely from experiments performed by the applicant; data available in the
scientific literature can also be used in the assessment. Regardless of the source,
data should be both relevant and of an acceptable scientific quality. The ERA may
be based on data from experiments previously performed for other purposes,
such as product characterization tests and nonclinical safety and toxicity studies.
Ideally, the ERA is based on quantitative data and expressed in quantitative
terms. However, much of the information that is available for an ERA may be
qualitative since quantification is often difficult to accomplish and may not be
104
Annex 2
necessary to make a decision. The level of detail and information required in the
ERA is also likely to vary according to the nature and the scale of the proposed
release. Information requirements may differ between licensure and clinical
development and according to whether studies will be carried out in a single
country or multiple countries.
Uncertainty is inherent in the concept of risk. Therefore, it is important
to identify and analyse areas of uncertainty in the risk assessment. Since there is
no universally accepted approach for addressing uncertainty, risk management
strategies may be considered. Precise data on the environmental fate of the live
vaccine in early clinical trials will in most cases be insufficient or lacking. However,
at the stage of market registration, the level of uncertainty is expected to be lower
as gaps identified in available data should already have been addressed.
The need for risk management measures should be based on the
estimated level of risk. If new information on the GMO becomes available, the
ERA may need to be re-performed to determine whether the estimated level of
risk has changed. This also holds true if the risks for the participating subjects
have changed, as these aspects can be translated to other individuals. It should be
noted that the ERA will not deal with medical benefit for the subject or scientific
issues such as proof of principle.
D.2 Procedure for environmental risk assessment

Risk assessment involves identification of novel characteristics of the GMO
that may have adverse effects (hazard), evaluation of the consequences of each
potential adverse effect, estimation of the likelihood of adverse effects occurring,
risk estimation, risk management and, in some methodologies, estimation of the
overall risk to the environment. These processes should identify the potential
adverse effects by comparing the properties of the GMO with those of non-
modified organisms under the same conditions and in the same receiving
environment. The principles and methodology of an ERA should be applicable
irrespective of the geographical location of the intended environmental release
of the GMO. However, the ERA should take into account the specificities
associated with the mosquito vector being endemic or non-endemic in the
region in which vaccine trials will be carried out, and/or where licensure is
being requested. Depending on local regulatory requirements, the ERA may be
undertaken by the applicant or by the competent local authority on the basis of
data supplied. In all cases, the competent local authority should use the ERA as
a basis for deciding whether any identified environmental risks are acceptable.
Nevertheless, the decision on whether any identified risks are acceptable may
vary from country to country. Several national and international documents
address ERA issues (59–62).
The general process for undertaking an ERA is shown in Figure A2.1 as
an example (60, 61).
105
Figure A2.1
Typical steps in an environmental risk assessment
D.3 Special considerations for live recombinant dengue vaccines

The ERA of live recombinant DENV vaccines should be conducted according to
the general principles described above, taking into consideration in particular the
vector responsible for disease transmission. Aspects which could be developed
include: the genetic stability of the live recombinant virus (including reversion
and recombination), potential transmission of the vaccine virus among hosts by
the vector, and the immune status of the population. These aspects are further
outlined below.
D.3.1 Genetic stability

DENV vaccines currently under clinical evaluation are attenuated DENV strains,
intertypic chimeric vaccines or DENV/yellow fever 17D vaccine chimeras. In
the intertypic approach, the structural genes of an attenuated strain of DENV of
a given serotype are replaced by the corresponding genes of a different DENV
serotype. In the dengue/yellow fever chimeras, the prM/E structural genes of
the dengue virus are cloned into the backbone of the yellow fever 17D vaccine,
replacing the corresponding structural yellow fever 17D genes.
D.3.1.1 Reversion
After vaccination, there is potential for reversion of attenuated live dengue virus
vaccines to a virulent form of the dengue virus, although this has not been seen
in clinical trials so far. The potential reversion is based on the stability of the
attenuating mutation(s), the number of attenuating mutations, and the nature of
attenuating mutation. Attenuating mutations that are dependent on a single base
change may be more susceptible to reversion than a mutation that is stabilized by
multiple base substitutions. In addition, attenuating mutations that are derived
by deletions of segments of RNA are generally more stable against reversion.
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Annex 2
Changes in virus genotype have the potential to influence disease transmission,

tropism of vector vaccine, virulence, and/or patterns of disease, resulting in a
virus with a previously unknown combination of properties. However, the
likelihood of such a reversion depends on the number of attenuation mutations
present and the viral genes involved in the vaccine virus (63).
D.3.1.2 Recombination
Whether or not recombination takes place among flaviviruses is controversial. In
theory, recombination between live DENV vaccines and wild-type flaviviruses
could produce a virus with an altered phenotype, but there is currently no
evidence to support this (64–70).
The potential for recombination within and between flaviviruses has
been widely discussed and challenged in the past, both on the basis of existing
literature (64–67, 69, 70) and also of data obtained in specific experiments.
In particular, a “recombination trap” has recently been designed to allow the
products of rare recombination events to be selected and amplified, in the case of
West Nile encephalitis, tick-borne encephalitis and Japanese encephalitis viruses
(69). Intergenomic but aberrant recombination was observed only in the case
of Japanese encephalitis virus, and not for West Nile or tick-borne encephalitis
viruses. Moreover, its frequency appeared to be very low and generated viruses
with impaired growth properties.
While their likelihood of appearance is very low, as stated above, the
potential adverse effects of recombined DENVs should be evaluated in the ERA.
In this respect, “worst-case” scenarios for chimeras have been constructed to
address that risk (65, 66, 70).
These different studies showed that such recombinants constructed
artificially from a wild-type flavivirus and a chimeric vaccine (70), or from two
wild-type viruses, such as highly virulent yellow fever Asibi virus and wild-type
DEN-4 virus (66), were highly attenuated compared to their parental viruses.
Attenuation was shown in culture in vitro, in mosquito vectors and in susceptible
animal models, including monkeys. These data provide experimental evidence
that the potential of recombinants, should they ever emerge, to cause disease or
spread would probably be very low. Dual infection laboratory studies between
vaccine and wild-type strains are not recommended because the predictive
clinical value of such studies would be low.
D.3.2 Vector transmission

The presence of the DENV vectors such as Ae. aegypti and Ae. albopictus play
a key role in the transmission of flaviviruses and potentially of live DENV
vaccines from the vaccinated subject to other individuals. Dengue does not
spread directly from person to person, except via blood transfusion in very
rare instances where a donor was dengue viraemic. Transmission of the dengue
107
vaccine in regions where the vector is absent is therefore highly unlikely.

Dengue is currently restricted to the tropical and a few subtropical regions. Due
to climate change there is the possibility of a geographical shift in mosquito
populations which could conceivably lead to the spread of dengue to areas that
are currently non-endemic.
Recombination between live DENV vaccines and wild-type flaviviruses
could theoretically occur in a vaccinee (see above) and possibly also within an
infected mosquito, although neither has been reported. A recombined DENV
could potentially, for instance in combination with climate change, use new vectors
for transmission, leading to previously unknown transmission characteristics.
Therefore, the presence of a relevant mosquito vector and a “dengue favourable
climate” in the vaccination region should be taken into account in the ERA of live
DENV vaccines.
To assess the likelihood of effective transmission of the vaccine from a
vaccinated individual, two parameters should be taken into consideration: the
level of viraemia in the vaccinated hosts, and the ability of the mosquito vectors to
transmit the live DENV vaccine to new hosts. The blood titre required for effective
transmission of dengue virus from human to mosquito via the bite has been
studied in a laboratory setting. The typical level and duration of vaccine viraemia
in inoculated volunteer subjects is also known for all live vaccines currently in
the clinical phase of development. These data show that peak titres of vaccine
viraemia are several orders of magnitude below those needed to infect a mosquito
(71). In addition, the ability of the live vaccine viruses to replicate in mosquitoes
and then to escape the midgut in order to render the mosquito infectious for
humans by entering the salivary glands is also very impaired compared to wild-
type dengue (18, 19, 26, 30, 54, 72, 73). Thus it is highly unlikely that vaccinated
subjects could ever spread vaccine virus via mosquito transmission.
The outcome of the ERA for clinical trials in regions where the vector is
absent is obviously that the environmental risk is negligibly small. The mosquito
vector is not present and therefore the vaccine, or theoretical de novo recombinant
viruses, cannot be transmitted to other people. However, in endemic areas, NRAs

should decide whether or not to perform an ERA.
D.3.3 Immune status

Live DENV vaccines are able to replicate in vaccinated persons. The immune
status against the vaccine antigens, the viral vectors and/or cross-reacting
flaviviruses in the vaccinee may be a confounding factor in the assessment of
the environmental risk of a live DENV vaccine. In general, the presence of pre-
existing immunity due to earlier exposure to DENVs will reduce the extent and
duration of vaccine virus replication and dissemination within a vaccinee. The
potential for transmission of the vaccine is therefore considered to be greater in
naive or immunocompromised individuals.
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Annex 2
An unvaccinated population with no pre-existing immunity will respond

differently upon exposure to the vaccine compared to a population in which
dengue is endemic. The immune status should therefore be taken into account in
the ERA as it can influence both the environmental impact of the vaccines and
the potential occurrence of adverse effects in contacts of the vaccinees. There is a
theoretical potential for pre-existing heterotypic antibody to cause higher levels
of vaccine virus. Enhanced illness in vaccine recipients who have pre-existing
DENV antibody (antibody-dependent enhancement) has not been observed in
clinical trials of live, attenuated dengue vaccines to date and was not observed
in a clinical trial of live, attenuated dengue vaccines designed to address this
possibility (74, 75).
Part E. Guidelines for NRAs

E.1 General
The general recommendations for NRAs and NCLs provided in the Guidelines
for national authorities on quality assurance for biological products (76) should
apply. In addition, the general recommendations for NRAs and NCLs provided
in the Guidelines for independent lot release of vaccines by regulatory authorities
(77) should be followed. These Guidelines specify that no new biological
substance should be released until consistency of manufacturing and quality,
as demonstrated by a consistent release of batches, has been established. The
detailed production and control procedures, and any significant changes in them,
should be discussed with and approved by the NRA. The NRA should obtain the
working reference from manufacturers to establish a national working Reference
Preparation until an international Reference Reagent is available.
E.2 Release and certification

A vaccine lot should be released only if it fulfils the national requirements and/
or Part A of the present Guidelines. A protocol based on the model given in
Appendix 1, signed by the responsible official of the manufacturing establishment,
should be prepared and submitted to the NRA in support of a request for release
of vaccine for use.
A statement signed by the appropriate official of the NRA should be
provided if requested by a manufacturing establishment, and should certify
whether or not the lot of vaccine in question meets all national requirements, as
well as Part A of these Guidelines. The certificate should also state the lot number,
the number under which the lot was released, and the number appearing on the
labels of the containers. In addition, the date of the last satisfactory determination
of antigen concentration, as well as the expiry date assigned on the basis of shelf-
life, should be stated. A copy of the official national release document should be
109
attached. The certificate should be based on the model given in Appendix 2. The
purpose of the certificate is to facilitate the exchange of dengue virus vaccines
between countries.
Authors
The scientific basis for the revision of the Guidelines for the production and
quality control of candidate tetravalent dengue virus vaccines (live) published
in WHO Technical Report Series, No. 932, was discussed at the meeting of the
WHO working group on technical specifications for manufacture and evaluation
of dengue vaccines, which met in Geneva, Switzerland, 11–12 May 2009 and was
attended by the following: Dr A. Barrett, University of Texas Medical Branch,
Galveston, TX, USA; Dr D. Bleijs, National Institute for Public Health and the
Environment, Bilthoven, the Netherlands; Dr F. Denamur, GlaxoSmithKline
Biologicals, Rixensart, Belgium; Dr A. Durbin, Johns Hopkins Bloomberg
School of Public Health, Baltimore, MD, USA; Dr K. Eckels, Walter Reed Army
Institute of Research, Silver Spring, MD, USA; Dr R. Edelman, University of
Maryland School of Medicine, Baltimore, MD, USA; Dr D. Francis, Global
Solutions for Infectious Diseases, South San Francisco, CA, USA; Dr M. Freire,
Instituto Oswaldo Cruz, Manguinhos, Rio de Janeiro, Brazil; Dr J. Hombach,
World Health Organization, Geneva, Switzerland; Dr H. Langar, World Health
Organization Regional Office for the Eastern Mediterranean, Cairo, Egypt; Dr I.
Knezevic, World Health Organization, Geneva, Switzerland; Dr C. Lecomte,
GlaxoSmithKline Biologicals, Wavre, Belgium; Dr L. Mallet, Sanofi Pasteur,
Marcy l'Étoile, France; Dr H. Margolis, International Vaccine Institute, Seoul,
Republic of Korea; Dr L. Markoff, Center for Biologics Evaluation and Research,
Food and Drug Administration, Bethesda, MD, USA; Dr P. Minor, National
Institute of Biological Standards and Control, Potters Bar, England (Chair); Dr S.
Nishioka, World Health Organization, Geneva, Switzerland; Dr K. Peden, Center
for Biologics Evaluation and Research, Food and Drug Administration, Bethesda,
MD, USA; Dr M. Powell, Medicines and Healthcare Products Regulatory Agency,
London, England; Dr J. Robertson, National Institute of Biological Standards
and Control, Potters Bar, England; Dr J. Roehrig, Centers for Disease Control
and Prevention, Fort Collins, CO, USA; Dr A. Sabouraud, Sanofi Pasteur, Marcy
l’Étoile, France; Dr J. Shin, World Health Organization, Geneva, Switzerland;
Mrs P. Thanaphollert, Food and Drug Administration, Ministry of Public
Health, Nonthaburi, Thailand; Dr D. Trent, University of Texas Medical Branch,
Galveston, TX, USA (Rapporteur); Dr D. Wood, World Health Organization,
Geneva, Switzerland.
The first draft of these Guidelines was developed by the following lead
authors for the part indicated: (1) Part A – Dr L. Mallet, Sanofi Pasteur, Canada
and Dr P. Minor, National Institute of Biological Standards and Control, Potters
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Annex 2
Bar, England; (2) Part B – Dr D. Trent, University of Texas Medical Branch,

Galveston, TX, USA; (3) Part C – Dr M. Powell, Medicines and Healthcare
Products Regulatory Agency, London, England; (4) Part D – Dr D. Bleijs, National
Institute for Public Health and the Environment, Bilthoven, the Netherlands and
Dr J. Robertson, National Institute of Biological Standards and Control, Potters
Bar, England.
The first draft was discussed in the meeting of the working group held on
29–30 April 2010 in Geneva, Switzerland attended by: Dr A. Barrett, University
of Texas Medical Branch, Galveston, TX, USA; Dr D. Bleijs, National Institute for
Public Health and the Environment, Bilthoven, the Netherlands; Dr F. Denamur,
GlaxoSmithKline Biologicals, Rixensart, Belgium; Dr A. Durbin, Johns Hopkins
Bloomberg School of Public Health, Baltimore, MD, USA; Dr K. Eckels, Walter
Reed Army Institute of Research, Silver Spring, MD, USA; Dr R. Edelman,
University of Maryland School of Medicine, Baltimore, MD, USA; Dr D. Francis,
Global Solutions for Infectious Diseases, South San Francisco, CA, USA; Dr M.
Freire, Instituto Oswaldo Cruz, Manguinhos, Rio de Janeiro, Brazil; Dr N. Gallina,
Buntan, Sao Paulo, Brazil; Mr M. Galves, National Agency of Health Surveillance,
Brasília-DF, Brazil; Mrs F. Garnier, Agence Française de Sécurité sanitaire
de Produits de Santé (French Agency for Safety of Health Products), Lyons,
France; Dr J. Hombach, World Health Organization, Geneva, Switzerland; Dr I.
Knezevic, World Health Organization, Geneva, Switzerland; Dr L. Mallet, Sanofi
Pasteur, Toronto, Canada; Dr P. Minor, National Institute of Biological Standards
and Control, Potters Bar, England (Chair); Dr L. Morgan, Sanofi Pasteur, Marcy
l'Étoile, France; Dr Le Van Phung, National Institute for Control of Vaccine and
Biologicals, Hanoi, Viet Nam; Dr L. Markoff, Center for Biologics Evaluation and
Research, Food and Drug Administration, Bethesda, MD, USA (Rapporteur for
clinical working group); Dr J. Korimbocus, Agence Française de Sécurité sanitaire
de Produits de Santé (French Agency for Safety of Health Products), Lyons, France;
Dr S. Nishioka, World Health Organization, Geneva, Switzerland; Dr K. Peden,
Center for Biologics Evaluation and Research, Food and Drug Administration,
Bethesda, MD, USA (Rapporteur for manufacture, nonclinical and environmental
risk assessment group); Dr M. Powell, Medicines and Healthcare Products
Regulatory Agency, London, England; Dr V. Quivy, GlaxoSmithKline Biologicals,
Wavre, Belgium; Dr J. Roehrig, Centers for Disease Control and Prevention, Fort
Collins, CO, USA; Ms M. Saville, Sanofi Pasteur, Marcy l’Étoile, France; Mrs P.
Thanaphollert, Food and Drug Administration, Ministry of Public Health,
Nonthaburi, Thailand; Dr C. Thomson, Inviragen, Capricorn, Singapore; Dr D.
Trent, University of Texas Medical Branch, Galveston, TX, USA; Dr J-W. van der
Laan, National Institute for Public Health and the Environment, Bilthoven, the
Netherlands; and Dr D. Wood, World Health Organization, Geneva, Switzerland.
On the basis of comments from the April 2010 meeting, a second draft
was prepared by Dr D. Bleijs of the National Institute for Public Health and
111
the Environment, Bilthoven, the Netherlands; Dr L. Mallet of Sanofi Pasteur,

Canada; Dr M. Powell of the Medicines and Healthcare Products Regulatory
Agency, London, England; and Dr D. Trent of the University of Texas Medical
Branch, Galveston, TX, USA. A modified draft of Part D of the Guidelines was
further developed by Dr D. Bleijs of the National Institute for Public Health and
the Environment, Bilthoven, the Netherlands, on the basis of comments from
teleconferences held in January and February 2011 with an informal workgroup
on environmental risk assessment for dengue vaccines in which additional
members were: Dr M. Dornbusch, Office of the Gene Technology Regulator,
Department of Health and Aging, Canberra, Australia; Dr A. Durbin, Johns
Hopkins Bloomberg School of Public Health, Baltimore, MD, USA; Dr K. Eckels,
Walter Reed Army Institute of Research, Silver Spring, MD, USA; Dr R. Edelman,
University of Maryland School of Medicine, Baltimore, MD, USA; Dr L. Morgan,
Sanofi Pasteur, Marcy l’Étoile, France; Dr J. Robertson, National Institute of
Biological Standards and Control, Potters Bar, England; Dr J. Shin, World
Health Organization, Geneva, Switzerland; and Dr V. Quivy, GlaxoSmithKline
Biologicals, Wavre, Belgium.
The third draft was prepared by Dr J. Shin, World Health Organization,
Geneva, Switzerland.
The fourth draft was prepared by Dr A. Barrett, University of Texas
Medical Branch, Galveston, TX, USA; Dr D. Bleijs, National Institute for Public
Health and the Environment, Bilthoven, the Netherlands; Dr P. Minor, National
Institute of Biological Standards and Control, Potters Bar, England; Dr M. Powell,
Medicines and Healthcare Products Regulatory Agency, London, England;
Dr J. Shin, World Health Organization, Geneva, Switzerland; and Dr D. Trent,
University of Texas Medical Branch, Galveston, TX, USA, taking into account
suggestions for modification and comments by the participants in the informal
consultation held on 11–12 April 2011 in Geneva, Switzerland, attended by: Dr B.
Barrere, Sanofi Pasteur, Marcy l'Étoile, France; Dr A. Barrett, University of Texas
Medical Branch, Galveston, TX, USA; Dr D. Bleijs, National Institute for Public
Health and the Environment, Bilthoven, the Netherlands; Dr A. Chawla, Greater

Noida, Uttar Pradesh, India; Dr K. Dobbelaere, GlaxoSmithKline Biologicals,
Rixensart, Belgium; Dr M. Dornbusch, Office of the Gene Technology Regulator,
Department of Health and Aging, Canberra, Australia; Dr A. Durbin, Johns
Hopkins Bloomberg School of Public Health, Baltimore, MD, USA; Dr K. Eckels,
Walter Reed Army Institute of Research, Silver Spring, MD, USA; Dr R. Edelman,
University of Maryland School of Medicine, Baltimore, MD, USA; Mr M. Galves,
National Agency of Health Surveillance, Brasília-DF, Brazil; Dr E. Griffiths,
Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Canada;
Dr J. Hombach, World Health Organization, Geneva, Switzerland; Dr I. Knezevic,
World Health Organization, Geneva, Switzerland; Dr L. Mallet, Sanofi Pasteur,
Toronto, Canada; Dr L. Markoff, Center for Biologics Evaluation and Research,
112
Annex 2
Food and Drug Administration, Bethesda, MD, USA; Dr P. Minor, National

Institute of Biological Standards and Control, Potters Bar, England (Chair);
Dr L. Morgan, Sanofi Pasteur, Marcy l'Étoile, France; Dr J. Korimbocus, Agence
Française de Sécurité sanitaire de Produits de Santé (French Agency for Safety
of Health Products), Lyons, France; Dr M. Powell, Medicines and Healthcare
Products Regulatory Agency, London, England; Dr A. Precioso, Butantan, Sao
Paulo, Brazil; Dr V. Quivy, GlaxoSmithKline Biologicals, Wavre, Belgium; Dr J.
Roehrig, Centers for Disease Control and Prevention, Fort Collins, CO, USA; Dr J.
Schmitz, World Health Organization, Geneva, Switzerland; Mrs P. Thanaphollert,
Food and Drug Administration, Ministry of Public Health, Nonthaburi, Thailand;
Dr D. Trent, University of Texas Medical Branch, Galveston, TX, USA; Dr J-W. van
der Laan, National Institute for Public Health and the Environment, Bilthoven,
the Netherlands; and Dr S. Viviani, Sanofi Pasteur, Marcy l'Étoile, France. This
fourth draft was posted on the WHO web site with a call for public comments
for one month from 22 May to 23 June 2011.
The fifth draft was prepared by the same members of the drafting
group that prepared the fourth, and was submitted to the Expert Committee
on Biological Standardization for consideration. This draft was posted on the
WHO web site with a call for public comments for two months from 21 July to
23 September 2011.
The document was further modified and then adopted by the WHO
Expert Committee on Biological Standardization in October 2011.
Acknowledgements
Acknowledgements are also due to the following experts for their written
comments on scientific and technical issues during the public consultations
following web publication of amended drafts from 22 May to 23 June 2011 and from
21 July to 23 September 2011: Dr M. Alali, Therapeutic Goods Administration,
Australian Capital Territory, Australia; Dr L. Bigger, International Federation
of Pharmaceutical Manufacturers and Associations, Geneva, Switzerland;
Dr R. Edelman, University of Maryland School of Medicine, Baltimore, MD,
USA; Dr J. Hombach, World Health Organization, Geneva, Switzerland; Dr J.
Korimbocus, Agence Française de Sécurité sanitaire de Produits de Santé (French
Agency for Safety of Health Products), Lyons, France; Dr R. Krause, International
Federation of Pharmaceutical Manufacturers and Associations, Geneva,
Switzerland; Dr L. Markoff, Center for Biologics Evaluation and Research,
Food and Drug Administration, Bethesda, MD, USA; Dr S. Morgeaux, Agence
Française de Sécurité sanitaire de Produits de Santé (French Agency for Safety of
Health Products), Lyons, France; Dr F. Mortiaux, GlaxoSmithKline Biologicals,
Rixensart, Belgium; Dr S. Phumiamorn, Ministry of Public Health, Nonthaburi,
113
Thailand; Dr J. Schmitz, World Health Organization, Geneva, Switzerland; Dr L.

Slamet, National Agency of Drug and Food Control, Jakarta Pusat, Indonesia;
Dr T.F. Tsai, Novartis Vaccines, Cambridge, MA, USA; Dr S. Whitehead, National
Institute of Allergy and Infectious Diseases, Bethesda, MD, USA; and Dr K. Zoon,
National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA.
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29. Whitehead SS et al. Prospects for a dengue virus vaccine. Nature Reviews Microbiology, 2007,
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38. WHO guidelines on tissue infectivity distribution in transmissible spongiform encephalopathies.
Geneva, World Health Organization, 2006 (http://www.who.int/bloodproducts/
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39. Requirements for the collection, processing and quality control of blood, blood components and
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40. Monath TP et al. Safety testing for neurovirulence of novel live, attenuated flavivirus vaccines:
infant mice provide an accurate surrogate for the test in monkeys. Biologicals, 2005, 33:131–144.
41. Guirakhoo F et al. Recombinant chimeric yellow fever-dengue type 2 virus is immunogenic and
protective in nonhuman primates. Journal of Virology, 2000, 74:5477–5485.
42. Guidelines for stability evaluation of vaccines. In: WHO Expert Committee on Biological
Standardization. Fifty-seventh report. Geneva, World Health Organization, 2011 (WHO Technical
43. Good manufacturing practices: main principles for pharmaceutical products. In: WHO Expert
Committee on Specifications for Pharmaceutical Preparations. Forty-fifth report. Geneva, World
44. Model guidance for the storage and transport of time- and temperature-sensitive pharmaceutical
products (jointly with the Expert Committee on Biological Standardization). In: WHO Expert
45. WHO guidelines on nonclinical evaluation of vaccines. In: WHO Expert Committee on Biological
Standardization. Fifty-fourth report. Geneva, World Health Organization, 2005 (WHO Technical
46. Durbin AP et al. rDEN4delta30, a live attenuated dengue virus type 4 vaccine candidate, is safe,
immunogenic, and highly infectious in healthy adult volunteers. Journal of Infectious Diseases,
2005, 191:710–718.
47. Edelman R et al. Phase I trial of 16 formulations of a tetravalent live-attenuated dengue vaccine.
48. Huang CY et al. Chimeric dengue type 2 (vaccine strain PDK-53)/dengue type 1 virus as a
potential candidate dengue type 1 virus vaccine. Journal of Virology, 2000, 74:3020–3028.
49. Lum LC et al. Dengue encephalitis: a true entity? American Journal of Tropical Medicine and
Hygiene, 1996, 54:256–259.
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50. Levenbook IS et al. The monkey safety test for neurovirulence of yellow fever vaccines: the
utility of quantitative clinical evaluation and histological examination. Journal of Biological
Standardization, 1987, 15:305–313.
51. Recommendations to assure the quality, safety and efficacy of live attenuated yellow fever
vaccines. In: WHO Expert Committee on Biological Standardization. Sixty-first report. Geneva,
World Health Organization, 2013 (WHO Technical Report Series, No. 978), Annex 5.
52. Maximova OA et al. High-throughput automated image analysis of neuroinflammation and
neurodegeneration enables quantitative assessment of virus neurovirulence. Vaccine, 2010,
28:8315–8326.
53. Miller BR et al. Dengue-2 vaccine: oral infection, transmission, and lack of evidence for reversion
in the mosquito, Aedes aegypti. American Journal of Tropical Medicine and Hygiene, 1982,
31:1232–1237.
54. Johnson BW et al. Analysis of the replication kinetics of the ChimeriVax-DEN 1, 2, 3, 4 tetravalent
virus mixture in Aedes aegypti by real-time reverse transcriptase-polymerase chain reaction.
55. Guidelines on clinical evaluation of vaccines: regulatory expectations. In: WHO Expert Committee
on Biological Standardization. Fifty-second report. Geneva, World Health Organization, 2004
(WHO Technical Report Series, No. 924), Annex 1.
56. Guidelines for the clinical evaluation of dengue vaccines in endemic areas. Geneva, World Health
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pdf, accessed 3 April 2012).
57. Guidelines for plaque-reduction neutralization testing of human antibodies to dengue viruses.
Geneva, World Health Organization, 2007 (http://whqlibdoc.who.int/hq/2007/WHO_IVB_07.07_
eng.pdf, accessed 3 April 2012).
58. Thomas SJ et al. Scientific consultation on cell mediated immunity (CMI) in dengue and dengue
vaccine development. Vaccine, 2009, 27:355–368.
59. The New Substances Notification Regulations of the Canadian Environmental Protection Act, 1999.
Gatineau, QC, Environment Canada, 1999.
60. Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the
deliberate release into the environment of genetically modified organisms and repealing Council
Directive 90/220/EEC. Official Journal of the European Communities, 2002, L106:1–39.
61. Commission Decision 2002/623/EC of 24 July 2002 establishing guidance notes supplementing
Annex II to Directive 2001/18/EC. Official Journal of the European Communities, 2002, L106:22–33.
62. Risk analysis framework. (Version 3, April 2009). Canberra, ACT, Commonwealth of Australia, 2009
(http://www.health.gov.au/internet/ogtr/publishing.nsf/Content/riskassessments-1, accessed 3
April 2012).
63. dos Santos CN et al. Complete nucleotide sequence of yellow fever virus vaccine strains 17DD
and 17D-213. Virus Research, 1995, 35:35–41.
64. Hombach J et al. Arguments for live flavivirus vaccines. Lancet, 2004, 364:498–499.
65. McGee CE et al. Recombinant chimeric virus with wild-type dengue 4 virus premembrane and
envelope and virulent yellow fever virus Asibi backbone sequences is dramatically attenuated in
nonhuman primates. Journal of Infectious Diseases, 2008, 197:693–697.
66. McGee CE et al. Substitution of wild-type yellow fever Asibi sequences for 17D vaccine sequences
in ChimeriVax-dengue 4 does not enhance infection of Aedes aegypti mosquitoes. Journal of
Infectious Diseases, 2008, 197:686–692.
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67. Monath TP et al. Recombination and flavivirus vaccines: a commentary. Vaccine, 2005, 23:2956–
2958.
68. Seligman SJ, Gould EA. Safety concerns with regard to live attenuated flavivirus vaccines.
Journal of Infectious Diseases, 2008, 198:794–795.
69. Taucher C et al. A trans-complementing recombination trap demonstrates a low propensity of
flaviviruses for intermolecular recombination. Journal of Virology, 2010, 84:599v611.
70. Pugachev KV et al. Construction and biological characterization of artificial recombinants
between a wild type flavivirus (Kunjin) and a live chimeric flavivirus vaccine (ChimeriVax-JE).
Vaccine, 2007, 25:6661–6671.
71. Morrison D et al. A novel tetravalent dengue vaccine is well tolerated and immunogenic against
all 4 serotypes in flavivirus-naive adults. Journal of Infectious Diseases, 2010, 201:370–377.
72. Blaney JE Jr et al. Genetically modified, live attenuated dengue virus type 3 vaccine candidates.
73. Whitehead SS et al. A live, attenuated dengue virus type 1 vaccine candidate with a 30-nucleotide
deletion in the 3ʹ untranslated region is highly attenuated and immunogenic in monkeys. Journal
of Virology, 2003, 77:1653–1657.
74. Durbin AP et al. Heterotypic dengue infection with live attenuated monotypic dengue virus
vaccines: implications for vaccination of populations in areas where dengue is endemic. Journal
of Infectious Diseases, 2011, 203:327–334.
75. Guy B et al. Development of Sanofi Pasteur tetravalent dengue vaccine. Human Vaccines, 2010, 6.
76. Guidelines for national authorities on quality assurance for biological products. In: WHO Expert
Committee on Biological Standardization. Forty-second report. Geneva, World Health Organization,
77. Guidelines for independent lot release of vaccines by regulatory authorities. In: WHO Expert
118
Annex 2
Appendix 1
Summary protocol for manufacturing and control of
dengue tetravalent vaccine (live, attenuated)
The following protocol is intended for guidance, and indicates the information
that should be provided as a minimum by the manufacturer to the NRA.
Information and tests may be added or omitted as necessary to be in line with
the marketing authorization approved by the NRA. It is therefore possible that
a protocol for a specific product may differ in detail from the model provided.
The essential point is that all relevant details demonstrating compliance with the
licence and with the relevant WHO guidelines on a particular product should
be given in the protocol submitted. The section concerning the final product
should be accompanied by a sample of the label and a copy of the leaflet that
accompanies the vaccine container. If the protocol is being submitted in support
of a request to permit importation, it should also be accompanied by a lot release
certificate from the NRA of the country in which the vaccine was produced and/
or released stating that the product meets national requirements as well as Part A
of the Guidelines of this document published by WHO.
1. Summary information on finished product (final vaccine lot)

International name:
Commercial name:
Product licence (marketing authorization) number:
Country:
Name and address of manufacturer:
Name and address of product licence
holder if different:
Virus strains:
Origin and short history:
Batch number(s):
Finished product (final lot):
Final bulk:
Type of container:
Number of filled containers in this final lot:
Number of doses per container:
Composition (antigen concentration)/
volume of single human dose:
Target group:
119
Expiry date:
Storage conditions:
2. Summary information on manufacture

Batch number of each monovalent bulk:
Site of manufacture of each monovalent bulk:
Date of manufacture of each monovalent bulk:
Batch number of final bulk:
Site of manufacture of final bulk:
Date of manufacture of final bulk:
Date of manufacture (filling or lyophilizing) of finished
product (final vaccine lot):
Date on which last determination of virus
concentration was started:
Shelf-life approved (months):
Storage conditions:
Volume of single dose:
Prescribed virus concentration per human dose:
Serotype 1:
Serotype 2:
Serotype 3:
Serotype 4:
A genealogy of the lot numbers of all vaccine components used in the formulation
of the final product will be informative.
The following sections are intended to report the results of the tests
performed during production of the vaccine.
3. Control of source materials

3.1 Cell cultures

3.1.1 General information on cell banking system
Information and results of characterization tests on the cell banking system
from cell seed (if applicable), master cell bank, working cell bank, end-of-
production cells or extended cell bank should be provided according to WHO’s
Recommendations for the evaluation of animal cell cultures as substrates for the
manufacture of biological medicinal products and for the characterization of
cell banks.
Name and identification of cell substrate:

Origin and short history (attach a flowchart
if necessary):
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Annex 2
Lot number and date of preparation for each bank:

Date each bank was established:
Date of approval by the NRA:
Total number of ampoules stored for each bank:
Passage/population doubling level of each bank:
Maximum passage/population doubling level
approved for each bank:
Storage conditions:
Date of approval of protocols indicating compliance with
the requirements of the relevant monographs and with
the marketing authorization:
3.1.2 Characterization tests on cell seed (if applicable), master cell bank,
working cell bank, end-of-production cells, or extended cell banks
A summary table for characterization tests on each bank should be provided.
Characterization tests performed on each bank

Methods:
Specifications:
Date tested:
Results:
3.1.3 Cell culture medium

Serum used in cell culture medium
Animal origin of serum:
Batch number:
Vendor:
Country of origin:
Certificate of TSE-free:
Tests performed on serum
Methods:
Specifications:
Date of test:
Results:
Trypsin used for preparation of cell cultures

Animal origin of trypsin:
Batch number:
Vendor:
Country of origin:
Certificate of TSE-free:
121
Tests performed on trypsin

Methods:
Specifications:
Date of test:
Results:
Antibiotics
Nature and concentration of antibiotics or selecting
agent(s) used in production cell culture maintenance
medium:
Other source material

Identification and source of starting materials used in
preparing production cells, including excipients and
preservatives (particularly any materials of human or animal
origin, e.g. albumin, serum):
3.2 Virus seeds

Vaccine virus strain(s) and serotype(s):
Substrate used for preparing seed lots:
Origin and short history:
Authority that approved virus strain(s):
Date approved:
3.2.1 Information on seed lot preparation

Virus master seed
Source of virus master seed lot:
Virus master seed lot number:
Passage level:
Date of inoculation:
Date of harvest:
Number of containers:
Conditions of storage:
Date of establishment:
Maximum passage level approved for virus master seed:
Date approved by the NRA:
Virus working seed

Virus working seed lot number:
122
Annex 2
Passage level from virus master seed lot:

Date of harvest:
Number of containers:
Conditions of storage:
Date of establishment:
3.2.2 Tests on virus seeds

Identity test
Method:
Specification:
Lot number of reference reagents:
Dates of test (start, end):
Result:
Genetic/phenotypic characterizations
Method:
Reference reagents:
Specification:
Result:
Tests for bacteria and fungi

Method:
Specification:
Media:
Number of containers tested:
Volume of inoculum per container:
Volume of medium per container:
Temperatures of incubation:
Result:
Test for mycoplasmas

Method:
Specification:
Media:
Volume tested:
Temperature of incubation:
Positive controls:
123

Result:
Test for mycobacteria

Method:
Specification:
Media:
Volume tested:
Result:
Adventitious agents
Volume of virus seed samples for neutralization
and testing:
Batch number(s) of antisera/antiserum used for
neutralization of virus seeds:
Test in tissue cultures for adventitious agents

Test in monkey cells
Type of monkey cells:
Quantity of neutralized sample inoculated:
Incubation conditions:
Method:
Specification:
Ratio of cultures viable at end of test:
Result:
Test in human cells

Type of human cells:
Method:
Specification:
Result:
Other cell types

Type of cells:
124
Annex 2

Method:
Specification:
Result:
Test in animals for adventitious agents

Method:
Specification:
Result:
Test by molecular methods for adventitious agents

Method:
Specification:
Result:
Tests in nonhuman primates (either master or working seed lot) for neurovirulence
For details, please see Recommendations for yellow fever vaccine (51)
Method:
Specification:
Result:
Tests in suckling mice (either master or working seed lot, where necessary)
for neurovirulence
(Detailed protocol should be developed)
Method:
Specification:
Result:
Virus titration for infectivity

Method:
Specification:
Result:
125
4. Control of vaccine production

4.1 Control of production cell cultures
4.1.1 Information on preparation
Lot number of master cell bank:
Lot number of working cell bank:
Date of thawing ampoule of working cell bank:
Passage number of production cells:
Date of preparation of control cell cultures:
Result of microscopic examination:
4.1.2 Tests on control cell cultures

Amount or ratio of control cultures to
production cell cultures:
Period of observation of cultures:
Dates started/ended:
Ratio of cultures discarded and reason:
Results of observation:
Date supernatant fluid collected:
Test for haemadsorbing viruses

Quantity of cells tested:
Method:
Specification:
Result:
Test for adventitious agents on supernatant culture fluids


Quantity of pooled sample inoculated:
Method:
Specification:
Result:
Test in human cells

126
Annex 2
Method:
Specification:
Result:
Other cell types

Type of cells:
Method:
Specification:
Result:
Identity test
Method:
Specification:
Result:
4.1.3 Cells used for vaccine production

Observation of cells used for production
Specification:
Date:
Result:
4.2 Monovalent virus harvest pools

4.2.1 Information on manufacture
Information on each monovalent virus harvest pool should be provided separately.
Batch number(s):
Date of harvesting:
Lot number of virus master seed lot:
Lot number of virus working seed lot:
Passage level from virus working seed lot:
Methods, date of purification if relevant:
Volume(s), storage temperature, storage time
and approved storage period:
127
4.2.2 Tests on monovalent virus harvest pools

Identity
Method:
Specification:
Lot number of reference reagents:
Specification:
Date of test:
Result:
Test for bacteria and fungi

Method:
Specification:
Media:
Result:
Test for mycoplasma

Method:
Specification:
Media:
Volume tested:
Positive controls:
Result:
Test for mycobacteria

Method:
Specification:
Media:
Volume tested:
Result:
Test for adventitious agents

128
Annex 2

Method:
Specification:
Result:
Test in human cells

Method:
Specification:
Result:
Other cell types

Type of cells:
Method:
Specification:
Result:
Virus titration for infectivity

Method:
Specification:
Result:
Test for host cell proteins

Method:
Specification:
Date of test:
Result:
Test for residual cellular DNA

Method:
129
Specification:
Date of test:
Result:
Consistency of virus characteristics

Method:
Specification:
Result:
4.3. Final tetravalent vaccine bulk

4.3.1 Information on manufacture
Batch number(s):
Date of formulation:
Total volume of final bulk formulated:
Monovalent virus pools used for formulation:
Serotype/lot number/volume added/
virus concentration:
Name and concentration of added substances
(e.g. diluent, stabilizer if relevant):
Volume(s), storage temperature, storage time
and approved storage period:
4.3.2 Tests on final tetravalent bulk lot

Residual animal serum protein
Method:
Specification:
Result:

Method:
Specification:
Media:
Result:
130
Annex 2
5. Filling and containers

Lot number:
Date of filling:
Type of container:
Volume of final bulk filled:
Filling volume per container:
Number of containers filled (gross):
Date of lyophilization:
Number of containers rejected during inspection:
Number of containers sampled:
Total number of containers (net):
Maximum period of storage approved:
Storage temperature and period:
6. Control tests on final vaccine lot

6.1 Tests on vaccine lot
Inspection of final containers
Appearance:
Specification:
Date of test:
Results:
Before reconstitution:
After reconstitution:
Diluent used:
Lot number of diluent used:
Test for pH
Method:
Specification:
Date of test:
Result:
Identity test (each serotype)

Method:
Specification:
Date of test:
Result:

Method:
Specification:
131
Media:
Volume tested:
Result:
Test for potency (each serotype)

Method:
Batch number of reference vaccine and
assigned potency:
Specification:
Result for each serotype:
Thermal stability (each serotype)

Method:
Specification:
Result for each serotype:
General safety (unless deletion authorized)

Tests in mice
Number of animals tested:
Volume and route of injection:
Observation period:
Specification:
Results (give details of deaths):
Tests in guinea-pigs
Number of animals tested:
Observation period:
Specification:
Residual moisture
Method:
Specification:
Date:
Result:
132
Annex 2
Residual antibiotics if applicable

Method:
Specification:
Date:
Result:
6.2 Diluent
Name and composition of diluent:
Lot number:
Date of filling:
Type of diluent container:
7. Certification by the manufacturer

Name of head of production (typed)
Certification by the person from the control laboratory of the manufacturing

company taking overall responsibility for the production and control of the vaccine.
I certify that lot no. of dengue vaccine, whose number

appears on the label of the final containers, meets all national requirements and
satisfies Part A1 of the WHO Guidelines on the quality, safety and efficacy of
dengue tetravalent vaccines (live, attenuated) (2013) 2 (if applicable)
Name (typed)
Signature
Date
8. Certification by the NRA

If the vaccine is to be exported, attach a certificate from the NRA (as shown in
Appendix 2), a label from a final container, and an instruction leaflet for users.
1
With the exception of provisions on distribution and shipping, which the NRA may not be in a position
to assess.
2
WHO Technical Report Series, No. 979, Annex 2.
133
Appendix 2
Model certificate for the release of dengue tetravalent
vaccine (live, attenuated) by NRAs
This certificate is to be provided by the NRA of the country where the vaccine
has been manufactured, on request by the manufacturer.
Lot release certificate

Certificate no.
The following lot(s) of dengue vaccine produced by 1
in , whose numbers appear on the labels of the final

2
containers, complies with the relevant specification in the marketing authorization

and provisions for the release of biological products3 and Part A4 of the WHO
Guidelines on the quality, safety and efficacy of dengue tetravalent vaccines (live,
attenuated) (2013)5 and comply with WHO good manufacturing practices: main
principles for pharmaceutical products; 6 Good manufacturing practices for
biological products; 7 and Guidelines for independent lot release of vaccines by
regulatory authorities.8
The release decision is based on 9
The certificate may include the following information:

■ name and address of manufacturer;
■ site(s) of manufacturing;
■ trade name and common name of product;
1
Name of manufacturer.
2
Country of origin.
3
If any national requirements are not met, specify which one(s) and indicate why release of the lot(s) has
nevertheless been authorized by the NRA.
4
With the exception of provisions on distribution and shipping, which the NRA may not be in a position to
assess.
5
6
7
8
9
Evaluation of summary protocol, independent laboratory testing, and/or specific procedures laid down in
defined document etc., as appropriate.
134
Annex 2
■ marketing authorization number;

■ lot number(s) (including sub-lot numbers, packaging lot numbers
if necessary);
■ type of container;
■ number of doses per container;
■ number of containers/lot size;
■ date of start of period of validity (e.g. manufacturing date) and/or
expiry date;
■ storage condition;
■ signature and function of the authorized person and authorized
agent to issue the certificate;
■ date of issue of certificate;
■ certificate number.
The Director of the NRA (or other authority as appropriate):
Name (typed)
Signature
Date
135
Annex 3
Recommendations to assure the quality, safety and
efficacy of BCG vaccines
Replacement of Annex 2 of WHO Technical Report Series, No. 745, and
Amendment to Annex 12 of WHO Technical Report Series, No. 771
Introduction 139
Special considerations 140
Scope of the Recommendations 141
BCG vaccine strains 141
Potency-related tests 142
Part A. Manufacturing recommendations 143
A.1 Definitions 143
A.2 General manufacturing recommendations 145
A.3 Control of source materials 147
A.4 Control of vaccine production 149
A.6 Control tests on final lot 152
A.7 Records 156
A.8 Retained samples 156
A.9 Labelling 156
A.10 Distribution and transport 157
Part B. Nonclinical evaluation of BCG vaccines 159
Part C. Clinical evaluation of BCG vaccines 160
C.1 General considerations 160
C.2 Special considerations 162
C.3 Post-marketing surveillance 164
Part D. Guidelines for NRAs 164
D.1 General 164
D.2 Release and certification 166
Authors and acknowledgements 166
References 170
137
Appendix 1
History and genealogy of BCG substrains 174
Appendix 2
Summary protocol for manufacturing and control of BCG vaccine 175
Appendix 3
Model certificate for the release of BCG vaccine by NRAs 184
Recommendations published by the WHO are intended to be

scientific and advisory in nature. Each of the following sections
constitutes guidance for national regulatory authorities (NRAs)
and for manufacturers of biological products. If an NRA so desires,
these Recommendations may be adopted as definitive national
requirements, or modifications may be justified and made by the NRA.
It is recommended that modifications to these Recommendations
be made only on condition that such modifications ensure that the
vaccine is at least as safe and efficacious as that prepared in accordance
with the recommendations set out below. The parts of each section
printed in small type are comments for additional guidance, intended
for the benefit of manufacturers and NRAs.
138
Annex 3
Introduction
The last revision of the Requirements for dried bacille Calmette–Guérin (BCG)
vaccine for human use was in 1985, and an amendment which updated the
section on the expiry date was published in 1988 (1, 2). Recent WHO consultation
meetings (3–6) have addressed issues concerning the improvement of vaccine
characterization and quality control assays of BCG vaccine to reflect current state-
of-the-art technology. In addition, a recommendation to replace the International
Reference Preparation for BCG vaccine by substrain-specific Reference Reagents
evaluated by collaborative studies has been proposed. This document provides:
recommendations for the production and control of BCG vaccines (Part A);
guidelines for nonclinical evaluation (Part B); guidelines for the content of the
clinical development programme applicable to BCG vaccines (Part C); and
recommendations for NRAs (Part D). The guidelines for nonclinical evaluation
apply to classic BCG vaccine products that are still in need of such evaluation,
including newly manufactured products requiring clinical trial studies or those
produced following changes in the manufacturing process. The clinical part of this
document aims to provide a basis for assessment of efficacy and safety of BCG
vaccines in pre-licensing clinical trials as well as in post-marketing surveillance,
monitoring consistency of production and clinical testing of new classic BCG
vaccine products. If important changes have been introduced to an authorized
production process, the need for preclinical and clinical testing should be
considered on a case-by-case basis in consultation with the NRA(s) concerned.
Tuberculosis (TB) was declared a global emergency by WHO in 1993, and
Mycobacterium tuberculosis (M. tuberculosis) is now considered to be responsible
for more adult deaths than any other pathogen. Vaccination with BCG still
remains the standard for TB prevention in most countries because of its efficacy
in preventing life-threatening forms of TB in infants and young children. It is
inexpensive and usually requires only one administration in either newborns or
adolescents (7, 8). As there is currently no suitable alternative, BCG will remain
in use for the foreseeable future and may continue to be used as a prime vaccine in
a prime-boost immunization schedule in conjunction with new TB vaccines (4).
BCG vaccine contains a live, attenuated strain of M. bovis that was
originally isolated from cattle with tuberculosis and cultured for a period of 13
years and a total of 231 passages (7). The BCG vaccine was first used to immunize
humans in 1921. Following its introduction into the WHO Expanded Programme
on Immunization (EPI) in 1974, the vaccine soon reached global coverage rates
exceeding 80% in countries endemic for TB (9).
139
Over the years, different BCG vaccine seed strains have evolved from the
original vaccine strain for production. A number of BCG vaccine strains that
are used worldwide differ in terms of their genetic and phenotypic properties,
and their reactogenicity and immunogenicity profile when given to infants
and children. With this background of a diversity of substrains, manufacturing
processes, immunization schedules and levels of exposure to environmental
mycobacteria and virulent M. tuberculosis infection, different levels of protective
efficacy of BCG vaccines in adult populations have been reported (10). However,
the data are insufficient to make recommendations on whether one strain should
be preferred over the other (11). The United Nations agencies are the largest
supplier of BCG vaccines, distributing more than 120 million doses each year to
more than 100 countries. Worldwide, the most commonly used vaccine strains
are currently Danish 1331, Tokyo 172-1 and Russian BCG-I because they are
supplied by the United Nations Children’s Fund (UNICEF) which purchases the
vaccines through a published prequalification process which determines their
eligibility for use in national immunization programmes (12).
There has been particular concern over the safety of BCG vaccination
in subjects infected with the human immunodeficiency virus (HIV) (8). WHO
previously recommended that in countries with a high burden of TB, a single
administration of BCG vaccine should be given to all healthy infants as soon as
possible after birth, unless the child presented a symptomatic HIV infection (9).
However, recent evidence shows that children who were HIV-infected when
vaccinated with BCG at birth, and who later developed acquired immunodeficiency
syndrome (AIDS), were at increased risk of developing disseminated BCG
disease. Among these children, the benefits of potential prevention of severe TB
are outweighed by the risks associated with the use of BCG vaccine; thus the use
of BCG vaccines at birth in relation to HIV-infected infants should follow the
recommendations of the Global Advisory Committee on Vaccine Safety (GACVS)
(13, 14).
Special considerations
The formulation of international requirements for freeze-dried BCG vaccine is
complicated by the following: (a) a number of different substrains derived from
the original strain of BCG are used in vaccine manufacture; (b) a number of
different manufacturing and testing procedures are employed; (c) it is difficult to
identify a link between significant differences in vitro and in vivo between different
BCG vaccine strains and any possible differences in protective efficacy against
TB in humans; (d) vaccines are produced with different total bacterial content
and numbers of culturable particles; and (e) vaccines intended for administration
by different routes are prepared. Therefore, the following considerations should
be borne in mind regarding the scope of these recommendations, BCG vaccine
strains, and potency-related tests.
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Scope of the Recommendations

These revised Recommendations refer to freeze-dried BCG vaccines prepared
from substrains derived from original BCG for use in the prevention of TB. Where
BCG vaccine is issued in liquid form, the application of these Recommendations
is entirely the responsibility of the NRA. In that case, only the relevant parts of
this document apply since the limited stability of liquid BCG limits the possibility
of completing the full recommended control test schedule. Although many of
the principles expressed in this document (e.g. manufacturing, quality control)
are expected to apply also to new recombinant BCG and other live, attenuated
mycobacterial vaccines modified by molecular biology techniques, these novel
vaccines are outside the scope of these Recommendations. The same pertains
to the use of BCG for immunotherapy (e.g. treatment of bladder cancer).
However, applicability of issues on nonclinical and clinical evaluations should
be considered on a case-by-case basis. These Recommendations have been
formulated primarily to cover vaccines intended for intradermal and percutaneous
administration. Although WHO recommends intradermal administration of the
vaccine, preferably in the deltoid region of the arm using syringe and needle,
other administration methods such as percutaneous application by the multiple
puncture technique are practised in some countries (9, 15–17).
BCG vaccine strains

The original BCG vaccine strain was formerly distributed by the Pasteur Institute
of Paris and subcultured in different countries using different culture conditions
that were not standardized. Over the years, more than 14 substrains of BCG
have evolved and have been used as BCG vaccine strains in different parts of
the world (see Appendix 1). Recently, the various substrains have been studied
by comparative genomics (18, 19). BCG vaccine strains were thus divided into
the “early” strains, in which the original characteristics of “authentic Pasteur”
were conserved with fewer deletions, insertions and mutations in the genome of
the bacilli than the “late” strains. “Early” strains are represented by BCG Russia
BCG-I, BCG Moreau-RJ, BCG Tokyo 172-1, BCG Sweden, and BCG Birkhaug;
and the “late” strains include BCG Pasteur 1173P2, BCG Danish 1331, BCG
Glaxo (Copenhagen 1077) and BCG Prague. The genomic sequences of BCG
Pasteur 1173P2 as a “late” strain, and BCG Tokyo 172-1 and BCG Moreau as
“early” strains were determined (18–20). There is insufficient direct evidence to
suggest that various BCG substrains differ significantly in their efficacy to protect
against TB in humans. However, evidence from animal and human studies
indicates differences in the immune responses induced by different BCG vaccine
strains (12, 21). Although the “early” strains may confer better protection against
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TB in some animal studies (18, 22), commonly administered BCG vaccine

strains including both evolutionary “early” and “late” strains induce comparable
protective immunity against TB (23).
Only master seed lots that have been shown to be acceptable by
laboratory and clinical tests on batches derived from them should be used for
the production of working seed lots and/or final product. A suitable seed lot of
BCG should yield vaccines that give protection in experimental animals, produce
a relatively high level of immunological responses to M. tuberculosis antigens
including tuberculin sensitivity in humans, and have an acceptably low frequency
of adverse reactions (see section A.3.1).
Some manufacturers of freeze-dried BCG vaccine have modified their
master seed lot strain to make it more suitable for their particular production
procedure. The seed lots prepared in this way may not retain the same
immunogenic properties, and should be used only with the approval of the NRA.
In practice, a product prepared from BCG seed lots may generally be
investigated in humans only for the properties of producing tuberculin sensitivity
and vaccination lesions. The former should be measured by the distribution of
tuberculin reactions according to size in persons vaccinated with a given dose
of BCG vaccine. A low dose of tuberculin should be employed (e.g. equivalent
to 5 IU of the First WHO International Standard for purified protein derivative
(PPD) of M. tuberculosis, or 2 tuberculin units (TU) of a batch of PPD RT23 with
Tween 80).
Currently three substrain-specific Reference Reagents for BCG vaccines
are available: BCG Danish 1331, Tokyo 172-1 and Russian BCG-I.
Potency-related tests
There is some evidence that BCG seed lots that have been shown to produce
vaccines with protective potency in laboratory animals and tuberculin sensitivity
in humans will give effective protection against TB in humans. It should be noted

that tuberculin sensitivity is a marker for cell-mediated immune responses to
mycobacteria and not a direct indicator of protective immunity. A number of
alternative laboratory tests have been developed primarily for research purposes
but, to date, none have been proved reliable indicators of protective immune
conversion following administration of different vaccines.
Studies in animals should include protection tests, tests of vaccination
lesions, and tests for tuberculin conversion. Immunizing efficacy should be
measured in terms of degree of protection afforded to the test animals against a
challenge with virulent M. tuberculosis. Sensitizing efficacy should be measured
by the average dose of vaccine that will convert a negative tuberculin reaction
in guinea-pigs to a positive one, as well as by the reaction time during which
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the conversion takes place. In these animal tests, the inclusion for comparative
purposes of an in-house reference BCG vaccine prepared from a seed lot known
to be effective in animals and humans is recommended.
Currently there is no biomarker which directly correlates to clinical
efficacy of BCG vaccine. These Recommendations are intended to be used for
ensuring the manufacture of consistent lots. This means that new lots should
not significantly differ from those that have already been shown to be safe and
effective in humans.
At present, for batch control purposes, much reliance is placed on tests
for the estimation of the total bacterial content and for the number of culturable
particles. It is not possible to specify single requirements for the total bacterial
content and for the number of culturable particles for all vaccines (24), since
different substrains and methods of manufacture may yield different specifications
for these parameters. For example, although the number of culturable bacteria in
a single human dose may differ for different vaccines, these vaccines may show
satisfactory properties as regards their ability to induce adequate sensitivity to
tuberculin and their safety in humans. It is therefore essential that clinical studies
for dose optimization in humans be carried out to estimate suitable total bacterial
contents and the number of culturable particles for a particular manufacturer’s
product. For a particular vaccine, the difference between the lower and upper
specification for the number of culturable particles should not be larger than
fourfold. In addition, it is necessary to perform animal experiments that give an
indication of the safety and efficacy of the vaccines to the satisfaction of the NRA.
Part A. Manufacturing recommendations

A.1 Definitions
The international name should be “freeze-dried BCG vaccine”. The proper name
should be the equivalent of the international name in the language of the country
of origin. The use of the international name should be limited to vaccines that
satisfy the recommendations formulated below.

Freeze-dried BCG vaccine is a freeze-dried preparation containing live bacteria
derived from a culture of the bacillus of Calmette and Guérin, known as BCG,
intended for intradermal injection. The name of the freeze-dried vaccine
intended for percutaneous vaccination, should be “freeze-dried BCG vaccine,
percutaneous”. The preparation should satisfy all the recommendations
formulated below.
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A.1.3 International Reference Preparations and Reference Reagents

The First WHO International Reference Preparation for BCG vaccine was
established in 1965 and the First WHO International Standard for PPD of
M. tuberculosis was established in 1951. Because of the age of these preparations,
the need for replacements has been recognized, especially for the First WHO
International Reference Preparation for BCG vaccine which is a live bacterial
preparation. WHO has initiated the development of replacements. These were
presented to the WHO Expert Committee on Biological Standardization in 2009
and 2010 as candidates for the First WHO International Reference Reagents
for BCG vaccines of substrain Danish 1331, Tokyo 172-1 and Russian BCG-I
(25, 26). These materials are available through the WHO web site (http://www.
who.int/entity/bloodproducts/catalogue/BlooFeb2013.pdf). These International
Reference Reagents cover the major proportion of BCG vaccine strains currently
used in production. The establishment of substrain Moreau-RJ as the WHO
International Reference Reagent for BCG vaccine is currently in progress and
is scheduled for submission to the Committee in 2012 for adoption. These
preparations are intended as International Reference Reagents, if required, for:
■ periodical consistency monitoring of quantitative assays such as
viability estimates (such as culturable particle count and modified
ATP assays);
■ residual virulence/local reactogenicity assays and protection assays
in animal models for nonclinical evaluation.
They are also intended:
■ as reference BCG substrains;
■ for identity tests using multiplex polymerase chain reaction (PCR)
as included in the collaborative study or in other molecular biology
techniques.
The National Institute for Biological Standards and Control (NIBSC),

England, distributes the WHO International Reference Reagents for BCG vaccines.
A.1.4 Terminology
The definitions given below apply to the terms as used in these Recommendations.
They may have different meanings in other contexts.
Final bulk: the homogeneous finished liquid vaccine present in a single
container from which the final containers are filled, either directly or through
one or more intermediate containers derived from the initial single container.
Final lot: a number of sealed, final containers that are equivalent with
respect to the risk of contamination during filling and, when it is performed,
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freeze-drying. A final lot should therefore have been filled from a single container
and freeze-dried in one continuous working session.
In-house reference: a batch of vaccine prepared from the same BCG
strain as the tested vaccine and used in parallel to the vaccine tested in:
■ quantitative assays such as viability estimates (such as culturable
particle count and modified ATP assays);
■ residual virulence assays.
Master seed lot: a bacterial suspension of a single substrain originated
from the bacillus of Calmette and Guérin that has been processed as a single lot
and is of uniform composition. A seed lot should be maintained in the freeze-
dried form stored at –20 °C or below (in the liquid form it is stored at –80 °C
or below) in order to maintain viability. In each manufacturing establishment, a
master seed lot is that from which material is drawn for inoculating media for the
preparation of working seed lots or single harvests.
Single harvest: the material obtained from one batch of cultures that
have been inoculated with the working seed lot (or with the inoculum derived
from it), harvested and processed together.
Working seed lot: a quantity of bacterial organisms of a single substrain
derived from the master seed lot by growing the organisms and maintaining
them in aliquots in the freeze-dried form stored at –20 °C or below (in the liquid
form stored at –80 °C or below). The working seed lot should be prepared from
the master seed lot by as few cultural passages as possible (e.g. 3–6 passages from
the master seed lot), having the same characteristics as the master seed lot and
intended for inoculating media for the preparation of single harvests.
A.2 General manufacturing recommendations

The general manufacturing recommendations for manufacturing establishments
contained in WHO’s Good manufacturing practices: main principles for
pharmaceutical products (27) and Good manufacturing practices for biological
products (28) should apply to establishments manufacturing BCG vaccine. In
addition, the compliance with current good manufacturing practices should
apply with the addition of the following.
Details of standard operating procedures for the preparation and testing of
BCG vaccines adopted by the manufacturer, together with evidence of appropriate
validation of each production step, should be submitted for the approval of the
NRA. As required, proposals for the modification of manufacturing and control
methods should also be submitted for approval to the NRA before they are
implemented.
The NRA should satisfy itself that adequate control of the manufacturing,
shipping and storage of the BCG vaccine has been achieved. NRAs may consider
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that a formal clinical lot-to-lot consistency study is not necessary if there are
adequate and satisfactory data provided to support consistency of manufacture.
However, several different lots of the product should be used in randomized
studies and should elicit comparable immune responses in similar populations.
The degree of consistency in producing satisfactory final lots is an
important factor in judging the efficacy and safety of a particular
manufacturer’s product.
The data that should be considered in determining the consistency of production

should include the results obtained with consecutive vaccine lots when tested as
described in Part A, section 6 (e.g. the test for viability in Part A, section 6.7, and
the thermal stability test in Part A, section 6.8).
More than two consecutive vaccine lots should have been satisfactorily
prepared before any vaccine from a given manufacturer, or resulting from a
new method of manufacture, is released. In subsequent routine production, if a
specified proportion of vaccine lots or a specified number of consecutive vaccine
lots fails to meet the requirements, the manufacture of BCG vaccine should be
discontinued and should not be resumed until a thorough investigation has been
made and the cause or causes of the failures determined to the satisfaction of
the NRA.
Conventionally, production of BCG vaccine should take place in a
dedicated area, completely separate from areas used for production of other
medicines or vaccines, and using dedicated separate equipment. Such areas
should be so situated and ventilated that the hazard of contamination is reduced
to a minimum. No animals should be permitted in the vaccine production areas.
Tests for the control of vaccine that require cultures to be made of contaminating
microorganisms should be carried out in a completely separate area. Tests in
which animals are used should also be carried out in a completely separate area.
For the purposes of these requirements, the processes of vaccine production
that should take place in dedicated facilities are all operations up to and including
the sealing of the vaccine in the final containers.
In some countries, the production of BCG vaccine – although isolated –
is carried out in a building in which other work takes place. This should
be done only after consultation with, and with the approval of, the NRA.
If production takes place in part of a building, the work carried out in
other parts of the building should be of such a nature that there is no
possibility of cross-contamination with the BCG vaccine.
No cultures of microorganisms other than the BCG vaccine strain approved

by the NRA for vaccine production should be introduced into the manufacturing
areas. In particular, no strains of other mycobacterial species, whether pathogenic
or not, should be permitted in the BCG vaccine production area.
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BCG is susceptible to sunlight. Therefore, the procedures for the

preparation of the vaccine should be so designed that all cultures and vaccines are
protected from direct sunlight and ultraviolet light at all stages of manufacture,
testing and storage, until the vaccine is issued.
BCG vaccine should be produced by a staff consisting of healthy persons
who do not work with other infectious agents; in particular, they should not work
with virulent strains of M. tuberculosis, nor should they be exposed to a known
risk of tuberculosis infection. Precautions should also be taken to ensure that
no worker should be employed in the preparation of BCG vaccine unless he or
she has been shown by medical examination to be free from TB. The scope and
nature of the medical examination should be at the discretion of the NRA. It may
include a radiological examination and/or a validated immunological blood assay
that should be repeated at intervals or when there is reason to suspect illness. The
frequency of radiological examination should be at the discretion of the NRA,
taking into consideration the incidence of TB in the country.
It is advisable to keep radiation exposure to a minimum, but the
examination should be of sufficient frequency to detect the appearance of
early active TB. It is estimated that, if workers in BCG vaccine laboratories
were given one or two conventional X-ray examinations of the chest each
year, not using fluoroscopic methods, and if the best available techniques
were employed to minimize the radiation dose, the doses received
would be considerably lower than the maximum permissible doses for
workers occupationally exposed to radiation that have been set by the
International Commission on Radiological Protection (29, 30).
Should an examination reveal signs of TB or suspected TB in a worker, he or

she should no longer be allowed to work in the production areas and the rest of
the staff should be examined for possible TB infection. In addition, all cultures
should be discarded and the production areas decontaminated. If it is confirmed
that the worker has TB, all vaccine made while he or she was in the production
areas should be discarded, and all distributed batches should be recalled.
Persons not normally employed in the production areas should be
excluded from them unless, after a medical examination, including radiological
examination, they are shown to be free from TB. In particular, persons working
with mycobacteria other than the BCG seed strain should be excluded at all times.
A.3 Control of source materials

A.3.1 Seed lot system
The production of vaccine should be based on the seed lot system. A seed lot
prepared from a strain approved by the NRA (see Part D, section 1.1) should be
prepared under conditions satisfying the requirements of Part A, sections 2, 3
and 4.
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The BCG vaccine strain used should be identified by historical records

that include information on its origin and subsequent manipulation. It would be
preferable for the master seed lot to have protection proven clinically through
clinical studies on a batch derived from it by a production process that is
representative of the commercial process. It is also recommended to use a batch
derived from such a clinically “validated” seed lot as an in-house reference in the
laboratory to help ensure consistency in production.
If a working seed lot is being used, the total number of passages for a
single production harvest should not exceed 12, including the passages necessary
for preparing the working seed lot.
A.3.2 Tests on seed lot

A.3.2.1 Antimicrobial sensitivity test
An antimicrobial sensitivity test should be carried out as part of the ongoing
characterization of BCG vaccine strains. It would be appropriate to test this
property at the level of master or working seed lot.
A.3.2.2 Delayed hypersensitivity test

When a new working seed lot is established, a suitable test for delayed
hypersensitivity in guinea-pigs is carried out; the vaccine is shown to be not
significantly different in activity from the in-house reference.
A.3.2.3 Identity test

The bacteria in the master and working seed lots are identified as M. bovis BCG
using microbiological techniques (e.g. morphological appearance of the bacilli
in stained smears and the characteristic appearance of the colonies grown on
solid media). Manufacturers are encouraged to carry out the test using molecular
biology techniques (e.g. PCR test) to identify the specific substrain of BCG. The
techniques will also provide relevant information to ensure genetic consistency

in production, from master seed through working seed and to final product (4).
A.3.2.4 Test for bacterial and fungal contamination

Each master and working seed lot should be tested for bacterial and fungal
contamination by appropriate tests, as specified in Part A, section 5.2 of General
requirements for the sterility of biological substances (31), or by the validated
methods approved by the NRA.
A.3.2.5 Test for absence of virulent mycobacteria

The test for absence of virulent mycobacteria, described in Part A, section 4.2.3,
should be made in at least 10 healthy guinea-pigs injected with a quantity of
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vaccine not less than 50 single human doses and should be observed for at least
six weeks. If none of the animals shows signs of progressive TB and at least 90%
survive the observation period (i.e. should one of the 10 animals die), the seed lot
should be considered to be free from virulent mycobacteria.
If more than 10% of the guinea-pigs die during the observation period
(i.e. should two out of 10 animals die) and freedom from progressive TB disease
is verified, the test should be repeated on at least 10 more guinea-pigs. On the
second occasion, the seed lot passes the test if not more than 10% of the animals
die during the observation period (i.e. should one of the 10 animals die) and the
autopsy does not reveal any sign of TB.
A.3.2.6 Test for excessive dermal reactivity

The test for excessive dermal reactivity, described in Part A, section 6.4.2,
should be made in six healthy guinea-pigs, each weighing not less than 250 g
and having received no treatment likely to interfere with the test. Each guinea-
pig should be injected intradermally, according to a randomized plan, with
0.1 ml of the reconstituted vaccine and of vaccine dilutions 1:10 and 1:100. The
same dilutions of the appropriate international Reference Reagent or in-house
reference should be injected into the same guinea-pigs at randomly selected sites.
The guinea-pigs should be observed for at least four weeks. The vaccine complies
with the test if the reactions it produces at the sites of injection are not markedly
different from those produced by the appropriate international Reference Reagent
or in-house reference.
A.3.3 Production culture medium

The production culture medium should contain no substances known to cause
toxic or allergic reactions in humans. The use of material originating from
animals should be discouraged. However, if constituents derived from animals
are necessary, approval of the NRA should be sought and the materials should
comply with current policy on TSEs (32–37). A risk assessment for TSE would
need to be included for the materials of the culture medium. WHO’s revised
Guidelines on transmissible spongiform encephalopathies in relation to biological
and pharmaceutical products (32) provide guidance on risk assessments for
master and working seeds and should be consulted. Substances used in that
medium should meet such specifications as the NRA may prescribe.
A.4 Control of vaccine production

A.4.1 Control of single harvests
All cultures should be examined visually, and any that have grown in an
uncharacteristic manner should not be used for vaccine production.
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A.4.2 Control of final bulk

A.4.2.1 Final bulk
The final bulk should be prepared from a single harvest or by pooling a number
of single harvests.
A.4.2.2 Test for bacterial and fungal contamination

The final bulk should be tested for bacterial and fungal contamination by
appropriate tests as specified in Part A, section 5.2 of the General requirements for
the sterility of biological substances (31), or by the validated methods approved
by the NRA. No vaccine lot should be passed for use unless the final bulk has
been shown to be free from such contamination.

The test for absence of virulent mycobacteria should be carried out on each final
bulk or final lot.
At least six healthy guinea-pigs, all of the same sex, each weighing
250–400 g should be used. They should not have received any treatment or diet,
such as antibiotics, that is likely to interfere with the test. A sample of the final
bulk intended for this test should be stored at 4 °C for not more than 72 hours
after harvest.
A dose of BCG organisms corresponding to at least 50 single human
doses of vaccine intended for intradermal injection should be injected into each
guinea-pig by the subcutaneous or intramuscular route.1 The guinea-pigs should
be observed for at least six weeks. If, during that time, they remain healthy, gain
weight, show no signs of progressive TB and not more than one dies, the final
bulk should be considered to be free from virulent mycobacteria.
At the end of the observation period, the animals should be killed and
examined postmortem for macroscopic evidence of progressive TB disease.

Similarly, any animals that die before the end of the observation period should
be subjected to a postmortem examination.
Should one third of the guinea-pigs die (i.e. should two out of six
animals die) during the observation period (and freedom from progressive TB
disease is verified), the test should be repeated on at least six more guinea-pigs.
On the second occasion, the vaccine lot passes the test if not more than one
1
When a more concentrated vaccine, intended for administration by the percutaneous route, is tested, a
dilution factor approved by the NRA should be applied so that the mass of BCG injected corresponds to at
least 50 human doses of intradermal vaccine.
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animal dies during the observation period and the autopsy does not reveal any
sign of TB.
Should a vaccine lot fail to satisfy the requirements of this test because
animals die from causes other than TB, the procedure to be followed by
the manufacturer should be determined with the approval of the NRA.
If signs of TB disease are seen, the vaccine lot should be rejected, all subsequent
vaccine lots should be withheld, and all current vaccine stocks should be held
pending further investigation. The manufacture of BCG vaccine should be
discontinued and it should not be resumed until a thorough investigation has
been made and the cause or causes of the failure determined and appropriate
actions have been taken. Production should be allowed to resume only upon the
approval of the NRA.
A.4.2.4 Test for bacterial concentration

The bacterial concentration of the final bulk should be estimated by a validated
method approved by the NRA and should have a value within a range approved
by the NRA (see Part D, section 1.2).
Based on manufacturers’ experience, the opacity method is the method
of choice. The International Reference Preparation of Opacity,2 or an
equivalent Reference Preparation approved by the NRA, may be employed
in comparative tests.
Clumping issues should be considered during validation of the assay.
A.4.2.5 Test for number of culturable particles

The number of culturable particles on a solid medium of each final bulk should
be determined by an appropriate method approved by the NRA. Alternatively,
a bioluminescense or other biochemical method can be used (38, 39), provided
that the method is properly validated against the culturable particle test for the
production step in question. If properly validated, such tests can be used as
equivalent methods. Regular calibration with the reference method as agreed
with the NRA would be relevant.
The medium used in this test should be such that the number of
culturable particles may be determined at an optimal time point (usually
3–5 weeks) after the medium has been inoculated with dilutions of the
vaccine.
2
The International Reference Preparation of Opacity is in the custody of the NIBSC, Potters Bar, England,
which supplies samples on request.
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There are various methods of determining the number of culturable

particles in BCG vaccine, and it is essential that only one culture
method be used for all the vaccine lots produced by a manufacturer (5).
It is also desirable for assay validation that the clumping issue should
be considered and that tests should be carried out in parallel with the
appropriate international Reference Reagent or in-house reference, e.g.
the same vaccine production that has been used in clinical trials and has
assured safety (including immunogenicity) and efficacy.
A.4.2.6 Substances added to the final bulk

Substances used in preparing the final bulk should meet such specifications as the
NRA may prescribe. In particular, the NRA should approve the source(s) of any
animal-derived raw materials which should comply with the WHO Guidelines
on tissue infectivity distribution in transmissible spongiform encephalopathies (34).
Substances added to improve the efficiency of the freeze-drying process
or to aid the stability of the freeze-dried product should be sterile and of high and
consistent quality, and should be used at suitable concentrations in the vaccine.

The general requirements concerning filling and containers given in Good
manufacturing practices for biological products (28) should apply to vaccine
filled in the final form.
The containers should be in a form that renders the process of
reconstitution as simple as possible. Their packaging should be such that
the reconstituted vaccine is protected from direct sunlight.
A.6 Control tests on final lot

Tests on the final lot should be performed after reconstitution, except for
appearance and residual moisture tests. The diluent supplied or recommended for
reconstitution should be used, unless such diluent would interfere with any of the
tests, in which case some other suitable fluid should be used. The vaccine should
be reconstituted to the concentration at which it is to be used for injection into
humans; however, an exception may be made in the case of the test for absence
of virulent mycobacteria (Part A, section 6.4.1), when a higher concentration
of reconstituted vaccine may be necessary. It would be appropriate to monitor
periodically the antimicrobial sensitivity in final lots.
A.6.1 Inspection of final containers

Every container in each final lot should be inspected visually, and those showing
abnormalities should be discarded.
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The appearance of the freeze-dried vaccine and the reconstituted vaccine

should be described with respect to form and colour. If reconstitution with the
product diluent does not allow for the detection of particulates, an alternative
diluent may be used.
A.6.2 Identity test

An identity test should be performed on samples of the vaccine from each final
lot. The identity test for final lots should be used to identify the product as BCG
as approved by the NRA. The identity of each final lot of vaccine should be
verified by the morphological appearance of the bacilli in stained smears and by
the characteristic appearance of the colonies grown on solid media. A validated
nucleic acid amplification technique (such as PCR) should preferably be used.
A.6.3 Test for bacterial and fungal contamination

Samples from each final lot should be tested for bacterial and fungal contamination
by appropriate tests as specified in Part A, section 5.2 of the General requirements
for the sterility of biological substances (31), or by the validated methods approved
by the NRA.
A.6.4 Safety tests

Provided the test for virulent mycobacteria has been carried out with satisfactory
results on the final bulk vaccine, it may be omitted on the final lot.
If the test for the absence of virulent mycobacteria, applied to the final
bulk, is unsatisfactory (and freedom from progressive TB disease is verified), it
should be repeated with a sample of a final lot (see Part A, section 4.2.3).
A.6.4.2 Test for excessive dermal reactivity

Provided the test has been carried out with satisfactory results on the working
seed lot and on at least three consecutive final lots produced from it, the test may
be omitted on the final lot.
Historically the omission of the test with satisfactory results on five
consecutive final lots has been accepted by the authorities (40).
A.6.5 Test for bacterial concentration

The total bacterial content of the reconstituted vaccine should be estimated for
each vaccine lot by a validated method approved by the NRA, and should have a
value within a range approved by the NRA (see Part D, section 1.2).
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The estimation of tota1 bacterial content may be made either directly,

by determining the dry weight of organisms, or indirectly by an opacity
method that has been calibrated in relation to the dry weight of the
organisms.
The clumping issue should be considered during validation of the assay.
A.6.6 Test for residual moisture

The average moisture content of a freeze-dried vaccine should be determined by
a validated method accepted by the NRA. Values should be within limits of the
preparations shown to be adequately stable in the stability studies of the vaccine.
A.6.7 Tests for viability

A.6.7.1 Test for number of culturable particles
The number of culturable particles of each final lot should be determined by an
appropriate method approved by the NRA (see Part A, section 4.2.5). The viable
count should have a value within a range approved by the NRA that should not
be wider than a fourfold difference between the lower and upper levels of the
specification for numbers of culturable particles (see Part D, section 1.2). By
comparison with the results of the test for number of culturable particles carried
out on final bulk, as described in Part A, section 4.2.5, the percentage survival
on freeze-drying may be calculated and this value should be not less than one
approved by the NRA. The appropriate international Reference Reagent or
in-house reference should be used for every test in order to validate the assay.
The purpose of including the appropriate international Reference Reagent
or in-house reference is to have a check on the quality and consistency
of the culture medium and the accuracy of the technique used for the
determination of the number of culturable particles. It is not intended
to adjust the count of the vaccine by comparison with the Reference

Preparation.
The clumping issue should be considered during validation of the assay.
The survival rate after freeze-drying is usually not less than 20%.
A.6.7.2 Rapid test for viability

As an alternative to the colony counting method, a bioluminescense or other
biochemical method can be used provided that the method is properly validated
against the culturable particle test for the production step in question. If properly
validated, such tests may be considered by the NRA to replace the culturable
particle test.
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The bioluminescence reaction occurring in fireflies depends on the

presence of adenosine triphosphate (ATP), luciferin luciferase, oxygen
and magnesium ions. This reaction can be reproduced in vitro by mixing
these components. If all components except ATP are present in excess,
the amount of light emitted is proportional to the amount of ATP coming
from the vaccine.
Since ATP is present in all living cells and is immediately destroyed when
the cell dies, ATP is a reliable marker for living cells.
Studies have shown that, if properly validated, measurement of ATP

using the bioluminescence reaction can be used to estimate the viable
count of freeze-dried BCG vaccine within 1–2 days as accurately as
other, more time-consuming methods, once the mean content of ATP
per culturable particle has been estimated for a given vaccine production.
A.6.8 Thermal stability test

The thermal stability test is part of the characterization and consistency
demonstration of vaccine production. The requirement for this test should be
at the discretion of the NRA and, if required, each final lot should be tested for
thermal stability by a validated method approved by the NRA. If the production
consistency is demonstrated, this test may be omitted on the final lot subject to
NRA approval (6).
If performed, the test should involve the determination of the number
of culturable particles before and after the samples have been held at appropriate
temperatures and for appropriate periods.
For example, the thermal stability test may be carried out by taking
samples of the vaccine and incubating them at 37 °C for 28 days.
The percentage decrease in the number of culturable particles is then compared

with that of samples of the same vaccine lot stored at 2–8 °C. The number of
culturable particles in the vaccine after heating should be not less than 20% of
that stored at 2–8 °C (41). The absolute value should be approved by the NRA.
The viability test should also be performed with the appropriate international
Reference Reagent or in-house reference for checking validity of the assay. One
method of determining the number of culturable particles should be adhered to,
as suggested in Part A, section 4.2.5.
The purpose of including the appropriate international Reference Reagent
or in-house reference is to check the quality and consistency of the
medium used for determination of the number of culturable particles. It
is not intended to adjust the count of the vaccine by comparison with
the Reference Preparation.
155
All manufacturers should keep their product for the approved storage period
and should determine the number of culturable particles from time to time to
demonstrate that the number is being maintained at an adequate level.
In some countries, the thermal stability test is carried out only after the
vaccine has been stored for 3–4 weeks after freeze-drying, since it is
considered that the degree of stability during the first three weeks may
not be related to the long-term stability of the product.
As a guide to stability, some manufacturers of freeze-dried BCG vaccine

determine the residual moisture content of the final vaccine, since failure
to achieve a certain degree of desiccation results in an unstable product.
However, such a test cannot be regarded as an alternative to tests involving
the determination of the number of culturable particles.
A.7 Records
The recommendations in section 8 of Good manufacturing practices for biological
products (28) should apply.
Written records should be kept of all seed lots, all cultures intended for
vaccine production, all single harvests, all final bulk vaccines, and all vaccine in
the final containers produced by the manufacturing establishments, including
all tests irrespective of their results.
The records should be of a type approved by the NRA. An example of a
suitable protocol is given in Appendix 2.
A.8 Retained samples

The recommendations in section 9.5 of Good manufacturing practices for
biological products (28) should apply.
It is desirable that samples should be retained for at least one year after the
expiry date of the final lot.
A.9 Labelling
The recommendations in section 7 of Good manufacturing practices for biological
products (28) should apply, including the following guidance.
The label, and/or the packaging insert in some countries, printed on or
affixed to each container should show the volume and nature of the diluent. Also,
this label, or the label on the carton holding several final containers, or the leaflet
accompanying the containers, should carry the following additional information:
■ the fact that the vaccine fulfils the requirements of this document;
■ instructions for use of the vaccine and information concerning
contraindications and the reactions that may follow vaccination;
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■ the volume and nature of the diluent to be added to reconstitute

the vaccine, specifying that only the diluent supplied by the
manufacturer should be used;
■ the conditions recommended during storage and transport, with
information on the reduced stability of the vaccine if exposed to
temperatures higher than that stated on the label;
■ warnings that the vaccine should be protected from direct sunlight;
■ a statement that the reconstituted vaccine should be used as soon as
possible, or should be stored at 2–8 °C, protected from direct sunlight
and used within six hours (42);
■ information on antimicrobial sensitivity.
The label for the diluent should state “Reconstituting fluid for BCG
vaccine [proprietary name]”.
A.10 Distribution and transport

The recommendations given in section 8 of Good manufacturing practices for
biological products (28) should be followed, along with the guidance provided
in Safe vaccine handling, cold chain and immunizations (43). Further guidance
is provided in Model guidance for the storage and transport of time- and
temperature-sensitive pharmaceutical products (44).
It is desirable that samples should be retained for at least one year after
the expiry date of the final lot.

A.11.1 Stability testing
Adequate stability studies form an essential part of vaccine development. The
recommendations provided in WHO’s Guidelines for stability evaluation of
vaccines should be followed (46). Stability testing should be performed at different
stages of production if stored for a given time period, namely as appropriate for
single harvests or pool of single harvests, final bulk or final lot. Stability-indicating
parameters should be defined or selected appropriately according to the stage of
production. It is advisable to assign a storage period to all in-process materials
during vaccine production, particularly intermediates such as single harvests and
final bulk, and a shelf-life period to the final lots.
BCG vaccines require special precautions to ensure sufficient stability.
In this connection the most important measures are lyophilization, the use of an
effective stabilizer, and proper sealing of vaccine containers.
157
Historically the use of ampoules sealed under vacuum was the most
common practice for increasing stability. However, vacuum-sealing is
difficult compared to sealing in the presence of inert gas. There were
no significant differences between BCG vaccines sealed under vacuum
and under nitrogen or carbon dioxide at either 4 °C or 37 °C (41).
Manufacturers now prepare BCG vaccines in vials/ampoules and, under
well-validated conditions, the product is adequately stable.

The Guideline for establishing or improving primary and intermediate vaccine
stores (47) should apply.
Storage conditions should be based on stability studies and approved by
the NRA. Before being distributed by the manufacturing establishment, or before
being issued from a depot for the storage of vaccine, all vaccines in their final
containers should be stored constantly at 2–8 °C (41, 48) and vaccine diluents
should be stored as recommended by the manufacturer. Freeze-dried BCG
vaccines, regardless of their substrain, are sensitive to ultraviolet and fluorescent
light. They should be protected from direct sunlight (41).
BCG vaccines are sensitive to light as well as to heat. Normally, these
vaccines are supplied in vials/ampoules made from dark brown glass,
which gives them some protection against light damage, but care should
still be taken to keep them covered and protected from strong light at all
times (48).
Freeze-dried BCG vaccines may be kept frozen at –15 °C to –25 °C if cold

chain space permits, but this is neither essential nor recommended (41).
Precautions should also be taken to maintain the vaccine during transport

and up to the time of use at the temperature and under the storage
conditions recommended by the manufacturer.
A.11.3 Expiry date

The expiry date should be approved by the NRA and should be based on the
stability of the final product, as well as on the results of the stability tests referred
to in section A.11.1 above. It is established for each batch by adding the shelf-life
period to the date of manufacture. Most freeze-dried BCG vaccines are stable at
temperatures of 2–8 °C for at least two years (41) from the date of manufacture.
The storage of final product at –20 °C to extend the shelf-life should be validated.
A.11.4 Expiry of reconstituted vaccine

Stability studies should be undertaken on reconstituted vaccine. Freeze-
dried BCG vaccines become much more heat-sensitive after they have been
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reconstituted with diluent (41). After multidose containers of freeze-dried BCG

have been reconstituted, the vaccine should be used as soon as possible. Any
reconstituted vaccine remaining should be stored at 2–8 °C until used, and the
expiry time should be defined by stability studies (4, 42, 45).
Part B. Nonclinical evaluation of BCG vaccines

Details on the design, conduct, analysis and evaluation of nonclinical studies are
available in the WHO Guidelines on nonclinical evaluation of vaccines (49).
Nonclinical testing of a new strain (i.e. a strain derived by selection from
existing BCG strains in Appendix 1) or of a strain from a new manufacturer
of a BCG vaccine is a prerequisite for initiation of clinical studies in humans.
Nonclinical testing includes immunogenicity, protection studies (proof of
concept) and safety testing in animals. The vaccine lots used in nonclinical studies
should be adequately representative of the formulation intended for clinical
investigation and, ideally, should be the same lots, manufactured according to
current good manufacturing practice (cGMP), as those used in clinical studies.
If this is not feasible, the lots used clinically should be comparable to those used in
the nonclinical studies with respect to potency, stability and other characteristics
of quality. The technical manufacturing consistency lots may often be used for
these purposes.
New manufacturers of BCG vaccine for human use will need to refer to
the range of nonclinical safety and characterization tests that are recommended
for existing licensed BCG vaccines. Although there is currently no requirement
for additional nonclinical testing beyond that already described for licensed
BCG vaccines, the development of new variants of BCG, the potential for new
fermentation technologies and the possibility of novel live vaccines against TB
have shown that additional nonclinical studies beyond that required for licensed
BCG vaccine can be helpful in demonstrating that a new BCG product has
satisfactory nonclinical efficacy, safety and stability.
Guideline example on protective potency testing: Hartley guinea-pigs
are used for potency testing. The guinea-pigs are vaccinated with a
small amount of BCG (~10 3 colony-forming units (CFU)). Eight weeks
after the vaccination, the guinea-pigs are challenged with virulent
M. tuberculosis H37Rv (ATCC 27294) by the pulmonary route with a
low dose (10–15 CFU) per animal. Five weeks after the infection, the
guinea-pigs are killed and the spleen and the lung lobes are removed.
These organs are homogenized separately. Appropriate dilutions are
inoculated on to duplicate solid medium and incubated at 37 °C for three
weeks. The number of M. tuberculosis H37Rv colonies is counted, and is
expressed as mean log10 CFU per tissue. The CFU results are compared
between the vaccinated and non-vaccinated groups (50).
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If there are two pharmacologically relevant species for the clinical candidate (one
rodent and one non-rodent), both species should be used for short-term (up to one
month duration) toxicology studies. If the toxicological findings from these studies
are similar in both species, longer-term studies in one species are usually considered
sufficient; the rodent species should be considered unless there is a rationale for
using non-rodents. Studies in two non-rodent species are not appropriate. Other
in vivo studies should address both potency (such as tuberculin sensitivity and
immunological tests) and safety issues (such as tests for excessive dermal reactivity
and absence of virulent mycobacteria) of the classical BCG vaccines.
It may be of benefit for new BCG vaccine developers to consider the
points raised in recent meetings establishing recommendations for new live
vaccines against TB (51, 52).
Part C. Clinical evaluation of BCG vaccines

Clinical trials should adhere to the principles described in the Guidelines for
good clinical practice (GCP) for trials on pharmaceutical products (53) and
the general principles described in WHO’s Guidelines on clinical evaluation of
vaccines: regulatory expectations (54). All clinical trials should be approved by the
relevant NRAs and local ethics committees. Continued licence of BCG vaccines
should be viewed in the light of ongoing post-marketing data on the safety,
immunogenicity and effectiveness of BCG vaccines in the target population.
Part C considers the provision of clinical data required (a) when a new
candidate “classical” BCG vaccine derived from (the same master seed of) one of
the recognized strains (see Appendix 1) is developed; (b) when there have been
major changes to the manufacturing process of an established vaccine, including
preparation of a new master seed lot of an established strain; (c) when technology
transfer of existing vaccine is planned to a new manufacturer; and (d) when
revalidation of existing vaccines used in national immunization programmes is
considered.
Vaccines manufactured using a “new strain” (i.e. a strain derived by

selection from existing BCG strains in Appendix 1) should require a full clinical
development programme that provides evidence of safety, efficacy and the
reactogenicity profile in all target age-groups.
Other vaccines against M. tuberculosis derived from M. bovis or other
mycobacterial strains cannot be considered as BCG. They would require a full
clinical development programme and are not included here.
C.1 General considerations

C.1.1 Comparative or placebo‑controlled clinical trials
It would not be considered ethical to conduct a placebo-controlled trial of
protective efficacy of a BCG vaccine in a TB-endemic area, particularly in
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infants. A comparative trial with a licensed, or internationally accepted (WHO

prequalified), BCG vaccine could be accepted.
C.1.2 Value of PPD response

It is recognized that the response to tuberculin PPD is not an indicator of a
protective immune response. Nonetheless this has been used for more than
50 years to indicate a cellular immune response to an infection with M. tuberculosis
or as evidence of “successful” BCG vaccination. At best, a PPD reaction is an
indicator of exposure to antigens of TB, and the generation of a cellular immune
response. Thus, it can be used in a PPD-naive population as an indicator of an
immune response to the BCG vaccine (55). Other immunological measures may
be more closely related to M. tuberculosis infection or vaccination, but currently
none has been agreed as a correlate of protection from infection or disease.
C.1.3 BCG in HIV‑infected infants

A very important safety consideration with regard to vaccination policy is to
establish, during clinical trials, the potential for disseminated BCG disease in
immunocompromised children. In this regard, the use of BCG vaccines at birth
should follow the recommendations from the GACVS (13, 14). The GACVS
recommendations consider the policies for immunization exclusion of infants
known to be infected with HIV, infants symptomatic for HIV infection, and
those infants born to mothers known to be HIV-infected and who therefore may
be infected.
C.1.4 Post‑vaccination reactions and complications

Vaccines intended for intradermal or percutaneous injection should be given
strictly intradermally or percutaneously, and vaccinators should be trained
accordingly. Incorrect vaccination technique can result in adverse reactions,
including discharging ulcers, abscesses and keloid scars.
Current BCG vaccines have a known reactogenicity profile after
intradermal inoculation (56). Local reaction at the vaccination site is normal
after a BCG vaccination. It may take the form of a nodule that, in many cases,
will break down and suppurate. The reaction developing at the vaccination
site usually subsides within 2–5 months and in practically all children leaves a
superficial scar of 2–10 mm in diameter. The nodule may persist and ulcerate.
Swelling of regional lymph nodes may also be seen, and this may be regarded as
a normal reaction, but the size should be limited.
Keloid and lupoid reactions may occur at the site of the vaccination.
Children with such reactions should not be revaccinated. Inadvertent subcutaneous
injections produce abscess formations and may lead to ugly retracted scars.
Among the major complications, suppurative lymphadenitis has been observed.
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In the case of certain vaccines, it has been revealed that there is a strong correlation
between the incidence of these complications in newborns and the number of
culturable particles in the vaccine.
The concentration of the vaccine should be shown to be effective and
tolerated in the age groups for which the vaccine is intended.
A reduction of the dose for newborns may be based on the evidence and
approved by the NRA (57).
The NRA should issue guidelines for the treatment of complications.
C.2 Special considerations

This section is limited to the clinical development of new “classical” BCG vaccines
manufactured following these Recommendations and using strains of BCG that
are derived from (the same master seed of) one of the strains recognized in
Appendix 1.
The use of comparative studies with a licensed BCG vaccine can provide
evidence of the similarity of safety and immune responses to a new classical BCG
vaccine product.
The target population for the vaccine would be newborns or infants
according to current recommendations on the use of BCG vaccines.
The nonclinical expectations for a new classical BCG vaccine are outlined
in Part B.
For such a new classical BCG vaccine, these nonclinical studies should be
conducted in comparison to an existing licensed BCG vaccine, preferably
derived from the same BCG substrain. It would be expected that the
results of preclinical studies would be similar for the new vaccine product
and for the comparator.
The clinical development programme should ideally be designed to show the

safety and protective efficacy of the vaccine. However, for such a new classical
BCG vaccine product, comparative studies with an existing licensed BCG vaccine,
using immunological responses as a marker for efficacy, may be acceptable to the
responsible NRA.
Comparable PPD response (proportion of PPD converters, intensity of
response) may be acceptable.
Clinical studies should provide evidence of safety in all the potential target
populations, including those with a high incidence of diseases that may affect the
safety or efficacy of the new vaccine product. In phase I and phase II studies this
should include evaluation of:
■ safety and reactogenicity in healthy adults (comparative);
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■ end-points;
■ safety and reactogenicity – can include healthy HIV-infected adults;
■ immune responses – non-inferior PPD response, and may include
other immunological markers.
These studies are difficult to interpret as adults will most likely have
received BCG vaccination at birth. Dose-finding studies may be considered
unnecessary for these vaccines. The safety in HIV-infected individuals
and in infants needs to be considered.
Dose-finding and age de-escalation can be included in these studies, but review
at each step by a suitable independent safety committee should be considered.
In phase III studies evaluation should be made of:
■ safety and reactogenicity in infants (comparative)
■ end-points
■ safety and reactogenicity
■ non-inferior PPD immune response.
Post‑marketing risk management

As it may not be practically possible to evaluate protective efficacy for a new
classical BCG vaccine, the responsible NRA in the country of manufacture
should require post-marketing surveillance activities for safety and effectiveness
in a suitable environment. Sentinel surveillance sites in an endemic country may
be considered.
In the past, the responsible NRA of a country of manufacture required a
demonstration that adequate control of BCG vaccine had been achieved,
by arranging for studies of some of the final lots to be performed at
regular intervals in children.
Studies of immunological responses to M. tuberculosis antigens should be

made, such as sensitivity to tuberculin. In at least 100 tuberculin-negative
persons per year, records of tuberculin-induced reaction (distribution of
tuberculin reactions by size) by a defined dose of tuberculin3 in vaccines,
local skin lesions (nature and size of reaction at injection site), and the
occurrence of untoward vaccination reactions should be obtained. Such
tests should be performed in parallel on two or more vaccine lots in
the same population group, one of the vaccine lots being preferably a
reference vaccine.
3
An intradermal test with a dose of tuberculin equivalent to 5 IU of tuberculin PPD is suitable. A description
of an appropriate method and a design for a study to assess BCG vaccines in humans are available on
application to the World Health Organization, 1211 Geneva 27, Switzerland.
163
C.3 Post‑marketing surveillance

The NRA in the country of manufacture may require periodic updates of reports
on BCG safety and immunogenicity.
C.3.1 BCG vaccine used in a national immunization programme

As in all immunization programmes, the adverse events following immunization
with BCG vaccines should be monitored and recorded.
The following events are important after BCG vaccination (58):
■ injection site abscesses

■ BCG lymphadenitis
■ disseminated BCG diseases
■ osteitis/osteomyelitis.
Appropriate training of health-care workers is important as some medical

incidents can be related to immunization even if they have a delayed onset.
C.3.2 WHO prequalified BCG vaccines

Prequalified vaccines may be used in a wide range of countries worldwide.
Periodic safety update reports supplied to WHO should include specific analysis
of countries where the vaccine has been used.
Part D. Guidelines for NRAs

D.1 General
The general recommendations for NRAs provided in the Guidelines for national
authorities on quality assurance for biological products (59) should apply. These
specify that no new biological substance should be released until consistency of

manufacturing and quality as demonstrated by a consistent release of batches
has been established. The detailed production and control procedures, as well
as any significant change in them that may affect the quality, safety or efficacy
of BCG vaccine, should be discussed with and approved by the NRA. For
control purposes, the NRA should obtain the WHO International Reference
Reagents as comparators for potency-related testing and, where necessary,
should establish national working Reference Preparation(s) calibrated against
the international reference.
In addition, the NRA should provide a reference vaccine or approve
one used by a manufacturer, and should give directions concerning the use of
the reference vaccine in specified tests. The NRA should also give directions to
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manufacturers concerning the BCG substrain to be used in vaccine production,

the total content of bacteria, the number of culturable particles, and the stability
required of the vaccine, and should specify the requirements to be fulfilled by
the manufacturer in accordance with the provisions of Part A of this document,
including those for consistency of quality in respect of the points referred to in
Part A, section 2.
D.1.1 BCG vaccine strain

The substrain of BCG (maintained in the form of a seed lot) used in the
production of vaccine should be derived from the original strain maintained by
Calmette and Guérin and should be identified by historical records that include
information on its origin and subsequent manipulation. On the basis of cultures
and biochemical and animal tests, the BCG seed lot should show characteristics
that conform to those of BCG and generally differ from those of other
mycobacteria. The identity test should be supplemented by molecular biology
techniques to identify the specific BCG substrain used. The seed lot should show
consistency in the morphological appearance of colonies and genetic stability on
serial subculture. It should also have been shown to yield vaccines that, upon
administration by intradermal injection to children and adults, induce relevant
immunological responses to M. tuberculosis antigens, including sensitivity to
tuberculin, and with a low frequency of untoward effects. In addition, the seed lot
should have been shown to give adequate protection against TB in experimental
animals in tests for protective potency.
D.1.2 Concentration of BCG vaccine

The concentration of BCG vaccine varies with different vaccine products and is
dependent on a number of factors, such as the substrain of BCG used and the
method of manufacture. It is therefore essential for each manufacturer, as well
as for each method of manufacture, for the optimum potency of vaccine to be
ascertained by trials in tuberculin-negative subjects (newborns, older children,
and adults) to determine the response to vaccination in respect of the induction
of relevant immunological responses to M. tuberculosis antigens – including
sensitivity to tuberculin, the production of acceptable local skin lesions, and the
occurrence of a low frequency of untoward reactions. As a result of such trials, the
NRA should give directions to the manufacturer concerning the total bacterial
content and the number of culturable particles required for the vaccine.
If a manufacturer changes its procedure for preparing BCG vaccine, and
if the NRA considers that the change might affect the final product, it may
be necessary to conduct further clinical trials in order to determine the
optimum content of BCG organisms in the new product.
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D.2 Release and certification

A vaccine lot should be released only if it fulfils the national requirements
and/or Part A of these Recommendations. Before any vaccine lot is released
from a manufacturing establishment, the recommendations for consistency
of production provided in the Guidelines for national authorities on quality
assurance for biological products (59) should be met. In addition, the general
recommendations for NRAs provided in the Guidelines for independent lot
release of vaccines by regulatory authorities (60) should be followed. A protocol
based on the model given in Appendix 2, signed by the responsible official of the
manufacturing establishment, should be prepared and submitted to the NRA in
support of a request for release of a vaccine for use.
A statement signed by the appropriate official of the NRA (or authority as
appropriate) should be provided if requested by a manufacturing establishment
and should certify whether or not the lot of vaccine in question meets all national
requirements, as well as Part A of these Recommendations. The certificate should
also state the date of manufacture, the lot number, the number under which
the lot was released, and the number appearing on the labels of the containers.
In addition, the date of the last satisfactory potency test, as well as the expiry
date assigned on the basis of shelf-life, should be stated. A copy of the official
national release document should be attached. The certificate should be based on
the model given in Appendix 3. The purpose of the certificate is to facilitate the
exchange of vaccines between countries.
Authors and acknowledgements

The scientific basis for the revision of the Requirements published in WHO
Technical Report Series, Nos. 745 and 771 was developed at meetings from 2003
to 2007 attended by:
Dr L. Barker, Aeras Global Tuberculosis Vaccine Foundation, Rockville,
MD, USA; Professor M. Behr, Montreal General Hospital, Quebec, Canada; Dr T.

Bektimirov, Tarassevich State Research Institute for Standardization and Control
of Medical Biological Preparations, Moscow, Russian Federation; Dr T. Brewer,
Brigham and Women’s Hospital, Boston, MA, USA; Dr R. Brosch, Institut
Pasteur, Paris, France; Dr L.R. Castello-Branco, Fundacao Ataulpho de Paiva,
Brazilian League Against Tuberculosis, Rio de Janeiro, Brazil; Dr M. Chouchkova,
National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria; Dr S. Cole,
Institut Pasteur, Paris, France; Dr M. Corbel, National Institute for Biological
Standards and Control, Potters Bar, England; Dr J. Darbyshire, MRC Clinical
Trials Unit, London, England; Dr J. Daviaud, World Health Organization,
Geneva, Switzerland; Dr H. Dockrell, London School of Hygiene and Tropical
Medicine, London, England; Dr U. Fruth, World Health Organization, Geneva,
Switzerland; Mr I. Fujita, Japan BCG Laboratory, Tokyo, Japan; Dr M. Gheorghiu,
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Annex 3
Institut Pasteur, Paris, France; Dr E. Griffiths, Health Canada, Ottawa, Canada;

Dr C. Gutierrez Pèrez, Institut Pasteur, Paris, France; Dr K. Haslov, Statens Serum
Institut, Copenhagen, Denmark; Dr M.M. Ho, National Institute for Biological
Standards and Control, Potters Bar, England; Dr S. Jadhav, Serum Institute of
India Ltd, Pune, India; Dr S.E. Jensen, Statens Serum Institut, Copenhagen,
Denmark; Dr I. Knezevic, World Health Organization, Geneva, Switzerland;
Dr M. Lagranderie, Institut Pasteur, Paris, France; Mr F. Leguellec, Aventis Pasteur,
Val de Reuil, France; Dr Y. Lopez Vidal, Universidad Nacional Autonoma de
Mexico, Mexico City, Mexico; Dr G. Marchal, Institut Pasteur, Paris, France; Dr H.
Mayanja-Kizza, Makarere Medical School, Kampala, Uganda; Dr S. Mostowy,
McGill University Health Centre, Montreal General Hospital, Montreal, Quebec,
Canada; Dr M. Roumaintzeff, Lyons, France; Dr M. Seki, BCG Laboratory,
Tokyo, Japan; Dr V. Vincent, Institut Pasteur, Paris, France; Dr F. Weichold, Aeras
Global Tuberculosis Vaccine Foundation, Rockville, MD, USA; Dr D. Wood,
World Health Organization, Geneva, Switzerland; Dr S. Yamamoto, National
Institute of Infectious Diseases, Tokyo, Japan; Dr I. Yano, Japan BCG Laboratory,
Tokyo, Japan; Dr D. Young, Imperial College of Science, Technology & Medicine,
London, England; Dr T. Zhou, World Health Organization, Geneva, Switzerland.
The first draft was prepared by Dr H-N. Kang, World Health Organization,
Geneva, Switzerland, taking into account comments received from:
Dr M. Andre, Agence Française de Sécurité Sanitaire des Produits de Santé,
Lyons, France; Dr I.S. Budiharto, Bio Farma, Bandung, Indonesia; Dr H. Chun,
Korea Food and Drug Administration, Seoul, Republic of Korea; Dr M. Corbel,
National Institute for Biological Standards and Control, Potters Bar, England;
Dr R. Dobbelaer, Lokeren, Belgium; Dr S. Gairola, Serum Institute of India,
Hadapsar, India; Dr L. Grode, Vakzine Projekt Management GmbH, Hannover,
Germany; Dr M.M. Ho, National Institute for Biological Standards and Control,
Potters Bar, England; Dr S. Jadhav, Serum Institute of India Ltd, Pune, India;
Professor D. Levi, Tarassevich State Research Institute for Standardization and
Control of Medical Biological Preparations, Moscow, Russian Federation; Dr S.
Morris, United States Food and Drug Administration, Silver Spring, MD, USA;
Dr V. Öppling, Paul-Ehrlich-Institute, Langen, Germany; Dr M. Roumiantzeff,
Lyons, France; Dr K. Shibayama, National Institute of Infectious Diseases,
Tokyo, Japan; Dr J. Southern, Cape Town, South Africa; Mr A. Supasansatorn,
Ministry of Public Health, Nonthaburi, Thailand; Dr S. Wahyuningsih, National
Agency of Drug and Food Control, Jakarta Pusat, Indonesia; Dr K.B. Walker,
National Institute for Biological Standards and Control, Potters Bar, England;
Dr S. Yamamoto, National Institute of Infectious Diseases, Tokyo, Japan; Mrs A.
Zhao, National Institute for the Control of Pharmaceutical & Biological Products,
Beijing, China.
Following the informal consultation meeting on standardization and
evaluation of BCG vaccines in September 2009, held in Geneva, Switzerland, draft
167
Recommendations were revised by Dr H.-N. Kang, World Health Organization,

Geneva, Switzerland, taking into account information on current manufacturing
and regulatory practice provided at the meeting attended by:
Dr M. Andre, Agence Française de Sécurité Sanitaire des Produits
de Santé, Lyons, France; Dr L.F. Barker, Aeras Global Tuberculosis Vaccine
Foundation, Rockville, MD, USA; Dr A. Bhardwaj, Central Research Institute,
Kasuli, Himachal Pradesh, India; Dr M. Brennan, Aeras Global TB Vaccine
Foundation, Rockville, MD, USA; Dr I.S. Budiharto, Bio Farma, Bandung,
Indonesia; Dr L.R. Castello-Branco, Fundacao Ataulpho de Paiva, Brazilian
League Against Tuberculosis, Rio de Janeiro, Brazil; Dr M. Chouchkova, National
Center of Infectious and Parasitic Diseases, Sofia, Bulgaria; Dr H. Chun, Korea
Food and Drug Administration, Seoul, Republic of Korea; Dr M. Corbel, National
Institute for Biological Standards and Control, Potters Bar, England; Dr B. Eisele,
Vakzine Projeckt Management GmbH, Hannover, Germany; Dr S. Gairola, Serum
Institute of India, Hadapsar, India; Dr L. Grode, Vakzine Projeckt Management
GmbH, Hannover, Germany; Dr K. Haslov, Statens Serum Institut, Copenhagen,
Denmark; Dr M.M. Ho, National Institute for Biological Standards and Control,
Potters Bar, England; Dr P. Hubrechts, Statens Serum Institut, Copenhagen,
Denmark; Dr G. Hussey, South African TB Vaccine Initiative, Cape Town, South
Africa; Dr H.-N. Kang, World Health Organization, Geneva, Switzerland; Dr I.
Knezevic, World Health Organization, Geneva, Switzerland; Professor D. Levi,
Tarassevich State Research Institute for Standardization and Control of Medical
Biological Preparations, Moscow, Russian Federation; Dr Y. Lopez Vidal,
Universidad Nacional Autonoma de Mexico, Copilco-Universidad, Mexico City,
Mexico; Dr K. Markey, National Institute for Biological Standards and Control,
Potters Bar, England; Dr C. Martin, Universidad de Zaragoza, Zaragoza, Spain;
Dr S. Morris, FDA/CBER, Bethesda, MD, USA; Dr V. Öppling, Paul-Ehrlich-
Institute, Langen, Germany; Dr M. Roumiantzeff, Lyons, France; Dr. M. Seki,
Japan BCG Laboratory, Tokyo, Japan; Dr K. Shibayama, National Institute of
Infectious Diseases, Tokyo, Japan; Dr J. Southern, Advisor to Medicines Control
Council in South Africa, Cape Town, South Africa; Mr A. Supasansatorn, Ministry

of Public Health, Nonthaburi, Thailand; Dr S. Wahyuningsih, National Agency
of Drug and Food Control, Jakarta Pusat, Indonesia; Dr K.B. Walker, National
Institute for Biological Standards and Control, Potters Bar, England; Dr S.
Yamamoto, Japan BCG Laboratory, Tokyo, Japan; Dr Z. Yan, Chengdu Institute
of Biological Products, Chengdu, China; Dr L. Zhang, Chengdu Institute of
Biological Products, Chengdu, China; Mrs A. Zhao, National Institute for the
Control of Pharmaceutical & Biological Products, Beijing, China.
Several draft Recommendations were subsequently prepared by Dr M.M.
Ho, National Institute for Biological Standards and Control, Potters Bar,
England, with support from the drafting group of Dr M. Corbel, Milton Keynes,
England; Dr R. Dobbelaer, Lokeren, Belgium; Dr H.-N. Kang, World Health
168
Annex 3
Organization, Geneva, Switzerland; Dr J. Southern, Cape Town, South Africa;

and Dr K.B. Walker, National Institute for Biological Standards and Control,
Potters Bar, England.
Following the meeting of the drafting group in March 2011, held in
Potters Bar, England, draft Recommendations were updated taking into account
comments received from:
Dr M. Andre, Agence Française de Sécurité Sanitaire des Produits de
Santé, Lyons, France; Dr M. Chouchkova, National Center of Infectious and
Parasitic Diseases, Sofia, Bulgaria; Dr H. Dockrell, London School of Hygiene
and Tropical Medicine, London, England; Dr S. Gairola, Serum Institute of
India, Hadapsar, India; Dr P. Hubrechts, Statens Serum Institut, Copenhagen,
Denmark; Dr J. Joung, Korea Food and Drug Administration, Chungchengbuk-do,
Republic of Korea; Dr M. Lagranderie, Institut Pasteur, Paris, France; Professor
D. Levi, Tarassevich State Research Institute for Standardization and Control of
Medical Biological Preparations, Moscow, Russian Federation; Dr Y. Lopez Vidal,
Universidad Nacional Autonoma de Mexico, Facultad de Medicina, Mexico
City, Mexico; Dr V. Öppling, Paul-Ehrlich-Institute, Langen, Germany; Mrs G.
Trisnasari, Bio Farma, Bandung, Indonesia; Dr V. Vincent, Institut Pasteur, Paris,
France; Dr S. Wahyuningsih, National Agency of Drug and Food Control, Jakarta
Pusat, Indonesia; Dr T. Ying, China National Biotec Group, Beijing, China; Mrs A.
Zhao, National Institute for the Control of Pharmaceutical & Biological Products,
Beijing, China.
The draft Recommendations were posted on the WHO Biologicals web
site for public consultation from 24 May to 23 June 2011. The WHO/BS/2011.2157
document was then prepared by Dr M.M. Ho, National Institute for Biological
Standards and Control, Potters Bar, England; Dr H.-N. Kang, World Health
Organization, Geneva, Switzerland; Dr J. Southern, Cape Town, South Africa;
Dr K.B. Walker, National Institute for Biological Standards and Control, Potters Bar,
England, taking into account comments received from the following reviewers:
Dr C. Ahn, Korea Food and Drug Administration, Chungchengbuk-do,
Republic of Korea; Dr M. Andre, Agence Française de Sécurité Sanitaire des
Produits de Santé, Lyons, France; Dr M. Brennan, Aeras Global TB Vaccine
Foundation, Rockville, MD, USA; Dr I.S. Budiharto, Bio Farma, Bandung,
Indonesia; Ms Y. Choi, Korea Food and Drug Administration, Chungchengbuk-do,
Republic of Korea; Dr M. Chouchkova, National Center of Infectious and Parasitic
Diseases, Sofia, Bulgaria; Dr S. Gairola, Serum Institute of India, Hadapsar, India;
Dr K. Haslov, Statens Serum Institut, Copenhagen, Denmark; Dr H. Hozouri,
Pasteur Institute of Iran, Tehran, Islamic Republic of Iran; Dr P. Hubrechts,
Statens Serum Institut, Copenhagen, Denmark; Dr J. Joung, Korea Food and Drug
Administration, Chungchengbuk-do, Republic of Korea; Mr H. Kim, Korea Food
and Drug Administration, Chungchengbuk-do, Republic of Korea; Mr J.-G. Kim,
Korea Food and Drug Administration, Chungchengbuk-do, Republic of Korea;
169
Ms Y.L. Kim, Korea Food and Drug Administration, Chungchengbuk-do, Republic

of Korea; Dr K. Lee, Korea Food and Drug Administration, Chungchengbuk-do,
Republic of Korea; Dr Y. Lopez Vidal, Universidad Nacional Autonoma de
Mexico, Copilco-Universidad, Mexico City, Mexico; Dr V. Öppling, Paul-Ehrlich-
Institute, Langen, Germany; Dr M. Roumiantzeff, Lyons, France; Dr M. Seki,
Japan BCG Laboratory, Tokyo, Japan; Dr J. Shin, World Health Organization,
Geneva, Switzerland; Mr S.-C. Shin, Green Cross, Yongin, Republic of Korea;
Dr V. Vincent, Institut Pasteur, Paris, France; Dr S. Yamamoto, Japan BCG
Laboratory, Tokyo, Japan.
Further changes were made to WHO/BS/2011.2157 by the Expert
Committee on Biological Standardization, resulting in the current document.
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2. Requirements for dried BCG vaccines. In: WHO Expert Committee on Biological Standardization.
Thirty-eighth report. Geneva, World Health Organization, 1988 (WHO Technical Report Series,
No. 771), Annex 12.
3. Corbel MJ et al. Report on a WHO consultation on the characterization of BCG strains, Imperial
College, London 15–16 December 2003. Vaccine, 2004, 22:2675–2680.
4. Ho MM et al. Report on a WHO consultation on the characterization of BCG vaccines held at
WHO Headquarters, Geneva, 8–9 December 2004. Vaccine, 2005, 23:5700–5704.
5. Knezevic I, Corbel MJ. WHO discussion on the improvement of the quality control of BCG vaccines
held at Pasteur Institute, Paris, France, 7 June 2005. Vaccine, 2006, 24:3874–3877.
6. Ho MM et al. Report on a WHO informal consultation on standardization and evaluation on BCG
vaccines, 22–23 September 2009, WHO, Geneva, Switzerland. Vaccine, 2010, 28:6945–6950.
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2006 (WHO/IVB/06.01; http://whqlibdoc.who.int/hq/2006/WHO_IVB_06.01_eng.pdf, accessed
6 February 2013).
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10. Colditz GA et al. Efficacy of BCG vaccine in the prevention of tuberculosis. Meta-analysis of the
published literature. Journal of the American Medical Association, 1994, 271:698–702.
11. Global Advisory Committee on Vaccine Safety, 12–13 June 2007. Weekly Epidemiological Record,
2007, 82:252–259.
12. Ritz N et al. Influence of BCG vaccine strain on the immune response and protection against
tuberculosis. FEMS Microbiology Review, 2008, 32:821–841.
13. Revised BCG vaccination guidelines for infants at risk for HIV infection. Weekly Epidemiological
Record, 2007, 82:193–196.
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14. Use of BCG vaccine in HIV-infected infants. Weekly Epidemiological Record, 2010, 85:32–33.
15. Birkhaug K. Protective value of the intracutaneous and percutaneous methods of BCG vaccination.
Acta Medica Scandinavica, 1944, CXVII, fasc. III–IV:274–312.
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Pédiatrie, 1976, 23:219–225.
17. Mori T, Yamauchi Y, Shiozawa K. Lymph node swelling due to bacilli Calmette-Guérin vaccination
with multipuncture method. Tubercle and Lung Disease, 1996, 77:269–273.
18. Brosch R et al. Genome plasticity of BCG and impact on vaccine efficacy. Proceedings of the
National Academy of Sciences, 2007, 104:5596–5601.
19. Seki M et al. Whole genome sequence analysis of Mycobacterium bovis bacillus Calmette-Guérin
(BCG) Tokyo 172: a comparative study of BCG vaccine substrains. Vaccine, 2009, 27:1710–1716.
20. Gomes LHF et al. Genome sequence of Mycobacterium bovis BCG Moreau, the Brazilian vaccine
strain against tuberculosis. Journal of Bacteriology, 2011, 193:5600–5601.
21. Castillo-Rodal AI et al. Mycobacterium bovis BCG substrains confer different levels of protection
against Mycobacterium tuberculosis infection in a BALB/c model of progressive pulmonary
tuberculosis. Infection and Immunity, 2006, 74:1718–1724.
22. Hayashi D et al. Comparable studies of immunostimulating activities in vitro among
Mycobacterium bovis bacillus Calmette-Guerin (BCG) substrains. FEMS Immunology and Medical
Microbiology, 2009, 56:116–123.
23. Horwitz MA et al. Commonly administered BCG strains including an evolutionarily early strain
and evolutionarily late strains of disparate genealogy induce comparable protective immunity
against tuberculosis. Vaccine, 2009, 27:441–445.
24. In vitro assays of BCG products. Geneva, World Health Organization, 1977 (WHO working
document WHO/TB/Technical guide/77.9).
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27. Good manufacturing practices: main principles for pharmaceutical products. In: WHO Expert
29. International Commission on Radiological Protection. Report of Committee II on permissible dose
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32. Guidelines on transmissible spongiform encephalopathies in relation to biological and
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33. Recommendations for the evaluation of animal cell cultures as substrates for the manufacture
of biological medicinal products and for the characterization of cell banks. In: WHO Expert
34. WHO Guidelines on tissue infectivity distribution in transmissible spongiform encephalopathies.
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encephalopathies. Geneva, World Health Organization, 2010 (http://www.who.int/bloodproducts/
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Commission, 2002 (http://ec.europa.eu/food/fs/sc/ssc/out296_en.pdf, accessed 11 April 2011).
37. Note for guidance on minimizing the risk of transmitting animal spongiform encephalopathy
agents via human and veterinary medicinal products (EMEA/410/01 Rev. 2 - October 2003)
adopted by the Committee for Proprietary Medicinal Products (CPMP) and by the Committee for
Veterinary Medicinal Products (CVMP). Official Journal of the European Union, 2004 (http://www.
emea.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003700.
pdf, accessed 24 February 2013).
38. Ho MM et al. Report of an international collaborative study to establish the suitability of using
modified ATP assay for viable count of BCG vaccine. Vaccine, 2008, 26(36):4754–4757.
39. Jensen SE et al. Development and validation of an ATP method for rapid estimation of viable units
in lyophilised BCG Danish 1331 vaccine. Biologicals, 2008, 36(5):308–314.
40. BCG vaccine, freeze-dried. In: European Pharmacopoeia, 6th ed. Strasbourg, Directorate for the
Quality of Medicines of the Council of Europe (EDQM), 2008 (01/2008:0163).
41. Temperature sensitivity of vaccines. Geneva, World Health Organization, 2006 (WHO/IVB/06.10).
42. WHO policy statement: the use of opened multi-dose vials of vaccine in subsequent immunization
sessions. Geneva, World Health Organization, 2000 (WHO/V&B/00.09; http://www.who.int/
vaccines-documents/DocsPDF99/www9924.pdf, accessed 11 April 2011).
43. Safe vaccine handling, cold chain and immunizations. Geneva, World Health Organization, 1998
(WHO/EPI/LHIS/98.02).
44. Model guidance for the storage and transport of time- and temperature-sensitive pharmaceutical
products (jointly with the Expert Committee on Biological Standardization). In: WHO Expert
45. Proper handling and reconstitution of vaccines avoids programme errors. Vaccines and Biologicals
Update, 2000, 34 (http://www.who.int/vaccines-documents/DoxNews/updates/updat34e.pdf,
accessed 11 April 2011).
47. Guideline for establishing or improving primary and intermediate vaccine stores. Geneva, World
Health Organization, 2002 (WHO/V&B/02.34; http://www.who.int/vaccines-documents/DocsPDF02/
www715.pdf, accessed 11 April 2011).
48. Immunization in practice: a practical resource guide for health staffs, 2004 update. Geneva, World
Health Organization, 2004 (WHO/IVB/04.06).
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50. Yamamoto T et al. Mycobacterium bovis BCG vaccination modulates TNF-a production after
pulmonary challenge with virulent Mycobacterium tuberculosis in guinea pigs. Tuberculosis, 2007,
87:155–165.
51. Walker KB et al. The second Geneva consensus: recommendations for novel live TB vaccines.
Vaccine, 2010, 28:2259–2270.
52. Kamath AT et al. New live mycobacterial vaccines: the Geneva consensus on essential steps
towards clinical development. Vaccine, 2005, 23:3753–3761.
53. Guidelines for good clinical practice (GCP) for trials on pharmaceutical products. In: WHO Expert
Committee on the Use of Essential Drugs. Sixth report. Geneva, World Health Organization, 1995
(WHO Technical Report Series, No. 850), Annex 3.
on Biological Standardization. Fifty-second report. Geneva, World Health Organization, 2004 (WHO
55. Roth A et al. Vaccination technique, PPD reaction and BCG scarring in a cohort of children born in
Guinea-Bissau 2000–2002. Vaccine, 2005, 23:3991–3998.
56. Hoft DF et al. Clinical reactogenicity of intradermal bacille Calmette-Guérin vaccination. Clinical
Infectious Disease, 1999, 28:785–790.
57. WHO Expert Committee on Tuberculosis. Ninth report. Geneva, World Health Organization, 1974
(WHO Technical Report Series, No. 552).
58. Surveillance of adverse events following immunization. Geneva, World Health Organization, 1997
(WHO/EPI/TRAM/93.02. Rev 1).
59. Guidelines for national authorities on quality assurance for biological products. In: WHO
Expert Committee on Biological Standardization. Forty-second report. Geneva, World Health
Organization, 1992 (WHO Technical Report Series, No. 822), Annex 2.
173
Appendix 1
History and genealogy of BCG substrains
Note: This diagram provides only a historical overview of the use of different substrains derived from BCG vaccine
strain. It does not indicate any WHO “qualification” or “approval” of the strains or vaccines in the context of this
document.
* Yamamoto S, Yamamoto T. Historical review of BCG vaccine in Japan. Japanese Journal of Infectious Disease, 2007,
60:331–336.
174
Annex 3
Appendix 2
Summary protocol for manufacturing and control of BCG
vaccine
The following protocol is intended for guidance, and indicates the information that
should be provided as a minimum by the manufacturer to the NRA. Information
and tests may be added or omitted as required by the NRA, if applicable.
It is thus possible that a protocol for a specific product may differ in
detail from the model provided. The essential point is that all relevant details
demonstrating compliance with the licence and with the relevant WHO
recommendations of a particular product should be given in the protocol
submitted.
The section concerning the final product must be accompanied by
a sample of the label and a copy of the leaflet that accompanies the vaccine
container. If the protocol is being submitted in support of a request to permit
importation, it must also be accompanied by a lot release certificate from the
NRA or NCL of the country in which the vaccine was produced stating that the
product meets national requirements as well as Part A of the recommendations
of this document published by WHO.
Summary information on the finished product (final lot)

International name:
Trade name:
Product licence (marketing authorization) number:
Country:
Site of manufacture of final lot:
Name and address of licence-holder
(if different):
BCG substrain:
Authority that approved the BCG substrain:
Date approved:
Final bulk number:
Volume of final bulk:
Final product:
Type of vaccine: Intradermal/Percutaneous/Other
Final lot number:
Type of container:
175

Number of filled containers in this final lot:
Date of manufacture of final lot:
Date on which last determination of the bacterial count
was started, or date of start of period of validity:
Shelf-life approved (months):
Expiry date:
Diluent:
Storage conditions:
Volume of single human dose:
Volume of vaccine per container:
Summary of the composition (include a summary of the
qualitative and quantitative composition of the
vaccine per human dose):
Release date:
Production information
A genealogy of the lot numbers of all vaccine components used in the formulation
of the final product will be informative.
The following sections are intended for the reporting of the results of
the tests performed during the production of the vaccine, so that the complete
document will provide evidence of consistency of production. Thus, if any test has
to be repeated, this must be indicated. Any abnormal results should be recorded
on a separate sheet.
Control of source materials (section A.3)

The information requested below is to be presented on each submission. Full details
on master and working seed lots are required on first submission only and whenever
a change has been introduced.
Master seed lot

Origin of seed lot:
Master seed lot number:
Passage level:
Date of preparation of seed lot:
Date of receipt of seed lot (if applicable):
Date of reconstitution of seed lot ampoule:
176
Annex 3
Working seed lot

Working seed lot number:
Passage level:
Date of reconstitution of seed lot ampoule:
Tests on working seed lot production (section A.3.2)

Antimicrobial sensitivity test (section A.3.2.1)
Method used:
Date test started:
Date test completed:
Results:
Delayed hypersensitivity test (section A.3.2.2)

Vaccine Reference vaccine
Method used:
Dilutions injected:
Inoculation route:
Number of guinea-pigs given injection:
Observation period (specification):
Date test started:
Result:
Identity test (section A.3.2.3)

Method used:
Date test started:
Results:
Test for bacterial and fungal contamination (section A.3.2.4)

Method used:
177
Incubation Media used Inoculum Date test Date test Results

began ended
20–25 °C
30–36 °C
Negative
control
Test for absence of virulent mycobacteria (section A.3.2.5)

Method used:
No. of human doses injected per guinea-pig:
Inoculation route:
No. of guinea-pigs given injection:
Weight range of guinea-pigs:
Date test started:
Health of animals during test:
Weight gains (losses):
Result:
Test for excessive dermal reactivity (section A.3.2.6)

Method used:
Dilutions injected:
Inoculation route:

Date test started:
Mean diameter of lesions (for
each dilution):
Result:
Production of culture medium (section A.3.3)

Any components of animal origin:
Certificate for BSE/TSE-free:
178
Annex 3
Control of vaccine production (section A.4)

Control of single harvests (section A.4.1)
Derived from master seed lot number:
Working seed lot number:
Passage level from master seed:
Culture medium:
Number and volume of containers inoculated:
Date of harvest:
Results of visual inspection:
Control of final bulk (section A.4.2)

Tests for bacterial and fungal contamination (section A.4.2.2)
Method used:

began ended
20–25 °C
30–36 °C
Negative
control

(if not performed on final lot)
Method used:
Inoculation route:
Date test started:
179

Result:
Test for bacterial concentration (section A.4.2.4)

Method used:
Date test started:
Specification:
Result:
Test for number of culturable particles (section A.4.2.5)

Method used:
Date test started:
Specification:
Result:
Details of working Reference Preparation:
Substances added (section A.4.2.6)

Any components of animal origin:
Certificate for BSE/TSE-free:
Filling and containers (section A.5)

Lot number:
Date of filling:
Volume of final bulk filled:
Number of containers filled (gross):
Date of freeze-drying:
Number of containers rejected during inspection:
Number of containers sampled:
Total number of containers (net):
Control tests on final lot (section A.6)

Inspection of final containers (section A.6.1)
Appearance:
Date of test:
Specification:
180
Annex 3
Result:
Recommended reconstitution fluid:
Volume of reconstitution fluid per final container:
Identity test (section A.6.2)

Method used:
Date test started:
Specification:
Result:
Tests for bacterial and fungal contamination (section A.6.3)

Method used:
Specification:

began ended
20–25 °C
30–36 °C
Negative
control
Safety tests (section A.6.4)

(if not performed on final bulk)
Method used:
Inoculation route:
Date test started:
181
Specification:
Result:
Test for excessive dermal reactivity (section A.6.4.2) if applicable

Method used:
Dilutions injected:
Inoculation route:
Date test started:
Mean diameter of lesions (for
each dilution):
Specification:
Result:
Test for bacterial concentration (section A.6.5)

Method used:
Date test started:
Specification:
Result:
Test for residual moisture (section A.6.6)

Method:
Date:
Specification:
Result:
Tests for viability (section A.6.7)

Test for number of culturable particles (section A.6.7.1)
Method used:
Medium:
Date test started:
Before lyophilization After lyophilization
No. of containers tested:
Mean count of culturable
particles per ml:
Mean survival rate (%):
182
Annex 3
Specification:
Result:
Rapid test for viability (section A.6.7.2) if applicable

Method:
Date:
Specification:
Result:
Thermal stability test (section A.6.8)

Method used:
Date test started:
Unheated containers Heated containers
No. of containers tested:
Culturable particles in each
container per ml:
Specification:
Result:
Submission addressed to NRA

Name of responsible person (typed)
Certification by the person from the control laboratory of the manufacturing

company taking over responsibility for the production and control of the vaccine:
I certify that lot no. of BCG vaccine, whose number
appears on the label of the final container, meets all national requirements and/
or satisfies Part A1 of the WHO Recommendations to assure the quality, safety
and efficacy of BCG vaccines (2013)2
Signature:
Name (typed):
Date:
1
to assess.
2
183
Appendix 3
Model certificate for the release of BCG vaccine by NRAs
Certificate no.
The following lot(s) of BCG vaccine produced by 1
in , whose numbers appear on the labels of the final

2
containers, complies with the relevant specification in the marketing authorization

and provisions for the release of biological products 3 and Part A4 of the WHO
Recommendations to assure the quality, safety and efficacy of BCG vaccines
(2013)5 and comply with WHO good manufacturing practices: main principles
for pharmaceutical products; 6 Good manufacturing practices for biological
products; 7 and Guidelines for independent lot release of vaccines by regulatory
authorities.8

■ name and address of manufacturer;
■ site(s) of manufacturing;
■ trade name and common name of product;
■ marketing authorization number;
■ lot number(s) (including sub-lot numbers, packaging lot numbers
if necessary);
1
2
Country of origin.
3
4
to assess.
5
6
7
8
9
defined document etc., as appropriate.
184
Annex 3
■ type of container;
■ number of doses per container;
■ number of containers/lot size;
■ date of start of period of validity (e.g. manufacturing date) and/or
expiry date;
■ storage condition;
■ signature and function of the authorized person and authorized
agent to issue the certificate;
■ date of issue of certificate;
■ certificate number.
Name (typed)
Signature
Date
185
Annex 4
Recommendations to assure the quality, safety and
efficacy of acellular pertussis vaccines
Introduction 189
Background 189
Scope 191
International Standards and Reference Preparations 194
Terminology 195
Part A. Manufacturing recommendations 196
A.1 Definitions 196
A.2 General manufacturing recommendations 197
A.3 Production control 197
A.5 Control of final product 207
A.6 Records 208
A.7 Samples 209
A.8 Labelling 209
A.9 Distribution and shipping 209
Part B. Nonclinical evaluation of acellular pertussis vaccines 211
B.1 Nonclinical characterization and testing of pertussis antigens and
in-process materials 212
B.2 Nonclinical characterization and testing of final vaccine formulation 215
Part C. Clinical evaluation of acellular pertussis vaccines 216
C.1 General considerations for clinical studies 218
C.2 Assessment of immune responses 221
C.3 Safety evaluation 226
C.4 Post-marketing studies and surveillance 227
Part D. Guidelines for NRAs 227
D.1 General 227
D.2 Official release and certification by the NRA 227
Authors 228
187
References 230
Appendix 1
Modified intracerebral challenge assay 236
Appendix 2
Histamine sensitization test by temperature measurement 238
Appendix 3
Histamine sensitization test by lethal end-point assay 239
Appendix 4
Mouse immunogenicity test 241
Appendix 5
Method for respiratory challenge 245
Appendix 6
Summary protocol for production and testing of acellular pertussis vaccine 248
Appendix 7
Model certificate for the release of acellular pertussis vaccine by NRAs 259
Recommendations published by WHO are intended to be scientific and

advisory in nature. The parts of each section printed in type of normal
size have been written in such a form that, should a national regulatory
authority (NRA) desire, they may be adopted as they stand as definitive
national requirements or used as the basis of such requirements.
Those parts of each section printed in small type are comments and
additional guidance. It is recommended that modifications be made

only on condition that the modifications ensure that the vaccine is at
least as safe and efficacious as that prepared in accordance with the
recommendations set out below.
188
Annex 4
Introduction
Pertussis immunization is an integral part of immunization programmes in all
regions of the world. It is recommended for all infants and children and in some
countries it is also recommended for adults and adolescents. Whole-cell pertussis
vaccines, which have been used for more than 50 years, have been shown to
provide protection against pertussis and still serve as the foundation of global
pertussis control. However, there is an increasing interest in acellular pertussis
vaccines which have also been shown to be safe and effective and which have
been successfully introduced into many national immunization programmes. A
detailed comparison of acellular and whole-cell pertussis vaccines is beyond the
scope of this document; however, these issues are discussed in detail in a WHO
position paper on pertussis vaccines (1). As a consequence of the increasing
demand for acellular pertussis vaccines, new manufacturers are entering the
field. The expansion in the number and use of acellular pertussis vaccines, the
development of new vaccines and advances in the standardization of quality-
control methods have prompted WHO to update its current Guidelines for
acellular pertussis vaccines (2).
These Guidelines were approved in 1996, with the recognition that further
improvements in the production and evaluation of these vaccines would follow.
Since then, stakeholders have gained additional experience with these vaccines,
and limitations in the original Guidelines have been identified (3–7). Acellular
pertussis vaccines are almost exclusively administered in combinations with
diphtheria and tetanus toxoid vaccines. Moreover, in recent years there has been
increased interest in the use of more complex combination vaccines – a trend
which increases the challenges of clinical evaluation. Furthermore, the evaluation
of the clinical efficacy of any new acellular pertussis vaccine formulations has
become increasingly difficult due to the decrease in the prevalence of pertussis
cases worldwide and for additional reasons discussed below in Part C. The goal
of this revision is to address these issues concerning the Guidelines in the light of
new information.
Background
The development of acellular pertussis vaccines was stimulated by scientific
advances that led to the identification of components such as toxins and surface
proteins of Bordetella pertussis that are believed to play a role in pathogenesis
and induction of protective immunity. The first acellular pertussis vaccines were
produced through a purification process that resulted in the enrichment of two
protein components – namely pertussis toxin (PT) and filamentous haemagglutinin
(FHA) – that were protective in animal models, and were introduced for routine
use in Japan around 1980 as a response to increasing public concern over adverse
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reactions to whole-cell pertussis vaccines. These vaccines were prepared from cell-
free B. pertussis culture supernatants by ultracentrifugation, and all contained
FHA and PT that were treated with formaldehyde to inactivate the PT. These
vaccines also contained various amounts of other B. pertussis proteins as minor
components, including pertactin (PRN or 69 kDa protein) and fimbriae (FIM).
Epidemiological evaluations indicated that these vaccines effectively controlled
pertussis disease when introduced for routine immunization (8–10).
An alternative approach to manufacturing employed individually purified
pertussis antigens which, after detoxification, were used to formulate vaccines of
defined composition. These purified component vaccines varied in the number
of antigens incorporated, and in the PT detoxification procedures and antigen-
purification processes used.
Between 1986 and 1996, several vaccines containing acellular pertussis,
including some composed of purified antigens and some composed of co-purified
antigens, were evaluated in a series of efficacy trials. These trials evaluated acellular
pertussis vaccines containing up to five pertussis components and utilized
different study designs, including randomized placebo-controlled cohort trials,
household contact studies, and case–control studies (11, 12). This series of trials
revealed that all the tested acellular pertussis vaccines provided some protection
against pertussis, although the studies suggested differences between the vaccines.
Additional detail is provided in Part C of these Recommendations. Unless
vaccines of different types were tested in parallel within the same trial, comparing
efficacy among different acellular vaccines must be done with caution as all the
trials varied with respect to design, case-ascertainment methodology and case
definition. Following the completion of these trials, many countries introduced
acellular pertussis vaccines into their routine immunization programmes.
In addition to heterogeneity in production and composition, there are
variations in the approaches used for control testing of acellular pertussis vaccines.
The testing methodology developed in Japan was based on modifications of the
tests used for whole-cell vaccines. This included a modification of the mouse
intracerebral challenge assay for potency, along with additional requirements

designed to monitor purity, content and residual toxin activities. In general,
these tests and specifications are not vaccine-specific and can be used for
evaluating new products with new antigen formulations. Alternative control
testing approaches were adopted initially in Europe and North America for
purified component vaccines based on the concept that the newly manufactured
lots should be comparable to those evaluated in pivotal clinical studies. Control
testing of these vaccines included in-process and final product tests for purity,
composition, residual toxin activity and immunogenicity. As the products
differ markedly from each other, specifications were product-specific and were
based on the concept that the newly manufactured lots should closely match
those evaluated in clinical studies. The immunogenicity of individual antigens
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using antibody-binding assays was used as one of the markers of production

consistency. The different methods used to evaluate potency and residual toxin
activity are designed to assess different characteristics of the vaccines. However,
acellular pertussis vaccines tested by both approaches have been used effectively.
Importantly, both testing approaches have been applied to purified component
vaccines and to co-purified vaccines. Further research is needed to develop
alternative methods and to standardize the current potency and safety tests used
for the evaluation of acellular pertussis vaccines.
Scope
These Recommendations apply to co-purified and purified component acellular
pertussis vaccines. The document covers only antigens produced by B. pertussis.
While other approaches are possible (e.g. antigens produced from B. bronchiseptica
or Escherichia coli) they are not considered in this document.
Although these Recommendations apply to the production and quality
control of acellular pertussis vaccines, the acellular pertussis component is
combined most commonly with other antigens (e.g. diphtheria and tetanus
toxoids, Haemophilus influenzae type b conjugate vaccine, inactivated polio
vaccine and hepatitis B). Therefore the tests recommended for the final bulk or
final product of acellular pertussis vaccines should be performed on the final
bulk or final product of the combined vaccines.
These revised Recommendations highlight the advances made in the
development, manufacturing and testing of acellular pertussis vaccines and aim
to provide guidance on the following issues:
■ improvement of quality control of existing vaccines on the basis of
new information and experience;
■ evaluation of new products and new combinations through control
of manufacturing;
■ evaluation of the vaccines in both nonclinical and clinical studies.
The main changes made to the WHO Guidelines published in 1998 are
as follows.
■ The title of the document is upgraded from “Guidelines” to
“Recommendations”.
■ Advice on clinical and nonclinical evaluation of acellular pertussis
vaccines is added to guide NRAs and vaccine manufacturers in
approaches that can be used to assess the safety, efficacy and quality
of vaccines.
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■ Details are provided for the modified intracerebral challenge assay

used to evaluate the potency of the acellular pertussis vaccine
(Appendix 1), and for the histamine sensitization test (HIST) based
on temperature measurement to determine the residual activity of
PT in the vaccine (Appendix 2).
■ Information is provided for the performance of the mouse
respiratory challenge method. Although the method is currently
not recommended for routine potency testing, it may have an
important role in nonclinical testing as discussed in Part B of these
Recommendations.
Written descriptions of detailed procedures or the standard operating procedures
used for the production and testing of the acellular pertussis vaccines or combined
vaccines containing acellular pertussis component(s), together with evidence of
appropriate validation of each production step and relevant control tests, should
be submitted for approval to the NRA as part of the licence application. Proposals
for any variations in manufacturing and/or control methods should be submitted
for approval to the NRA according to national regulatory requirements before
they are implemented.
There is as yet no consensus on the ideal antigenic composition of acellular
pertussis vaccines. Currently, various acellular pertussis vaccine products are
available from diverse manufacturers, differing in the number of components, their
concentrations and their degree of adsorption to different adjuvants. In addition,
these individual antigens have been derived from different strains of B. pertussis,
purified by different methods and treated with different detoxification agents.
All currently available acellular pertussis vaccines contain detoxified PT (PTxd)
and some vaccines formulated with PTxd alone have been shown to provide a
significant degree of protection. However, clinical and laboratory studies have

suggested that the protective efficacy of PT may be enhanced by other antigens
(11–19). Ongoing research is essential to identify the protective mechanisms, to
identify immunological markers of protection against pertussis, and to develop
and improve relevant laboratory models. Additionally, because all current
acellular pertussis vaccines are administered in more complex combination
vaccines, research is encouraged on models that allow the concurrent testing of
multiple components (e.g. diphtheria and tetanus toxoids).
Manufacturers should demonstrate consistency in manufacturing and
formulation and should adhere strictly to the production process used for the
manufacture of the vaccine lots used in the clinical trials supporting regulatory
approval. In addition, laboratory tests should show consistency in the safety,
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potency, and physicochemical and immunological characteristics of new vaccine

lots compared with lots evaluated in clinical studies. Special care should be taken
in the validation of those test procedures that are used for ensuring consistency
of production lots for specificity, sensitivity and accuracy. Manufacturers should
ensure that sufficient quantities of reference vaccine of adequate stability are
available for routine in-house testing and for confirmatory tests undertaken by
the NRA.
An in-house standard, when used, should be assigned a value so that
trend analyses can be monitored and quality-control testing limits can be defined.
When an International Standard exists, the in-house standard should be calibrated
against that standard.
Use of a suitable freeze-dried vaccine preparation as an in-house reference
may offer advantages of stability. A successful example of conditions
which have been used for diphtheria vaccines (adsorbed) is as follows:
vaccine lyophilized in the presence of 3.5% polygeline (1:1) under freeze-
drying cycle at –50 °C load, –50 °C freeze over 2.5 hours, then primary
drying at –35 °C (100 µbar vacuum) and secondary drying at 30 °C
(30 µbar vacuum) (20).
There are no laboratory tests, animal models and/or human immune responses
that can provide complete assurance that a newly developed acellular pertussis
vaccine will be adequately safe and effective. Within these limitations, these
Recommendations describe a sequential approach to the collection of supporting
evidence, beginning with a comprehensive programme of nonclinical testing
and followed by a progression of clinical evaluations. For the purpose of this
document, a newly developed vaccine would be any vaccine that contains either
a novel antigen or one of the antigens in the currently licensed products (i.e.
PTxd, FHA, PRN, FIM type 2 and FIM type 3) that is produced from a new
strain, new process and/or new manufacturer. As described in Part B, nonclinical
characterization studies should include evaluations of purity, residual toxin
activity, bioactivity, reactivity with specific antibodies, induction of binding and
functional antibodies, and induction of protective activity in animal models.
Whenever an additional antigen is added, studies should be undertaken to
characterize its interaction with other components in the product. If the antigen is
novel, more extensive characterization studies would be expected. This document
assumes that only those vaccines that have already undergone extensive nonclinical
testing would be considered for clinical evaluation, with agreement with the local
NRA responsible for evaluating adequacy of nonclinical information. Although
efficacy trials appear very difficult, if not impossible, safety and immunogenicity
trials of adequate design and size are possible and should be conducted. In Part C,
the Recommendations provide guidance on issues related to the design and
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evaluation of the clinical studies. Most studies are expected to be comparative

studies. Thus the choice of a comparator vaccine is a particularly important issue
because the potential comparator vaccines differ substantially in formulation
and composition. Finally, because the tools for clinical evaluation are limited,
rigorous post-marketing monitoring of the vaccines will be needed in order to
determine if they are achieving acceptable levels of clinical safety and efficacy.
Given these limitations, some caution is appropriate when considering a
transition from whole-cell to acellular pertussis vaccines. Specifically, whole-cell
pertussis vaccines are safe and effective, and offer some advantages as described
in the WHO position paper (1). Thus, although these Recommendations describe
an approach for approval of new acellular pertussis vaccines, the path for approval
of new acellular pertussis vaccines requires considerable effort and includes
significant challenges.
International Standards and Reference Preparations

Subsequent sections of this document refer to WHO reference materials that may
be used in laboratory or clinical evaluations. The WHO catalogue of international
biological standards should be consulted for the latest list of appropriate WHO
International Standards and reference materials (see: http://www.who.int/
bloodproducts/catalogue/en/index.html). Key standards and reference materials
include:
■ the First WHO International Standard for acellular pertussis vaccine
for use in modified mouse intracerebral challenge assay (MICA) and
other protection assays (Code no. JNIH 3 with assigned unitage per
ampoule of 34 IU);
■ the First WHO International Standard for pertussis toxin for
standardization of assays used to monitor the residual PT activity
in pertussis vaccines, e.g. histamine sensitization tests and Chinese

hamster ovary (CHO) cell assay (Code no. JNIH 5 with assigned
unitage per ampoule of 10 000 IU);
■ the First WHO International Standard for pertussis antiserum
(human) (Code no. 06/140 with assigned unitage per ampoule of
335 IU anti-PT IgG and 65 IU IgA anti-PT; 130 IU IgG anti-FHA and
65 IU IgA anti-FHA; 65 IU IgG anti-69K and 42 IU IgA anti-69K);
■ the First WHO International Reference Reagent for standardization
of clinical serology assays (Code no.06/142 with assigned unitage per
ampoule of 106 IU anti-PT IgG and 18 IU IgA anti-PT; 122 IU IgG
anti-FHA and 86 IU IgA anti-FHA; 39 IU IgG anti-69K and 38 IU IgA
anti-69K);
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■ the First WHO International Standard for monoclonal antibody

to B. pertussis fimbriae type 2 for the determination of serotype of
B. pertussis strains (Code no. 06/124);
■ the First WHO International Standard for monoclonal antibody
to B. pertussis fimbriae type 3 for the determination of serotype of
B. pertussis strains (Code no. 06/128);
■ the First WHO International Reference Reagent for pertussis
antiserum (mouse) (Code no. 97/642 with assigned unitage per vial
of 17 units of anti-PT, 143 units of anti-FHA, 30 units of anti-PRN
and 32 units of anti-FIM 2 and 3).
The above-mentioned WHO International Standards and reference

materials and other reagents from the WHO International Standards Laboratory
are in the custody of the National Institute for Biological Standards and Control
(NIBSC), Health Protection Agency, Potters Bar, England (see: http://www.nibsc.
ac.uk).
These Reference Preparations are available for the calibration and
establishment of regional, national or in-house reference materials. Samples are
distributed free of charge, on request, to NCLs.
Terminology
The definitions given below apply to some common terminology used throughout
this document. The terms may have different meanings in other contexts.
Master seed lot: a quantity of bacterial suspension that is derived from a
single strain, has been processed as a single lot and has a uniform composition. It
is used for inoculating media for the preparation of working seed lot.
Working seed lot: a quantity of bacterial suspension of a single substrain
derived from the master seed lot by growing the organisms and maintaining them
in aliquots in the frozen form or in the lyophilized form, stored at –20 ˚C or below
(in the liquid form stored at –80 ˚C or below). The working seed lot should be
prepared from the master seed lot by as few cultural passages as possible, having
the same characteristics as the master seed lot and intended for inoculating media
for the preparation of single harvests.
Single harvest: the culture filtrate or the suspension of bacteria obtained
from one batch of cultures that have been inoculated with the working seed lot
(or with the inoculums derived from it), harvested and processed together.
Purified antigen(s) bulk material: the processed purified material
prepared using pertussis antigen preparations processed either in a single run or
a pool of those prepared in multiple runs. In some cases, purified antigen bulk
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material may be adsorbed to or mixed with adjuvant and a preservative may be

added. It is the parent material from which the final bulk is prepared.
Final bulk: the homogeneous finished vaccine present in a single container
from which the final containers are filled either directly or through one or more
intermediate containers.
Final lot or final product: a collection of sealed final containers that are
homogeneous with respect to the risk of contamination during filling. A final
lot must therefore have been filled from a single container in one continuous
working session.
Individually purified antigen: each of the pertussis antigens that are
individually isolated and purified using combinations of several physicochemical
separation methods.
Co-purified antigen: two or more pertussis antigens that have been
isolated and purified using combinations of several physicochemical separation
methods (e.g. ammonium sulfate precipitation and density gradient centrifugation).
Comparator vaccine: an approved vaccine with established efficacy or
with traceability to a vaccine with established efficacy that is tested in parallel
with an experimental vaccine and serves as an active control in nonclinical or
clinical testing.
Part A. Manufacturing recommendations

A.1 Definitions
The international name should be acellular pertussis vaccine. The proper name
should be the equivalent of the international name in the language of the country
of origin.
The use of the international name should be limited to vaccines that
satisfy the recommendations formulated below.

Acellular pertussis vaccine is a preparation of purified or co-purified antigenic
component(s) of Bordetella pertussis that have been appropriately treated by
chemical means or obtained by genetic manipulation to minimize toxicity
and retain potency. The preparations for human use should satisfy all the
recommendations formulated below.
Currently licensed vaccines contain either PTxd alone or PTxd in
combination with one or more other antigens (FHA, PRN, fimbriae type
2 [FIM-2] and fimbriae type 3 [FIM-3]).
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A.2 General manufacturing recommendations

The general manufacturing requirements contained in good manufacturing
practices (GMP) for pharmaceutical (21) and biological products (22) apply to
the production of the acellular pertussis vaccines.
A.3 Production control

These Recommendations pertain to antigen production and purification from
B. pertussis.
A.3.1 Control of source materials

A.3.1.1 Strains of Bordetella pertussis
Strains of B. pertussis used in preparing vaccine should be identified by a full
record of their history, including origin and characteristics on isolation, and
particulars of all tests made periodically to verify strain characteristics. If
genetically modified B. pertussis is used, all relevant modified DNA sequences
should be clearly delineated and fully characterized. The strains of B. pertussis
used should be approved by the NRA.
A.3.1.2 Seed lot system

The production of the acellular pertussis component of monovalent or combined
vaccines should be based on a well-established seed lot system. Cultures from
the working seed lot should have the same characteristics as cultures from the
master seed lot. If genetically modified B. pertussis is used, the relevant modified
DNA sequences should be reconfirmed for each new working seed lot. The
strains should be maintained by a method approved by the NRA and able to
preserve the ability of the seed to yield potent vaccine in terms of the quality of
the antigens produced.
Freeze-drying or storage in liquid nitrogen are satisfactory methods of
maintaining strains, subject to suitable validation. In some countries, glycerol
stocks are also used for this purpose, but this method would require extensive
validation and approval of the NRA.
A.3.1.3 Culture media for production

B. pertussis should be cultured in culture media suitable to support its growth
and the production of relevant antigens with consistent yields. Media used
should be free from adventitious agents. Moreover, medium components that
are known to cause allergic reactions should be avoided. Human blood or blood
products should not be used in culture media either for seed lots or for vaccine
production. The acceptability of the source(s) of any components of bovine,
sheep or goat origin used in culture media should be approved by the NRA.
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If any materials of animal origin are used in seed preparation or preservation,

or in production, they should comply with the WHO guidelines on transmissible
spongiform encephalopathies in relation to biological and pharmaceutical products
(23) and national regulatory requirements. When animal blood or blood
products are used, they should be removed in the process of production by
appropriate methods.
Any change of media should be submitted for approval to the NRA.
A.3.2 Control of manufacturing process

A.3.2.1 Control of production cultures
Production cultures should be shown to be consistent with respect to growth
rate, rate of change of pH, rate of production of the desired antigen(s), and
additional parameters as agreed with the NRA. Acceptance specifications should
be established and agreed with the NRA.
A.3.2.2 Control of bacterial purity

Samples of individual cultures should be tested for microbial purity by microscopic
examination of stained smears, by inoculation of appropriate culture media, or by
any other suitable procedure. For microscopic examination, several fields should
be examined at high magnification. If a contaminant is found, the culture and any
product derived from it should be discarded.
In some countries the individual cultures are tested for microbial purity
by a minimum of two suitable and approved procedures.
A.3.2.3 Control of antigen purification

Cultures should be processed for further antigen purification in a way that
minimizes contamination of crude materials with undesirable molecules, such
as lipooligosaccharide (LOS), dermonecrotic toxin, adenylate cyclase toxin
(ACT), and tracheal cytotoxin (TCT). Cells of B. pertussis may be separated

from fermentation fluid by filtration or centrifugation, and should be suitably
inactivated before their further processing or disposal. Absence of cells of
B. pertussis from crude antigen solutions should be confirmed at this stage using
appropriate methods approved by the NRA.
Two approaches have been followed for the purification of pertussis
antigens for vaccine manufacturing. In the first approach, antigens have been
co-purified by repeated cycles of ammonium sulfate precipitation and density
gradient centrifugation to yield preparations enriched in certain proteins –
mainly PT, FHA and PRN – but depleted of endotoxin (LOS). The number and
proportion of each antigen in a given vaccine type may vary widely depending
on the process followed, but should be reproducible for each specific product.
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In the second approach each antigen is individually purified using combinations

of several physicochemical separation methods.
The tests used for determination of consistency of yield and purity and
their performance characteristics should be approved by the NRA.
It is advisable to sterilize purified antigens by membrane filtration or
other suitable sterilizing grade filtration before further processing.
A.3.2.3.1 Tests undertaken prior to detoxification/chemical treatment

Characterization of antigens: rigorous characterization of the antigens by
physicochemical, immunological or functional (biological) assays, as appropriate,
is essential before any step is undertaken that is capable of modifying their
original characteristics. Particular attention should be given to employing a
range of analytical techniques based on different principles. Immunoblot analysis
with specific monoclonal or polyclonal antibodies may be used to characterize
antigens or antigen subunits. The specific properties of each antigen component
should be determined in comparison with Reference Preparations. Specifications
should be established for each individual antigen. Suitable assays include sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), single radial
immunodiffusion, immunoblotting, the CHO cell test for detection of active PT,
haemagglutination and high-performance liquid chromatography (HPLC). The
specific activity of PT (IU/ng) should be determined.
Antigen purity: the purity of the individual or co-purified antigens claimed to
contribute to vaccine efficacy should be determined by SDS-PAGE, HPLC or other
appropriate analyses before detoxification. It is important that the techniques
used are based on as wide a range of properties of the vaccine components as
possible. Limits should be specified for all impurities detected.
The purity of the individual or co-purified antigens should be within the
range of values established for each product as found for vaccine lots shown to
be safe and effective in clinical trials or other lots used in support of licensing.
Specifications should be set during the process of product development and
validation and should be established by agreement with the NRA.
Residual levels of endotoxin: the antigens should be tested for residual endotoxin
content by means of the Limulus amoebocyte lysate (LAL) test or other appropriate
assay. This test may be carried out at a later stage. Endotoxin content should
be consistent with levels found in vaccine lots shown to be safe and effective in
clinical trials or for lots used in support of licensing. Specifications should be
established in agreement with the NRA.
Antigen content: if it is necessary at this stage, the amount of individually purified
antigens that have been characterized and purified, as appropriate, should be
estimated by means of a validated quantitative assay of sufficient sensitivity,
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such as an assay for protein content and, where available, a suitable quantitative
immunoassay. Antigen content can be determined by ELISA; active PT content
can be determined by CHO cell assay.
In cases where two or more antigens are co-purified, the proportion of
each antigen claimed to contribute to vaccine efficacy should be measured by a
suitable method (e.g. SDS-PAGE, HPLC, electrophoresis on non-denaturing gels,
or densitometry) and should be shown to be within the range of values found
for vaccine lots shown to be safe and effective in clinical trials or other lots used
in support of licensing. Specifications should be set during the validation and
established in agreement with the NRA.
Sterility test: bacterial and mycotic sterility for each antigen lot should be
determined in accordance with the requirements of Part A, section 5, of the
revised Requirements for biological substances, No. 6 (General requirements for
the sterility of biological substances) (24), or by a method approved by the NRA.
If appropriate, this test may be carried out at a later stage.
If a preservative is added, appropriate measures should be taken to
prevent interference with the sterility test.
A.3.2.4 Detoxification
The purified PT, if it is not genetically detoxified, or co-purified antigens that
contain this toxin should be subjected to appropriate detoxification methods.
Other antigens may also be treated with agents to detoxify any residual PT in the
preparation. The residual detoxifying agents should be removed by an appropriate
method. Different chemicals are used to detoxify PT. These include, but are not
limited to, formaldehyde, glutaraldehyde, a combination of both, or hydrogen
peroxide. Different detoxification processes yield distinct products.
The detoxification method/process should be validated for the ability
to consistently produce antigens that have acceptably low levels of biologically
active PT and retain acceptable levels of immunogenicity, as measured on final

formulation. In addition, the detoxification method should be validated for the
ability to produce detoxified PT that does not revert to biologically active PT
upon storage. If any aggregation of antigens has occurred following detoxification,
the aggregates should be homogenized by an appropriate procedure such as
sonication followed by filtration to remove the larger clumps.
A.3.3 Control of pertussis antigen bulk materials

Bulk materials should be prepared using antigen preparations processed either
in a single run or in a pool of those prepared in multiple runs. With the approval
of the NRA, the bulk materials may be adsorbed to/mixed with adjuvant and a
preservative may be added.
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Antigen content: the amount of each individual antigen or of co-purified antigens

should be estimated by means of a validated quantitative assay, such as an assay
for protein content and, where available, suitable quantitative immunoassays
for individual antigens. For co-purified antigens, the ratio of antigens should
be defined. When no adequate procedure is available for assessing individual
antigens following chemical detoxification, a validated suitable procedure may
be used to estimate the amount of individual antigens based on the amounts
measured before detoxification. Specifications for antigen content should be
set during the process of product development and validation and should be
approved by the NRA.
Residual activity of pertussis toxin: the amount of residual biologically active
PT in the individually or co-purified antigens should be estimated after
detoxification by means of a sufficiently sensitive test such as the HIST or the
CHO cell assay. Adjuvants and other vaccine components may interfere with the
adequate performance of the CHO cell assay (25) and special care must be taken
to ensure that they do not interfere with the tests (e.g. by adequate dilution of test
solutions). At the concentration of vaccine final formulation, the total amount of
residual bioactive PT from all pertussis antigens should not exceed that found
in vaccine lots shown to be safe in clinical trials or other lots used in support of
licensing. Specification should be established in agreement with the NRA.
Residual levels of endotoxin: the bulk material or antigens should be tested for
residual endotoxin content by means of the LAL test or other appropriate assay.
At the concentration of vaccine final formulation, the total amount of residual
endotoxin should not exceed that found in vaccine lots shown to be safe in
clinical trials or other lots used in support of licensing. The limits applied to the
vaccine concentration of individual components should be agreed with the NRA.
Sterility test: each purified antigen bulk should be tested for bacterial and
mycotic sterility in accordance with the requirements of Part A, section 5, of the
revised Requirements for biological substances, No. 6 (General requirements for
the sterility of biological substances) (24), or by a method approved by the NRA.
If a preservative is added, appropriate measures should be taken to prevent
interference with the sterility test.
A.3.4 Control of final bulk

Currently there is no stand-alone acellular pertussis vaccine, so the tests described
below are undertaken at the final bulk stage of combined vaccines.
If a stand-alone acellular pertussis vaccine were to be developed, the
procedures described here could be adapted for such a product.
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A.3.4.1 Preparation
The antigen bulk materials should be pooled to prepare the pertussis bulk.
Current preparations may contain PTxd alone or together with FHA, with or
without PRN and FIM 2 and 3, to produce one, two, three, four or five component
acellular pertussis vaccines. The bulk of acellular pertussis vaccine may be
adsorbed to/mixed with aluminium hydroxide or phosphate gel or another
appropriate adjuvant prior to or after blending with other components (e.g.
diphtheria toxoid, tetanus toxoid, inactivated polio vaccine (IPV)) to produce the
final formulation (final bulk). A suitable antimicrobial preservative may be added.
A.3.4.2 Control tests

The following control tests on final bulk may be performed on the final product
in agreement with the NRA.
A.3.4.2.1 Detoxifying agents

The content of residual detoxifying agent in the final bulk should be determined.
The method and limits should be approved by the NRA.
If formaldehyde has been used, the residual content should not exceed
0.2 g/l. The residual content of glutaraldehyde should not exceed 0.1 g/l.
A.3.4.2.2 Preservative
Consideration should be given to the effect of the preservative on the stability
of the vaccine formulation and possible interactions between the vaccine
components and the preservative. The content of preservative should be
determined by a method approved by the NRA. The amount of preservative in
the vaccine dose should be shown to have no deleterious effect on the antigen(s)
and should not impair the safety of the product for humans. The preservative,
its use at different stages of the manufacturing process, and its concentration
or residual amount should be approved by the NRA. If any modification of

preservative content in already licensed vaccine is made, general principles for
vaccine evaluation described in the WHO Guidelines on regulatory expectations
related to the elimination, reduction or replacement of thiomersal in vaccines
should be followed (26).
Phenol should not be used as a preservative.
A.3.4.2.3 Adjuvant
The nature, purity and concentration of the adjuvant or adjuvants added to
the vaccine should be determined by a method approved by the NRA. When
aluminium compounds are used as adjuvant the concentration of aluminium
should not exceed 1.25 mg per single human dose. When calcium adjuvants
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are used, calcium should not exceed 1.3 mg per single human dose. If other
substances are used as adjuvants, specifications should be set and agreed with the
NRA. The formulation should be such that the suspension appears homogeneous
after shaking and remains as such for a defined period (e.g. the time needed
for vaccine administration). Adsorption of antigens to the adjuvant should be
investigated, when possible, by tests designed to determine which, and how much
of each, are adsorbed. Consistency of adsorption is important, and the adsorption
of the antigen in production lots should be demonstrated to be within the range
of values found for vaccine lots which have been shown to be clinically safe and
effective, or for lots used in support of licensing.
A.3.4.2.4 Sterility
Each final bulk should be tested for bacterial and fungal sterility in accordance
with the requirements given in Part A, section 5, of the revised Requirements for
biological substances, No. 6 (General requirements for the sterility of biological
substances) (24) or by a method approved by the NRA.
If a preservative has been added to the vaccine, appropriate measures
should be taken to prevent it from interfering with the test.
A.3.4.2.5 Residual activity of pertussis toxin

Each final bulk of vaccine should be tested for active PT using a HIST or another
test that is sufficiently sensitive to detect the level of toxin activity agreed with
the NRA. For products containing genetically detoxified PT, this test may not
be necessary if agreed with the NRA. Currently the HIST method used for the
determination of residual bioactive PT in licensed acellular pertussis vaccines is
based on the response to a histamine challenge of mice injected with the test
vaccine. Two possible test outcomes (end-points) are in use. One is based on the
lethal effect of the histamine challenge dose and the other on the decrease in body
temperature following the histamine challenge. If an alternative assay is used, it
should be at least as sensitive and specific as a validated HIST assay (either of
the two tests mentioned above). The alternative method(s) should be approved
by the NRA.
The susceptibility to histamine sensitization of the mice used in each test
should be established using a suitable reference or control preparation of PT. The
specific activity of the standard or positive control should be calibrated using the
WHO International Standard (currently JNIH-5) and should be expressed in IU
because the nominal protein masses of the toxin preparations do not necessarily
predict their biological activity in HIST. The detection limit for the PT of the test
should be defined and accepted by the NRA.
The acceptance criteria for content of residual bioactive PT should be
consistent with the results for lots shown to be safe and effective in clinical trials
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or other lots used in support of licensing. Specifications should be established in

agreement with the NRA.
There is at present limited information about the relation between
the level of residual active PT in an acellular pertussis vaccine and its
clinical safety. Therefore the residual toxicity should be reduced as far
as is feasible without undue compromise of immunogenicity. There is
no internationally agreed upper limit for active PT in acellular pertussis
vaccines. In some countries, upper limits of PT per single vaccine dose are
a requirement for DTaP vaccines (27, 28). A recent collaborative study has
provided preliminary data on the content of bioactive PT in DTaP-based
combination vaccines using the HIST lethal end-point method (29).
The HIST lethal end-point method (see Appendix 3) measures the proportion of
animals that die upon histamine challenge due to sensitization with residual PT
in the vaccine. Assay sensitivity is verified by titration of a PT standard (calibrated
in IU). Once linearity has been established by repeated experiments, the assay
may be simplified to include in each test only a single dose of PT standard to
ensure assay sensitivity.
Some laboratories include a standard toxin at a concentration near the
acceptance limit to verify assay sensitivity.
The HIST based on measurement of the reduction in temperature (either rectal or

dermal) produced by histamine challenge (see Appendix 2) has been successfully
used in some countries. It has also been optimized to provide a quantitative
estimate of the activity of a test vaccine relative to the activity of a PT standard.
Although the CHO cell assay is highly sensitive for detection of PT
activity, the test may not be suitable for the final bulk vaccine because of
possible interference (e.g. presence of adjuvant or inactivating agents).
Development of an alternative to HIST is encouraged. An in vitro

assay system comprising an enzymatic HPLC (E-HPLC) assay and a
carbohydrate binding assay is under evaluation as a potential alternative
to the HIST (30). Any alternative assay to the current HIST to determine
the residual PT activity should be validated and approved by the NRA.
A.3.4.2.6 Reversion to toxicity

Accelerated reversion testing, consisting of HIST performance on final bulk or
the final lot incubated for at least four weeks at 37 °C, may be used to demonstrate
that it is unlikely that the chemically inactivated PT will regain some of its
toxicity before the vaccine expiry date. Some NRAs may not require this test for
the release of each new lot but only as part of process validation. For products
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containing genetically detoxified PT, this test may not be necessary as agreed
with the NRA.
A.3.4.2.7 Immunological activity

Two methods are currently used in the lot release procedure to assess the
immunogenicity or potency in mice after vaccination with acellular pertussis
vaccine: the mouse immunogenicity test (MIT) and the MICA. Importantly,
none of these assays can be considered as an index of clinical efficacy. Use of any
of these tests requires validation and agreement with the NRA. Active PT in the
vaccine (if any) may enhance the potency of vaccines obtained in these assays,
with the degree of enhancement depending on the immunizing antigens, mouse
strain, test method and assay conditions (31, 32).
The MIT is a non-lethal animal model designed to evaluate antibody responses
in immunized mice to all the antigens claimed to contribute to vaccine efficacy.
Currently, ELISA is used to measure the binding activity rather than the functional
activity of antibody for each of the antigens. An international mouse reference
serum containing antibodies to five antigens is available to monitor consistency
of the ELISA stage.
The MIT is designed to assess consistency of manufacture by evaluating
whether the results of the MIT for lots manufactured post-licensure are consistent
with the results of lots with acceptable performance in clinical trials. Due to the
different composition of different vaccine products, specifications are product-
specific. Additionally, to ensure adequate performance, MIT requires the use of
a product-specific reference or control vaccine analogous in composition to the
test vaccine.
In the absence of an international reference vaccine for MIT, each
manufacturer is responsible for the development of a reference vaccine that
can allow for meaningful assessment of the immunogenic activity of the test
vaccine with respect to the established specifications. Although a clinical lot with
established efficacy may be considered as the reference vaccine, this is usually
impractical due to considerations such as availability and long-term stability.
However, the vaccine lot selected as reference should be sufficiently similar to
the clinical lots in composition, manufacturing process, immunogenicity and/
or protective effect. Stabilization of the reference vaccine is recommended, but
careful attention should be given to any effects the stabilizing procedure may
have on its activity.
The manufacturer is responsible for monitoring the stability of the
reference and for replacement as needed. When monitoring stability of
the reference or testing a candidate replacement for the reference, testing
approaches that allow for higher precision (e.g. more tests, more animals
per test, or more dilutions per test) are encouraged in order to improve
the ability to detect changes in activity.
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Establishment of the specifications for each product should be based on the

response observed in the test for vaccine clinical lots and other lots used in
support of licensing. The specifications for the antibody response to each antigen
claimed to contribute to efficacy should be established and approved by the NRA.
Additional details on the method are provided in Appendix 4.
Tests which show only that the test product does not differ significantly
from the reference vaccine are not recommended. Specifications must be
carefully justified and should take into account the precision of the test
and the maximum allowable deviation from the reference vaccine.
The MICA: is a lethal challenge model in mice which detects mouse protective
activity provided by the vaccine. The potency of each final bulk is expressed as a
relative potency to a reference vaccine. That reference vaccine should be calibrated
against the WHO International Standard for acellular pertussis vaccines (currently
JNIH-3) (33) and the protective activity should be expressed in IU.
The assay method and the specifications should be approved by the NRA.
Additional details on the method are provided in Appendix 1.
The specifications, where used currently, are that the vaccine passes
the potency test if the result of a statistically valid assay shows that the
estimated potency of the vaccine is not less than 4.0 IU in the volume
recommended for a single human dose and if the lower fiducial limit
(p = 0.95) of the estimated potency is not less than 2.0 IU for primary
immunization vaccine.
Active PT has been shown to enhance the relative potency of vaccines

obtained in this assay, with the degree of enhancement depending on
mouse strain, assay conditions etc. (32).
Other assays
The development of alternative assays to MIT and MICA is encouraged.
An alternative assay – the guinea-pig immunogenicity test – has been

adopted in some countries (34, 35). The assay allows immunogenicity
testing of the acellular pertussis components and the diphtheria and
tetanus toxoid components using the same group of animals. Adoption
of this or another alternative method for routine lot release would require
validation and approval by the NRA.
Respiratory challenge method(s) such as the mouse intranasal challenge

assay (INCA) (see Appendix 5) have been evaluated in WHO international
collaborative studies. They have discriminated between vaccines
with different protective capacity in mice. The current respiratory
challenge assays are not optimized or designed for use as a routine test
for determining vaccine potency. Nevertheless, they can be used to
assess potential impact of changing the manufacturing process and/or
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formulation, the activity and stability of new antigens and formulations,

or the potential interactions in new combinations. This model is an
important tool in new product development as described in Part B of
this document.

The requirements concerning filling and containers given in Good manufacturing
practices for biological products (22) should apply to vaccine filled in the final form.
Single dose and multiple dose containers may be used. Vaccine in
multidose containers should contain a suitable antimicrobial preservative.
A.5 Control of final product

Quality control procedures and tests should be validated and approved by the
NRA to ensure that the final containers contain the appropriate amounts of each
of the vaccine antigens, as designed for the acellular vaccine formulation.
Unless otherwise justified and authorized, the following tests should
be performed on labelled containers from each final lot by means of validated
methods approved by the NRA.
A.5.1 Identity
An identity test should be performed on at least one container from each final lot
by means of a validated method approved by the NRA.
A.5.2 Sterility
Final containers should be tested for sterility by a method approved by the
NRA. Many countries have regulations governing the sterility testing of the final
product. Where these do not exist, the Requirements published by WHO should
be met (24). If a preservative has been added to the vaccine, appropriate measures
should be taken to prevent it from interfering with the test.
A.5.3 Adjuvant content

The content of adjuvant should be determined by a method approved by the
NRA. When aluminium compounds are used as adjuvants, the concentration
of aluminium should not exceed 1.25 mg per single human dose. If a calcium
adjuvant is used, the concentration of calcium should not exceed 1.3 mg per single
human dose. If other substances are used as adjuvants, appropriate specifications
should be set for the substance with adjuvant effect.
A.5.4 Preservative content

The content of preservative(s) should be determined by methods approved by
the NRA. The amount of preservative per dose should be shown not to have
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any deleterious effect on the antigen(s) nor cause untoward adverse reactions in
humans. The preservative and its concentration or residual amount should be
approved by the NRA. If any modification of thiomersal content in an already
licensed vaccine is made, general principles for vaccine evaluation described
in the WHO Guidelines on regulatory expectations related to the elimination,
reduction or replacement of thiomersal in vaccines should be followed (26).
A.5.5 pH
The pH of each final lot should be within the range of values found for vaccine
lots shown to be clinically safe and effective.
In some countries, determinations for osmolality and withdrawable
content are also required.
A.5.6 Endotoxin
In some countries, determination of endotoxin content may be required with
specifications approved by the NRA.
A.5.7 Innocuity test

Each final lot should be tested for unexpected toxicity (sometimes called abnormal
toxicity or innocuity) by a method approved by the NRA.
This test may be omitted for routine lot release once consistency of
production has been well established to the satisfaction of the NRA
and when GMP is in place. Each lot, if tested, should pass a test for
unexpected toxicity.
A.5.8 Immunological activity

An immunological activity test (MIT, MICA, or approved alternative) should be
carried out as described in section A.3.4.2.7, on each final lot, if such a test has
not been conducted on the final bulk.
A.5.9 Inspection of final containers

Each container in each final lot should be inspected visually or mechanically, and
those showing abnormalities should be discarded.
A.6 Records
A model of a suitable summary protocol to be used for pertussis vaccines
is given in Appendix 6.
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A.7 Samples
A.8 Labelling
products (22) should apply, with the addition of the following:
■ the words acellular pertussis vaccine;
■ the word “adsorbed”;
■ the name and address of the manufacturer;
■ the recommended storage temperature and the expiry date if kept at
that temperature;
■ the recommended single human dose and route of administration.
In addition, the label printed on or affixed to the container, or the label on
the carton, or the leaflet accompanying the container should contain the following:
■ a statement that the vaccine satisfies the recommendations of this
document;
■ the nature and amount of any preservative present in the vaccine
(if there is no preservative in single-dose containers, this should be
stated);
■ the nature and amount of the adsorbing agent, if applicable;
■ the nature and amount of any substances added to the vaccine;
■ the recommended conditions for storage and transport;
■ a warning that the vaccine should not be frozen;
■ a warning that the vaccine should be shaken before use;
■ instructions for the use of the vaccine, and information on
contraindications and the reactions that may follow vaccination.
A.9 Distribution and shipping


A.10.1 Stability
Stability evaluation is an important part of the quality assessment. The purpose
of stability studies is to ensure that the vaccine at the end of its shelf-life, storage
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period or period of use, still has the required characteristics supporting quality,
safety and efficacy. The recommendations given in the WHO Guidelines for
stability evaluation of vaccines should apply (36).
Adequate stability studies form an essential part of vaccine development.
The stability of the vaccine in its final containers, maintained at the recommended
storage temperature, should be demonstrated to the satisfaction of the NRA.
For each of the antigens claimed to contribute to protective efficacy, real-
time stability studies should support immunological activity and lack of specific
toxicity of the product up to the expiry date.
The product must be manufactured in such a way that reversion to toxicity
of the inactivated PT in the vaccine does not occur during the period of validity
provided that the product is stored under the conditions stated on the label.
The desorption of antigens from the adjuvant, which may occur over
time, should be investigated and, where possible, limits should be agreed with
the NRA.
Accelerated stability studies may provide additional evidence of product
stability, but cannot replace real-time studies.
When any changes that may affect the stability of the product are made
in the production procedure, the stability of the vaccine produced by the new
method should be demonstrated.
A.10.1.1 Stability for licensure

Studies that support stability of a vaccine for the purpose of licensure have to be
performed as real-time studies under the intended storage conditions. Stability-
indicating parameters should be carefully selected. They should always include,
but should not be limited to, the tests for immunological activity. Tests should
be conducted at appropriate time intervals during storage to determine the loss
of immunological activity. Final containers from at least three lots of vaccine
derived from different bulks should be tested on the expiry date to demonstrate
stability during storage.
Accelerated stability data for products stored for limited periods at

temperatures that may affect stability may support preliminary data from ongoing
real-time stability studies but should not replace them. Any modification of the
shelf-life approved as part of licensure requires additional stability data to support
proposed modification and should be approved by the NRA. Following licensure,
stability should be monitored throughout the proposed shelf-life.
A.10.1.2 Stability at different stages of the manufacturing process

Stability testing should be performed at different stages of production, namely
single harvests, bulk materials, final bulk and final lot on at least three lots each.
Suitable parameters indicating stability should be selected according to the stage
of production. Manufacturers are encouraged to assign a shelf-life to all materials
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during vaccine production, particularly intermediates such as single harvests,

purified antigen bulk and final bulk.
A.10.1.3 Stability for clinical trial approval

For vaccines under development, stability data, such as those described above,
are expected for the purpose of clinical trial approval. However, for such vaccines
under development, the stability data are generally available for a limited period.
Appropriate documentation to support the stability profile of a vaccine should be
submitted to the competent NRA at all stages mentioned above.

Recommended storage conditions and defined maximum duration of storage
should be based on stability studies, as described in section A.10.1 above, and
approved by the NRA. For acellular pertussis vaccines, a temperature of 2–8 °C
is generally considered to be satisfactory. This should ensure that the minimum
potency specified on the label of the container or package will be maintained
after release and until the end of the shelf-life if the conditions under which the
vaccine is stored are in accordance with what is stated on the label.
The manufacturer should recommend conditions of storage and transport
that ensure that the vaccine satisfies the potency requirements until the expiry
date stated on the label.
The vaccine must not be frozen.
A.10.3 Expiry date

The expiry date should be defined on the basis of shelf-life supported by the
stability studies, as described in section A.10.1 above, and approved by the NRA.
Part B. Nonclinical evaluation of acellular

pertussis vaccines
Nonclinical testing of vaccines is a prerequisite for initiation of clinical studies
in humans. There is no laboratory test or series of tests that will unequivocally
assure that a newly developed acellular pertussis vaccine will be adequately
safe and effective. With this limitation, these Recommendations describe a
sequential approach to the collection of supporting evidence, beginning with a
comprehensive programme of nonclinical testing, followed by a progression of
clinical evaluations. This part describes the recommended nonclinical testing.
The extent to which nonclinical studies will be required depends on the clinical
experience that has already been gained with the different vaccine components.
Animal studies, which aim to provide evidence that the vaccines induce functional
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immune responses (e.g. induction of PT neutralizing antibodies, protection

against bacterial challenge), form an essential part of the development of the
vaccines. For vaccines containing acellular pertussis components that have not
previously been evaluated for efficacy in clinical trials, the results of nonclinical
testing represent only a part of the aggregate of data that needs to be considered
when assessing the likelihood that the vaccine will prove to be effective when used
in the clinical setting. Other considerations include manufacturing methods,
control of the manufacturing process and clinical immunogenicity of the vaccine.
New vaccine formulations that have not been evaluated in safety and
efficacy trials require extensive characterization, including assessment in
vaccination/challenge studies in animal models (proof of concept) and safety
testing in animals. However, extensive nonclinical testing may not be required
for vaccines that use pertussis antigens that are the same (i.e. from the same
manufacturer and produced by the same methods) as those in vaccines that have
already been approved on the basis of their safety and efficacy.
For vaccines based on novel pertussis antigens or on formulations for
which the pertussis components are produced using a new manufacturing process
that is different from the established one, the characterization should include
detailed evaluation and testing of: 1) the purified antigens prior to chemical
treatment (e.g. detoxification), 2) the individual antigens prior to formulation,
and 3) the formulated product. Although characterization is more difficult for
pertussis antigens that are co-purified than for those that are individually purified,
co-purified antigens should undergo similar evaluation and testing before and
after chemical treatment (e.g. detoxification) and in the final formulated product.
Lots of individual antigens and formulated vaccine used in nonclinical
studies should be adequately representative of those intended for clinical
investigation and, ideally, should be the same lots as the ones to be used in clinical
studies. If this is not feasible, then the lots used clinically should be comparable
to those used in the nonclinical studies with respect to manufacturing,
immunological activity, stability and other characteristics of quality. Details of
the design, conduct, analysis and evaluation of nonclinical studies are available in
the WHO Guidelines on nonclinical evaluation of vaccines (37).
B.1 Nonclinical characterization and testing of

pertussis antigens and in‑process materials
In the case of vaccines for which the acellular pertussis antigen is new (in terms
of range of antigens and/or manufacturing processes for one or more antigens)
an extensive preclinical evaluation should be undertaken. This should include
thorough characterization of purified antigens before and after any chemical
treatment, as well as any relevant in-process intermediates and final product
materials. Characterization should evaluate purity, integrity and functional
activity using a variety of approaches including physicochemical evaluation,
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bioassays, measurement of residual toxicity/lack of reversion to toxicity, and

active protection models. Given the complexity of acellular pertussis vaccines
produced by co-purification methods, additional testing to fully define and
specify the composition should also be conducted. For example, an assessment of
the proportion of each antigen (e.g. the PT:FHA ratio) should be established to
characterize clinical lots and to monitor product consistency.
The inclusion of a novel antigen – i.e. an antigen other than those that have
already been tested in previous clinical efficacy trials (i.e. PT, FHA, PRN, FIM) –
would require additional considerations. So would, for instance, the inclusion of
the PRN antigen purified from B. bronchiseptica since this is not the same antigen as
PRN from B. pertussis. When possible, the individual antigens should be evaluated
in active protection animal models (16, 18, 19, 38, 39). Pertussis toxoid (PTxd)
is effective in most active protection models, and therefore the demonstration of
an additional benefit for antigens mixed with PTxd may be challenging. In such
cases, the antigen should be examined in protection models with either no PTxd
or suboptimal amounts of PTxd. The other antigens (FHA, PRN and FIM) are
not necessarily active in all protection models, so it is important to consult the
literature (see Table A4.1 below) to identify relevant models for each antigen.
The following describes the testing strategy that could be considered for
the antigens that are contained in currently approved acellular pertussis vaccines,
either produced by co-purification processes or by individual purifications of each
component (PTxd, FHA, PRN, FIM2 and FIM3). When included in an acellular
pertussis vaccine, the FIM2 and FIM3 are typically co-purified and processed as
a single antigen (FIM2/3).
PT: purity and bioactivity of PT before chemical treatment, residual
bioactivity after chemical treatment, lack of reversion to toxicity, activity
in animal protection models, binding and functional activity of antibodies
induced in animals, detection of known epitopes using monoclonal
antibodies.
FHA: purity and functional integrity (e.g. haemagglutinating activity) of

FHA before chemical treatment, residual activity after chemical treatment,
activity in animal protection models, binding activity of antibodies
induced in animals, detection of known epitopes using monoclonal
antibodies.
PRN: purity of PRN, activity in animal protection models, binding activity

of antibodies from immunized animals, detection of known epitopes
using monoclonal antibodies.
FIM2, FIM3, or FIM2/3: purity of FIM, relative content of FIM2:FIM3,

activity in animal protection models, binding and whole-cell agglutinating
activity of antibodies from immunized animals and detection of known
epitopes using monoclonal antibodies.
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Special consideration should be given to vaccines based on genetically

inactivated PT. Characterization studies of these vaccines should include
evaluation of genetic stability of the production strain, consistency of the genetic
sequence and attenuation of the toxic bioactivity (40).
Table A4.1 provides an overview of results from the published literature.
The specific references should be consulted for details and additional information.
Table A4.1
Summary of published studies evaluating the ability of purified pertussis antigens to
protect in mouse challenge models
Animal model protection studies
Respiratory challenge Intracerebral challenge models

models
Antigen Purified antigen

plus small,
Purified antigen Purified antigen nonprotective,
amount of active
PT
Active Passive Active Passive Active
immuniza‑ immuniza‑ immuniza‑ immuniza‑ immunization
tion tion tion tion
Yes Yes Yes Yes Yes
References: References: References: References: Reference: 31
PTxd
18, 19, 17, 43–51 17, 19, 31, 48, 49
41–47 42, 44, 45
Yes Yes No No Yes
References: References: References: Reference: Reference: 31
FHA 18, 19, 17, 44, 45, 17, 19, 31, 39

44–46, 51, 55 44–46
52–57
Yes Yes
PRN References: Reference: ? ? ?
53, 58–61 61
Yes Yes No Yes
FIM References: Reference: References: ? References: 31, 65
45, 62–65 45 31, 45, 65
PT = pertussis toxin; PTxd = pertussis toxoid; FHA = filamentous haemagglutinin; PRN = pertactin;
FIM = fimbriae; “Yes”= protection was demonstrated; “No” = protection was not observed; “?” = no information
was found.
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Despite advances in knowledge regarding the mechanisms of toxicity

of PT and other potentially reactogenic components produced by B. pertussis,
uncertainty remains concerning the exact role played by these substances in
the pathogenesis of pertussis and in vaccine reactions. This lack of information
has hampered the establishment of scientifically sound limits for the residual
activity of these components in vaccines containing pertussis antigens. However,
vaccines containing chemically or genetically inactivated PT require thorough
characterization to assess residual PT activity and, where appropriate, the possible
reversion of this toxoid during storage. Manufacturers should demonstrate to the
satisfaction of the NRA that chemically inactivated PT present in the final bulk
does not revert to its toxic form before the vaccine expiry date. In addition, as part
of the validation of the manufacturing process, manufacturers are required to
submit evidence that the purification steps reduce the levels of LOS endotoxin,
as well as the residual activities of heat-labile (dermonecrotic) toxin, tracheal
cytotoxin and adenylate cyclase toxin, to acceptable levels.
In some countries, during development of the vaccine, the production
process should be validated to demonstrate that it yields consistently
an antigenic fraction that complies with the purity requirements listed
below. After demonstration of consistency, the tests need not be applied
routinely to each lot (66).
Adenylate cyclase: not more than 500 ng in the equivalent of 1 dose of

the final vaccine, determined by immunoblot analysis or another suitable
method.
Tracheal cytotoxin: not more than 2 pmol in the equivalent of 1 dose of

the final vaccine, determined by a suitable method such as a biological
assay or liquid chromatography.
Absence of residual dermonecrotic toxin: inject intradermally into each

of three unweaned mice, in a volume of 0.1 ml, the amount of antigenic
fraction equivalent to 1 dose of the final vaccine. Observe for 48 hours.
No dermonecrotic reaction is demonstrable.
The mouse weight-gain test and the leukocytosis promotion test, which are
currently used to monitor the toxicity of whole-cell pertussis vaccines, are
considered to be of insufficient sensitivity to demonstrate residual PT activity
in acellular pertussis vaccines. Specific tests for residual PT activity (see sections
A.3.3 and A.3.4.2.5) are preferred.
B.2 Nonclinical characterization and testing

of final vaccine formulation
Given that there are currently no stand-alone acellular pertussis vaccines,
the following studies should be carried out using the final formulation (i.e.
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formulation that includes diphtheria and tetanus toxoid and other components).
The capacity of an acellular pertussis vaccine to protect mice against a B. pertussis
challenge may be used to establish a nonclinical proof of concept for new vaccine
formulations. Two models have been developed to assess acellular pertussis
vaccines: the MICA (Part A and Appendix 1) and the INCA (by instillation or
by aerosol) (Part A and Appendix 5). However, as noted above (section B.1),
PTxd is an effective antigen in both models; therefore, for vaccines that contain
PTxd, the contribution of antigens other than PTxd may be difficult to discern
in these models. Moreover, because residual active PT can influence the outcome
of the MICA (32), care should be taken in interpreting the results of that assay.
The assessment of functional antibodies, such as PT-neutralizing antibodies
as evaluated in the CHO cell assay (for anti-PT) or whole-cell agglutinating
antibodies (for anti-FIM), would provide further nonclinical evidence of the
potential protective efficacy against B. pertussis in humans.
Additional toxicity and other testing should follow the recommendations
outlined in the WHO guidelines on nonclinical evaluation of vaccines (37).
Part C. Clinical evaluation of acellular pertussis vaccines

This part of the Recommendations provides guidance on issues related to
the design and evaluation of clinical studies. Most studies are expected to be
comparative in nature, so the choice of a comparator vaccine is discussed in some
detail. Importantly, only those vaccines with adequate nonclinical testing should
be considered for clinical evaluation, with the local NRA being responsible for
evaluating adequacy of nonclinical information. Because efficacy trials appear
very difficult, the trials for assessing safety and immunogenicity are emphasized.
Clinical evaluations conducted over the past 30 years provide models for
the clinical evaluations of new vaccines. Most importantly, in the period between
1986 and 1996, several acellular pertussis-containing vaccines, including both

vaccine types (vaccines composed of purified antigens and vaccines composed
of co-purified antigens) were evaluated in a series of efficacy trials. In the first
acellular pertussis vaccine efficacy trial in Sweden (1986–1987), a PTxd and a
PTxd/FHA vaccine from the same Japanese manufacturer were evaluated (67).
The efficacy estimates for the primary case definition (culture-confirmed pertussis
with at least one day of cough) were 69% (95% CI 47–82) for the two-component
vaccine and 54% (95% CI 26–72) for the PTxd alone. Secondary analyses of this
trial revealed the critical importance of case definition, in particular the marked
influence on vaccine efficacy estimates of the laboratory and clinical criteria used
to define a case. For example, markedly different efficacy estimates for the one- and
two-component vaccines could be obtained depending on whether or not mild
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clinical cases were included (68). To address the problems with case definition,
WHO convened an expert group in 1991 to recommend case definitions that
could be used for subsequent efficacy studies (69). The recommended primary
case definition required 21 days of paroxysmal cough and laboratory confirmation
by culture, serology or household contact with a confirmed case. However,
because this primary case definition provided incomplete information, evaluation
of secondary end-points was strongly encouraged. The evaluation of efficacy
against milder illness (e.g. fewer than 21 days of paroxysmal cough) was considered
of particular importance.
Additional trials were conducted between 1991 and 1996 (11, 12, 70–79).
In these trials, DTaP vaccines containing 1–5 pertussis components were
investigated. Different study designs were employed in these trials, namely:
1) randomized placebo-controlled cohort trials, 2) household contact studies,
and 3) case–control studies. The different calculated vaccine efficacies were
affected by study design as well as case definition. The most reliable estimates of
absolute vaccine efficacies were obtained for those trials that used a double-
blind format with an unvaccinated control group (80). Blinding was not possible
in the case–control studies and in most of the household contact studies, and thus
the efficacy estimates for such trials have more potential for bias. The exceptions
were household contact studies which were nested within some randomized
controlled cohort trials.
This series of trials revealed that all the tested acellular pertussis vaccines
protected children against pertussis to at least some degree (11, 12). However,
unless the vaccines were tested in parallel within the same trial, comparing the
efficacy of the different acellular vaccines must be done with caution, as all the
trials varied with respect to design, case ascertainment methodology, and case
definition. For instance, in placebo-controlled cohort trials, culture-confirmation
was more likely to occur in unvaccinated than in vaccinated subjects, leading
to inflated vaccine efficacy estimates. This bias was overcome to a great extent
by employing appropriate serological tests to confirm the cases. Similarly, mild
cases were proportionally more frequent in vaccinees than in controls; thus
efficacy estimates were inflated when milder cases were excluded or were deflated
when they were included. Some of the randomized placebo-controlled cohort
trials investigated two different acellular pertussis vaccines and, from these,
some comparisons could be made. In two studies, an acellular pertussis vaccine
containing five components provided better protection than the specific (and
never licensed) two-component vaccine included in that trial (72, 75). However,
the vaccine composition that optimally protects against both mild and severe
disease remains uncertain. Epidemiological investigations have shown that disease
has been controlled by vaccines of varying composition (1, 10, 81, 82).
217
Several of these trials included both whole-cell and acellular pertussis

vaccines. Some tested whole-cell vaccines provided less protection than the
acellular vaccines (71, 72). However, in other trials other whole-cell vaccines
appeared to be more efficacious than most acellular vaccines, particularly against
mild pertussis (75, 77). Heterogeneity among whole-cell vaccines has been
reported since the 1950s, and emphasizes the importance of monitoring the
efficacy of any whole-cell or acellular pertussis immunization programme.
Two of the efficacy trials were designed to determine the antibody values
at the time of exposure, and thus were able to evaluate whether the presence of
specific antibodies was correlated with protection from disease (13, 14). Both
studies showed that the presence of antibodies to PRN, PT and fimbriae correlated
with protection. Neither study, however, found a correlation with antibody to
FHA. However, a role for an immune mechanism other than serum antibody
cannot be ruled out for this antigen.
Following the completion of these trials, many countries began exclusive
use of acellular pertussis vaccines. Several studies have attempted to evaluate the
duration of protection (82–84). To date, studies support a conclusion that efficacy
is retained for at least five years after a three- or four-dose series of acellular
pertussis vaccine. Further evaluations should be able to define the duration of
protection more clearly and thus provide guidance to public health officials on
the optimal time for administration of booster doses.
With respect to safety, several head-to-head studies have demonstrated
that primary immunization with DTaP vaccines caused fewer local reactions
and less fever than DTwP vaccines (11, 12). However, no clinically significant
differences in safety have been demonstrated among acellular pertussis vaccines
with differing numbers of components. Several studies evaluating booster doses
have indicated that the frequency of significant redness and swelling (e.g. redness
and/or swelling greater than five centimetres or swelling of the entire limb)
increases in those subjects who have received multiple doses of DTaP vaccines.
With respect to more serious events, the literature provides no reliable basis for a
causal relationship between vaccination and the handful of other serious adverse
effects described in case reports or national adverse event reports (12).
C.1 General considerations for clinical studies

This section addresses some issues that are specific to, or particularly
relevant to, the clinical development of new acellular pertussis vaccines. The
recommendations made should be considered in conjunction with the general
principles described in the WHO Guidelines on clinical evaluation of vaccines:
regulatory expectations (85).
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These Recommendations should be viewed in the light of further data

on the safety, immunogenicity and effectiveness or any relevant data on other
types of acellular pertussis vaccines that may become available in the future.
Manufacturers should provide justification for the choice of the vaccine
formulation (e.g. the pertussis components included in the formulation) and
the design of the clinical development programme used to evaluate the vaccine,
including the size of studies and the end-points for evaluation.
For vaccines that contain new acellular pertussis vaccine components
rather than established components – with established components defined
as the same pertussis purified antigen(s) manufactured by the same company,
using the same process, and formulated in the same way as components tested
in clinical efficacy studies – the immunogenicity data obtained in clinical studies
should be considered along with manufacturing information and nonclinical
data when assessing the likelihood that the vaccine will prove to be effective in
the clinical setting. Clinical investigations should be initiated only with vaccines
that have undergone thorough nonclinical evaluations, as described in Part B.
Consistency of manufacturing for the vaccine lots used in clinical trials should be
demonstrated and well documented. It is expected that several lots with the same
formulation intended for marketing will be used in the late stages of the clinical
development programme.
C.1.1 Scope of the studies

Placebo-controlled protective efficacy studies are no longer feasible for ethical
reasons, and trials designed to measure efficacy relative to that of a licensed
acellular pertussis vaccine with proven efficacy would need to be very large in
order to provide adequate precision in the relative efficacy estimates. Therefore,
the approval of any new acellular pertussis vaccines would be likely to rely on
data from comparative immunogenicity and safety trials of adequate size and
design to provide reasonable assurance of their clinical benefit. When applicable
and when relevant, studies documenting the performance of the investigational
vaccine when co-administered with other routinely recommended infant and
toddler vaccines should be performed.
Infants less than three months of age are at highest risk of hospitalization
and death from pertussis. There is a growing interest in approaches that could
provide improved protection for these very young infants. Such approaches,
include, for example, the development of stand-alone acellular pertussis
vaccines to be used as a birth dose and the immunization of mothers before
or during pregnancy. However, the unique considerations that apply to the
clinical evaluation of vaccines in these special populations are not included in
this document.
219
C.1.2 Comparator vaccine

The predictive relationship between the concentration of antibody induced and
protection against pertussis has not been established for each antigen. Therefore,
one of the critical aspects when designing clinical trials for licensure is the
choice of a comparator vaccine. When doing this, the points made below should
be considered.
The choice of the comparator vaccine will vary according to the
characteristics of the new acellular pertussis vaccine under development. However,
the comparator should in general be the vaccine most similar to the new vaccine
with respect to content and composition of the acellular pertussis component.
Three potential scenarios are proposed when assessing a new vaccine formulation:
■ Scenario 1 – the new acellular pertussis combination vaccine

contains an established acellular pertussis component (i.e. same
purified pertussis antigen(s) manufactured by the same company
using the same processes, and formulated the same way) that has been
found suitably efficacious in a clinical efficacy trial. In this scenario,
the most appropriate comparator vaccine would be the most similar
licensed product from same manufacturer. This scenario applies to
the evaluation of different combination vaccines based on the same
DTaP components (e.g. DTaP-IPV, DTaP-HepB, DTaP-HepB-Hib
etc.) or DTaP with different amounts of pertussis components (e.g.
booster formulations Tdap). The evaluation of a combination vaccine
in this scenario is based on a non-inferiority clinical trial of immune
responses relative to the separately administered licensed DTaP or
DTaP-based combination vaccine.
■ Scenario 2 – the new acellular pertussis-containing vaccine has
a composition which is the same as, or very similar to, that of an
established acellular pertussis component that has been found suitable

in a clinical efficacy trial. However, some or all antigens are made by a
different manufacturer and/or by a different process than the vaccine
tested in a previous protective efficacy study. In this scenario, the
most appropriate comparator would be the licensed product (with
proven efficacy) with matching composition (same acellular pertussis
antigens, similar amounts).
NOTE: Vaccines evaluated in efficacy studies include one-component
(PTxd) (67, 70), two-component (PTxd/FHA) (67, 72, 74, 75, 77),
three-component (PTxd/FHA/PRN) (71, 73, 75), four-component
(PTxd/FHA/PRN/FIM2) (78), and five-component (PTxd/FHA/PRN/
FIM2/FIM3) (72, 75) acellular pertussis vaccines.
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Annex 4
■ Scenario 3 – the new acellular pertussis-containing vaccine has an

acellular pertussis antigen composition that is not the same as that
of an already licensed acellular pertussis vaccine that has been found
suitably efficacious in a clinical efficacy trial. There are at least two
ways this could occur, namely: 1) the vaccine could be based on
the currently used antigens but present in different combinations
such as PTxd/PRN or PTxd/PRN/FIM2/FIM3, or 2) the vaccine
could include novel antigens in combination with one or more of
the currently used antigens. In this scenario, the most appropriate
comparator would be the licensed product (with proven efficacy) with
the most similar composition.
It is important to highlight that, for scenarios 2 and 3, manufacturers
should justify the choice of comparator vaccine and the non-inferiority margin
used, particularly when there are differences in the content and composition of
the acellular pertussis components.
C.2 Assessment of immune responses

C.2.1 Assays to assess antibody responses
Serological assays used in clinical immunogenicity studies in support of vaccine
licensure require validation (85). An international reference pertussis antiserum
has been established to assist in the standardization of serological methods (86).
Thus, to ensure the comparability and acceptability of the serological data across
trials, results of immunogenicity should be expressed in IU in reference to this
International Standard for pertussis antiserum. A rigorous assessment of assay
specificity is essential prior to initiating validation studies. Formal validation
should assess all appropriate performance criteria – including accuracy, linearity,
precision and range – and robustness studies are also recommended. The
validation studies should be designed to demonstrate that the range (including
the lower limit of quantitation) is suitable for the clinical study, and should
consider the way in which the vaccines are to be compared to each other
(e.g. whether the criteria for evaluation are based on percentages with post-
primary series titres above a threshold, seroconversion rates or geometric mean
antibody titres).
The immune response in clinical trials should be assessed by using a small
range of validated assays. Selection of the assays for evaluation of the immune
response to the vaccine should be justified by the vaccine developer. When
feasible, assays that measure functional immune responses should be employed.
Suitable assays are unlikely to be commercially available.
Specific antibody responses to different components of the vaccine (e.g.
PT, PRN, FIM etc.) can be assessed by methods that measure the concentration
221
of antibody binding to a specific antigen (e.g. ELISA) or, when applicable, the
functional biological activity by measuring the PT neutralizing titre (e.g. CHO
cell assay) (87) or the B. pertussis agglutination titre (88–90).
Cell-mediated immune (CMI) responses play a role in protecting against
B. pertussis infection. However, immunological assays to evaluate CMI responses
following immunization have not been standardized and have not been used to
support licensure. Nevertheless, the exploratory assessment of CMI should be
encouraged in order to enlarge the body of knowledge regarding all aspects of the
immune response to pertussis antigens.
C.2.1.1 ELISA to assess antibody responses to acellular pertussis components

The assessment of antibody responses to specific pertussis components included
in the vaccine should be regarded as the primary means of assessing the immune
response to new acellular pertussis vaccines. The standardization of serological
methods for B. pertussis has been pursued not only for the purpose of licensure
of new pertussis vaccines but also for clinical diagnosis of pertussis infection
and for seroepidemiological studies (91). However, it is important to note that
assays developed and optimized for diagnostic and epidemiological purposes,
including most commercially available ELISA kits, are unlikely to have the
performance characteristics needed for vaccine immunogenicity studies. For
instance, diagnostic kits are unlikely to provide the specificity required to
assess each of the pertussis components individually (e.g. PT, FHA, etc.) and the
accuracy to determine geometric mean concentration (GMC) (92).
Collection, recording, analysis and interpretation of data should be
conducted according to good clinical practice guidelines (93). Methodological
and statistical considerations described in WHO guidelines should be taken into
account (85).
C.2.1.2 Assessment of functional antibody titres

Functional activities of antibodies against pertussis components have been

identified as important additional parameters to consider, particularly when
evaluating new formulations containing PTxd and FIM which are known
to induce antibodies with functional activity such as toxin neutralization and
bacterial agglutination respectively (89). Assays to measure PT neutralizing and
whole-cell B. pertussis agglutinating antibodies have been established (87, 88, 90).
Although no functional thresholds have been found to correlate directly with
the protective efficacy of pertussis vaccines, there are nevertheless important
immune parameters to determine as part of the overall comparison of new
vaccine formulations to those proven to be safe and effective.
When feasible, functional antibodies should be measured, at a minimum,
in a subpopulation of the comparator and test vaccine groups.
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C.2.2 Criteria for evaluation of immune responses

The preferred method for evaluating new vaccine formulations is the direct
clinical comparison of licensed vaccines with proven pre-licensure clinical efficacy,
with the new product through randomized controlled trials.
C.2.2.1 Primary immunization of infants and young children

In comparative studies of post-primary immune responses, the main analysis will
be based on demonstrating that the response in subjects immunized with the test
vaccine is not inferior to that in the comparative vaccine group(s). The selection
of the primary parameter for the assessment of non-inferiority, the predefined
margin of non-inferiority and hence the total sample size for a comparative
study, will need careful justification (85). Although studies that compare immune
responses between candidate and licensed acellular pertussis vaccines are essential,
comparisons with historical data that were generated during previous protective
efficacy studies using similar assays may be used to provide supportive evidence.
While a demonstration that the new candidate vaccine is immunogenic in humans
is important, the data should be interpreted with some caution. In particular,
when evaluating immunogenicity data comparing vaccines produced by different
manufacturers or produced using different methods, equivalent efficacy cannot
be directly inferred from equivalent immunogenicity.
The study objectives must be taken into account when defining appropriate
time intervals for assessing the immune response. In most cases, clinical studies
for new vaccines for infants are designed to determine the antibody response to
acellular pertussis components at approximately four weeks following the final
dose. Predefined non-inferiority criteria using an appropriate acceptability limit
are used to compare the responses in subjects immunized with the test vaccine
versus the licensed comparator vaccine (see section C.1.2) using the end-points
described below.
The following co-primary analyses are recommended:
■ Percentage of responders – in one primary analysis, the percentage

of responders with a significant increase (e.g. fourfold increase) above
pre-immunization for each of the acellular pertussis components is
compared between subjects immunized with test vaccine and the
licensed comparator vaccine. Alternative definitions for responders
could be considered if well justified. The groups should be compared
using an appropriate predefined non-inferiority limit; generally
the lower bound of the two-sided 95% confidence interval of the
observed difference should not be less than the criterion approved
by the NRA, most commonly 10 percentage points.
223
■ Magnitude of the response – in a second primary analysis, the

magnitude of the response is compared – on the basis of the GMC
induced by the new vaccine and the licensed comparator using a
predefined margin of non-inferiority – to evaluate the antibody
response (GMC ratios) to each acellular pertussis component.
The lower bound of the two-sided 95% confidence interval of the
observed ratio of the GMC of the new vaccine relative to the control
should not be less than the criterion approved by the NRA (most
commonly 0.50 or 0.67).
In case of failure to meet the predefined non-inferiority criteria, detailed
investigation of the immune response and the reasons for not meeting the criteria
may be considered. In particular, the NRA may take into consideration the results
from the antibody responses to each of the antigens, as well as any differences
in composition between the test and the comparator vaccines, and the available
information about the contribution of that antigen (i.e., the antigen to which the
antibodies are directed) in protection.
C.2.2.1.1 Secondary analysis

Functional antibody response. When evaluating new vaccine formulations, it is
important to assess as many immune parameters as possible. Therefore, functional
antibody responses (i.e. PT neutralizing titres or B. pertussis agglutination titre if
PTxd or FIM are part of the formulation, respectively) should be determined in a
randomized subset of vaccinated subjects within some or all of the clinical studies.
At present, the interpretation of functional antibody data is made difficult by the
fact that a titre that might correlate with protection against pertussis infection
is unknown. For this reason it is recommended that comparisons of functional
antibody titres between the new vaccine and the licensed comparator should
focus on GMT ratios.
C.2.2.1.2 Additional information

Reverse cumulative distribution (RCD) curves (94). Use of RCD curves which
display the accumulated proportion of individuals with an antibody concentration
greater than or equal to a given level have been shown to be useful when
comparing the response to the test and licensed comparator vaccines. RCD plots
should be generated for ELISA data and for functional antibody response. The
review of these data should be viewed as exploratory.
It is recommended that subsets of subjects are identified for longer-term
follow-up of persistence of immunity. These data may be provided after first
approval. Waning of antibody concentrations over time is inevitable and should
not be interpreted per se to indicate the need for a booster dose. It is important
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Annex 4
that longer-term antibody concentrations are viewed in conjunction with

observed effectiveness data in order to assess the potential need for additional
doses later in life to maintain protection.
C.2.2.2 Booster immunization of older children, adolescents and adults

In most instances, the emphasis for initial use and evaluation will be for the
primary immunization of infants. However, acellular pertussis vaccines may
also be used for booster doses in the second year of life, and in preschool
children, adolescents and adults. Currently, different immunization schedules
are employed in different countries for primary and booster immunization. In all
cases, the chosen schedule should be supported by appropriate immunogenicity
studies. Previous experience has demonstrated that vaccines intended solely
for booster immunization may require lower amounts of one or more of the
pertussis antigens.
An active comparator vaccine may not be needed when evaluating the
immunogenicity of the acellular pertussis components used as a booster vaccine,
such as in older children and adults. In such cases, it may be possible to compare
the immune response of adolescents and adults following a single dose to that
of infants who received primary immunization with the corresponding DTaP
vaccine (historical comparator). In addition, the ability of these vaccines to
induce immunological memory assessed by an anamnestic response following
immunization should be evaluated for each acellular component.
C.2.3 Combined vaccines and concomitant administration with other vaccines

C.2.3.1 Combined vaccines
In the case of combination of acellular pertussis components with other antigens,
potential interference between the pertussis components and the other antigens
and/or excipients should be investigated, as described in the WHO Guidelines on
clinical evaluation of vaccines: regulatory expectations (85).
In particular, it has been demonstrated that some acellular pertussis
components can have a negative impact on immune responses to some
polysaccharide conjugated antigens when administered in pre-formulated
products or when vaccines are mixed only immediately before injection (e.g.
Haemophilus influenzae type b conjugate vaccine responses in some combination
products containing PRP-T and some acellular pertussis components). Therefore,
the immune responses to all the antigens in the final combined formulation should
be shown to be satisfactory through well-designed randomized comparative trials.
If there is any immune interference observed with respect to any of the combined
antigens, the possible clinical implications should be carefully considered before
proceeding with clinical development.
225
C.2.3.2 Concomitant administration with other vaccines

In recent years, it has become apparent that concomitant administration of
acellular pertussis components with other vaccines in routine use, including
conjugated vaccines, may give rise in some situations to detectable immune
interference, although the clinical significance of the observed phenomena is not
always clear. Examples include decreased antibody responses to Haemophilus
influenzae type b conjugate vaccine and to meningococcal C monovalent vaccine.
Thus it is important that immune responses to candidate acellular pertussis
vaccines should be evaluated on co-administration with other vaccines that are
representative of types that, for convenience and compliance reasons, are very
likely to be given at the same clinic visits. Responses to other co-administered
antigens should also be evaluated. The approach to these studies is based primarily
on demonstrating non-inferiority of responses to antigens when vaccines are
co-administered, compared to each vaccine given alone, with careful justification
of predefined non-inferiority margins.
These studies might compare concomitant administration with
administrations made in a staggered fashion (e.g. together at two, four and six
months compared to the usual antigens at this schedule and the new vaccine at
three, five and seven months).
C.3 Safety evaluation

As stated in section C.1.1, placebo-controlled efficacy studies which would also
deliver a large safety database are not feasible. Nevertheless, the pre-licensure
assessment of vaccine safety is a critically important part of the clinical programme,
and should be developed to meet the general principles described in the WHO
Guidelines on clinical evaluation of vaccines: regulatory expectations (85). A
comparison of rates of adverse events between test and approved comparator
vaccines is commonly a predefined secondary end-point in study protocols. The
minimum acceptable size of the safety database at the time of approval should
take into account the vaccine composition (including all antigens and adjuvants),
the presence of novel antigens, and any past experience with vaccines with the
same or similar composition of the acellular pertussis component.
For new vaccines, a total safety database (combined from all trials in
the same targeted age group) of approximately 3000–5000 subjects is
commonly expected because this allows for the evaluation of uncommon
adverse events – i.e. those that occur at a rate between 1 in 100 and 1
in 1000 subjects (85). However, depending on the data available for the
vaccine, the NRA may accept a smaller number or may request a larger
database prior to first approval.
Information on adverse events such as extensive limb swelling syndrome

should be carefully monitored in studies evaluating the safety of booster doses.
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Annex 4
C.4 Post‑marketing studies and surveillance

Every effort should be made to improve current scientific understanding of the
protection in humans afforded by acellular pertussis vaccines by providing data
from active post-marketing surveillance. Vaccine effectiveness in the population
should be reported wherever possible. In addition, given that limited safety data
are obtained in pre-licensure studies, all relevant safety-indicating parameters
should be monitored as part of post-marketing surveillance programmes. In
particular, the impact of routine vaccination on pertussis infection needs to be
assessed in comprehensive studies of vaccine performance. Ongoing surveillance
programmes should be in place to monitor for longer-term protection and for
evidence of any changes in vaccine effectiveness.
In reality, sound and comprehensive safety and effectiveness data cannot
be collected by the manufacturers alone. Therefore, there should be discussions
between vaccine manufacturers responsible for placing the product on the market
and national and international public health bodies regarding the feasibility
of estimating effectiveness and safety in the post-marketing period. Reliable
estimates of effectiveness can be obtained only in geographical locations where
appropriate vaccine campaigns are initiated and where there is already a suitable
infrastructure in place to identify cases of pertussis disease.
General WHO guidelines for continued oversight of vaccines after
licensure should be followed (85). All data collected should be submitted to the
responsible NRAs at regular intervals so that any implications for the marketing
authorization can be assessed.
Part D. Guidelines for NRAs

D.1 General
The general recommendations for control laboratories contained in the Guidelines
for national authorities on quality assurance for biological products (95) and
Guidelines for independent lot release of vaccines by regulatory authorities (96)
should apply.
The detailed production and control procedures and any significant
changes in them should be discussed with and approved by the NRA.
Consistency of production has been recognized as an essential component
in the quality assurance of acellular pertussis vaccines. In particular, the NRA
should carefully monitor production records and quality control test results for
clinical lots as well as a series of consecutive lots of the final bulk.
D.2 Official release and certification by the NRA

A vaccine should be released only if it fulfills national requirements and/or
satisfies Part A of these Recommendations.
227
A statement signed by the appropriate official of the NRA should be

provided at the request of the manufacturing establishment and should certify
that the lot of vaccine in question satisfies all national requirements as well as
Part A of the present Recommendations. The certificate should state the number
under which the lot was released by the NRA, and the number appearing on
the labels of the containers. The official national release document should be
provided to importers of pertussis vaccines.
The purpose of the certificate is to facilitate exchange of pertussis vaccines
between countries. A model of a suitable certificate is given in Appendix 7.
Authors
The first draft of these Recommendations was prepared by: Dr M. Baca-Estrada,
World Health Organization, Switzerland; Dr M. Corbel, United Kingdom;
Dr R. Gaines-Das, Consultant in Biostatistics, United Kingdom; Dr Y. Horiuchi,
Pharmaceuticals and Medical Devices Agency, Japan; Dr I. Knezevic, World
Health Organization, Switzerland; Dr D. Lei, World Health Organization,
Switzerland; Dr B. Meade, Meade Biologics, LLC, USA; Dr P. Olin, Swedish
Institute for Infectious Disease Control, Sweden; Dr M. Powell, Medicines and
Healthcare Products Regulatory Agency, England; Dr D. Xing, National Institute
for Biological Standards and Control, England; and Dr S.-M. Zhang, National
Institute for the Control of Pharmaceutical and Biological Products, China.
The second draft was prepared by Dr D. Lei, World Health Organization,
Switzerland; Dr B. Meade, Meade Biologics, LLC, USA; and Dr D. Xing, National
Institute for Biological Standards and Control, England, following a WHO
informal consultation held in Geneva, Switzerland in November 2009.
Acknowledgements
Acknowledgements are due to the following experts for their comments and
advice on the revision of these Recommendations during the WHO informal
consultation held in Geneva on 9–10 November 2009 and thereafter: Dr J.L.
Arciniega, United States Food and Drug Administration Center for Biologics
Evaluation and Research, USA; Dr M. Baca-Estrada, World Health Organization,
Switzerland; Dr D.L. Burns, United States Food and Drug Administration Center
for Biologics Evaluation and Research, USA; Dr C. Conrad, Paul-Ehrlich-Institute,
Germany; Dr N. Dellepiane, World Health Organization, Switzerland; Dr R.
Dobbelear, Belgium; Dr R. Dominguez Morales, Centro para el Control Estatal
de la Calidad de los Medicamentos, Cuba; Ms D. Felnerova, Crucell, Switzerland;
Dr M. Ferguson, Horning, England; Dr R. Gaines-Das, Consultant in Biostatistics,
228
Annex 4
United Kingdom; Dr R. Gibert, Agence Française de Sécurité Sanitaire des

Produits de Santé, France; Dr Y. Horiuchi, Pharmaceuticals and Medical Devices
Agency, Japan; Dr P. Hubrechts, Staten Serum Institut, Pharmaceuticals and
Medical Devices Agency, Denmark; Dr R. Isbrucker, Health Canada, Canada;
Dr M. Iwaki, National Institute of Infectious Diseases, Japan; Dr J-M. Jacquet,
GlaxoSmithKline Biologicals, Belgium; Mrs T. Jivapaisarnpong, Department of
Medical Sciences, Ministry of Public Health, Thailand; Dr I. Knezevic, World
Health Organization, Switzerland; Dr D. Kusmiaty, National Quality Control
Laboratory of Drug and Food, National Agency of Drug and Food Control,
Indonesia; Dr H. Langar, WHO Regional Office for the Eastern Mediterranean,
Egypt; Dr H. Lechner, Paul-Ehrlich-Institute, Germany; Dr D. Lei, World Health
Organization, Switzerland; Dr A. Maes, Scientific Institute of Public Health,
Belgium; Dr F. Mawas-Kossaibati, National Institute for Biological Standards
and Control, England; Dr B. Meade, Meade Biologics, LLC, USA; Dr P.V.V.S.
Murthy, Biological E Limited, India; Mr L. Nencioni, Crucell, Switzerland; Dr S.
Nishioka, World Health Organization, Switzerland; Dr P. Olin, Swedish Institute
for Infectious Disease Control, Sweden; Dr S.-R. Pakzad, Vaccine Potency and
Standardization Section, Food and Drug Control Laboratory, Islamic Republic
of Iran; Ms I. Pierard, GlaxoSmithKline Biologicals, Belgium; Dr M. Powell,
Medicines and Healthcare Products Regulatory Agency, England; Dr S. Prieur,
Agence Française de Sécurite Sanitaire de Produits de Santé, France; Dr C.
Rodriguez, World Health Organization, Switzerland; Dr H.C. Song, Korea Food
& Drug Administration, Republic of Korea; Dr V.K. Srinivas, Bharat Biotech
International Ltd, India; Dr A.L. Sterling, Centro para el Control Estatal de la
Calidad de los Medicamentos, Cuba; Dr P. Stickings, National Institute for
Biological Standards and Control, England; Dr S. Uhlrich, Sanofi Pasteur, France;
Mr O. Van Opstal, GlaxoSmithKline Biologicals, Belgium; Dr E. Vidor, Sanofi
Pasteur, France; Dr S. Wahyuningsih, National Quality Control Laboratory of
Drug and Food, Indonesia; Dr D. Wilkinson, National Institute for Biological
Standards and Control, England; Dr D. Wood, World Health Organization,
Switzerland; Dr D. Xing, National Institute for Biological Standards and Control,
England; Professor X. Yang, Wuhan Institute of Biological Products, China;
and Dr S.-M. Zhang, National Institute for the Control of Pharmaceutical and
Biological Products, China.
Thanks are also due to the following for their critical review and
comments on the revised Recommendations: Dr D. Burns, United States Food
and Drug Administration Center for Biologics Evaluation and Research, USA;
Dr J. Cowell, USA; Dr R. Dobbelaer, Belgium; Dr K. Farizo, United States Food
and Drug Administration Center for Biologics Evaluation and Research, USA;
Dr E. Griffiths, Health Canada, Canada; Dr N. Guiso, Institut Pasteur, France;
and Dr H. Kreeftenberg, the Netherlands.
229
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235
Appendix 1
Modified intracerebral challenge assay
1. Materials
Strain 18323 of Bordetella pertussis (hereafter referred to as the challenge strain
in this appendix) should be used for challenge. The diluent for the test sample
and standard should be sterile physiological saline (0.85% NaCl).
The challenge strain should be cultured on Bordet–Gengou medium
for approximately 24 hours and suspended in 1% w/v casamino acid solution
containing 0.6% w/v of sodium chloride (pH 7.0–7.2) to a concentration of
approximately 200 LD50 per 0.025 ml (or approximately 1×105 organism/challenge
dose) to serve as the suspension for challenge. Alternatively, a stable frozen stock
can be used for direct challenge after appropriate dilution.
2. Test procedures
The test sample and standard should be diluted serially to make at least three
levels of fourfold or other suitable logarithmic dilutions. Each dilution should
be given by intraperitoneal injection at a dose of 0.5 ml to at least 16 mice aged
approximately four weeks. The animals should be of the same sex or both sexes
in equal numbers for each dose. The challenge suspension should be given
by intracerebral injection into the animals at a dose of 0.025 ml 21 days after
immunization. The animals should be observed for 14 days. Any animals dying
within three days after challenge should be excluded from the test. Any animals
showing paralysis or swelling of the head at the end of the observation period
should be counted as deaths.
At least three appropriate serial dilutions of the challenge suspension
should be injected into a group of at least 10 mice to titrate the virulence. The
bacterial count for the LD50 per 0.025 ml of the challenge suspension should be
no more than 300 CFU.
3. Criterion for judgement

Assay data are analysed using parallel line analysis following probit transformation
of the proportion of mice responding. The dose–response curves of the test
and reference vaccines are checked for significant deviations from linearity
and parallelism. If there is a significant (p < 0.05) regression of probit response
on log dose and there are no significant deviations (p > 0.05) from linearity
or parallelism, the potency and its 95% limits are calculated. The First WHO
International Standard for acellular pertussis vaccine (JNIH-3) has been
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Annex 4
established and an in-house reference used in this test should be calibrated in

terms of the International Standard. The specifications should be established with
the agreement of the NRA. Where used currently, the potency of the test sample
is considered to be passed if it is no less than 8 IU/ml (4 IU/human dose) upon
statistical analysis.
4. Retest
If a test vaccine did not pass in the first test, the test should be repeated using the
same number or an increased number of mice. Results for all statistically valid
assays should be combined. Weighted mean log potency should be calculated for
homogeneous results using log potencies obtained in repeated tests using inverse
of variance estimate for each log potency value as weight.
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Appendix 2
Histamine sensitization test by temperature measurement
Groups of mice (with no fewer than 10 mice each) defined with respect to strain,
sex and age, should be randomly allocated to the different treatments. Samples
should include the test sample(s) and a Reference Preparation. If reversion to
toxicity testing is required by the NRA the test also should include a sample of
the test vaccine incubated at 37 °C for four weeks. All mice should be challenged
post-sensitization with histamine dihydrochloride. The reference and histamine
dihydrochloride should be diluted with physiological saline. If PT is used as
reference, physiological saline or phosphate buffered saline, both containing
0.2% (w/v) gelatin, should be used as diluent to prevent possible loss of PT activity
by adsorption to the container. Appropriate thermometers with recommended
precision of 0.1 °C and capable of measuring temperatures between 25 °C and
40 °C should be used for the test.
The Reference Preparation should be diluted to give a suitable dose-
response. The test sample (usually a single human dose) and each dilution of the
Reference Preparation in a volume of 0.5 ml should be given by intraperitoneal
injection to each group of mice. Four days after injection, 4 mg of histamine
dihydrochloride should be intraperitoneally injected into each mouse. The rectal
or dermal temperature should be measured 30 minutes after histamine injection
for all mice. Temperature should be recorded for all mice, including those that die
within the 30-minute observation period.
Temperature responses are analysed using a suitable statistical method to
give an estimate of the residual activity of PT in the test vaccine, in relation to the
Reference Preparation.
The test lot passes the test if the estimated residual activity of PT in the
test group is not higher than the value specified by agreement with the NRA.
There is currently no internationally agreed upper limit for active PT in acellular
pertussis vaccines. In some countries an upper limit of 1.09 or 2.18 IU (0.2 or
0.4 HSU) of PT per single vaccine dose is a requirement for DTaP vaccines.
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Appendix 3
Histamine sensitization test by lethal end-point assay
Groups of mice, each of an appropriate number defined with respect to strain,
sex and age, should be randomly allocated to the different treatments. For assays
performed for validation purposes and initially after vaccine licensure, the
positive control set consists of groups that should be injected intraperitoneally
with three or more serial dilutions of a Reference Preparation of PT of suitable
specific activity (IU/ng). The dilution factor should be chosen so as to obtain a
graded response; however, it should be no greater than five. An additional group
of mice (the negative control group) should be injected intraperitoneally with
diluent. One group should be injected intraperitoneally with the test vaccine and,
if reversion to toxicity testing is required, another group should receive the test
sample incubated at 37 °C for four weeks. A single human dose (some methods
allow up to two single human doses) of the final bulk is used as the test dose for
both groups. The position of the cages on the shelves during the testing period
should be allocated at random in order to reduce the influence of positional
effects on the assay outcome. All mice should be challenged by injection with a
defined dose of histamine (1 or 2 mg of histamine base is most commonly used)
at four or five days after sensitization or injection with diluent. The histamine
challenge dose may be administered intraperitoneally or intravenously; however,
the injection route should be defined and the same route should be used for all
testing within the laboratory. Histamine challenge should follow the place order
of the cages on the shelves. Deaths within 24 hours of histamine injection should
be recorded.
The assay sensitivity and other validity criteria should be defined in
agreement with the NRA. For the assay to be considered valid, mice injected with
diluent (negative control) must not show, in general, sensitization to the lethal
effect of histamine. However, experience has shown that, with low frequency, a
small percentage of mice (i.e. less than 5%) in the negative control group may
die following histamine challenge. Thus some laboratories consider a test valid
if there is no more than one death in a negative control group of 20 or more.
Each test should also meet the criteria set to demonstrate its sensitivity. Several
strains of mouse (all with Swiss-Webster ancestry) are highly responsive to
histamine sensitization but a number of strains, both inbred and outbred, are
weakly responsive. The susceptibility to sensitization of the strain chosen for the
test should be defined during assay validation studies and approved by the NRA.
Adequate susceptibility of mice used in a HIST should be verified by demonstrating
that the sensitizing dose of the PT control meets criteria established during assay
239
validation. Once linearity has been established by repeated experiments using

multiple doses of a control PT, a suitable dose of the reference toxin, chosen
in the linear region of the dose–response curve and giving a positive response
considered appropriate by the NRA, should subsequently be included in each
assay as the positive control group to demonstrate assay sensitivity.
In some laboratories, the test also includes a reference group of mice
injected with PT at a dose previously set as the allowable upper limit of PT in the
product or with a reference vaccine with established clinical safety.
A lot passes the test if the proportion of animals that die following
sensitization with the test dose of vaccine and the subsequent histamine challenge
do not surpass the maximum proportion approved by the NRA. This proportion
should be related to the performance in the test of lots shown to be safe and
effective in clinical trials or those used in support of licensure. When a reference
group is included, the lot passes the test if the percentage of deaths in the test
group is not greater than that in the reference group.
If a vaccine lot fails in a single test, it should pass two additional,
consecutive and independent assays to be considered suitable for release.
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Annex 4
Appendix 4
Mouse immunogenicity test
The mouse immunogenicity test (MIT) for an acellular pertussis vaccine is an
assay designed to demonstrate consistency between vaccine lots on the basis of
the induction of antibody in mice by each antigen in the vaccine. This test is
product-specific and a suitable product-specific reference (or control) vaccine is
required for a meaningful assay. Immunogenicity can be measured either in terms
of the amount of antibody produced in mice injected with a defined test dose, or
as the dose of antigen that induces a defined measurable antibody response in a
certain proportion of mice (e.g. the median effective immunizing dose, ED50).
For each antibody, the linear-response region of the dose–response curve
(vaccine dose versus antibody production) should be determined. In the first
method, a group of mice is injected once with a preselected dose of vaccine that is
within the linear-response region. For preparations containing multiple pertussis
antigens, more than one test dose of vaccine, and therefore more than one group
of mice per lot, may be required because of the differential immunogenicity of
these antigens in mice. In the second method, groups of mice are injected with a
suitable range of dilutions of vaccine, and the proportion of responding animals
is determined at each dose. After consistency in manufacturing and testing has
been demonstrated to the satisfaction of the NRA, the serial-dose method may be
simplified to an appropriate single-dose (e.g. ED50 for the antigen) assay.
Regardless of test design, the antibody content of test sera is calculated
relative to a stabilized reference serum by means of a validated ELISA.
For all antigens, reproducibility of the antibody response in the chosen
strain of mice should be verified in every test by the inclusion of group(s) of
mice injected with homologous reference (or control) vaccine. The reference (or
control) vaccine ensures that the test mice respond in a way that is consistent
with previous testing. The stability of the reference (or control) vaccine should
be monitored. Appropriate stabilization of the reference (or control) vaccine,
preferably by lyophilization, is recommended. An example of conditions for
lyophilization that have been successfully used are as follows: 3.5% polygelin
(1:1) under freeze-drying cycle at –50 °C load, –50 °C freeze over 2.5 hours,
then primary drying at 35 °C (100 µbar vacuum) and secondary drying at 30 °C
(30 µbar vacuum). The reference vaccine does not need to be a clinical lot because
acceptance criteria are values reflecting the behaviour in the test of clinical
lots or those lots used in support of licensure, either in absolute terms or in
terms relative to the reference vaccine. However, the reference vaccine should
be sufficiently similar to the clinical lots in composition and manufacture to
241
serve as an adequate control in the test. The response of the test vaccine may be
reported either in absolute terms or in terms relative to the reference vaccine.
Calibration of replacement reference vaccines for the MIT should make use of
sound statistical principles to prevent drift in the efficacy of acellular pertussis
vaccines in the market.
The specifications for evaluating vaccines containing acellular pertussis
are product-specific and are based on an appropriate statistical analysis of the
responses observed in the MIT test for clinical vaccine or other lots used in
support of licensure. Specifications must be carefully justified and defined with the
agreement of the NRA. Specifications should be defined for each antigen claimed
to contribute to vaccine efficacy. Specifications based on a simple failure to reject
the null hypothesis of equivalency of immunogenicity between a reference lot
and a manufacturing lot, or between two consecutively manufactured lots, are
not recommended.
Two components of the test require careful attention:
Mouse: strains of mouse (if necessary more than one) should be selected so that
a sufficient antibody response is obtained for each antigen. The optimal age for
mouse immunization (e.g. more than five weeks of age), the optimal time for
bleeding (e.g. 4–6 weeks after immunization), and the isotype of the antibody
response should be thoroughly studied. The test design should be agreed with
the NRA.
Antibody detection system: the ELISA used for the detection of antibodies
should be subjected to thorough validation and standardization studies.
These studies should include determination of the biochemical integrity and
immunological purity of antigens used for coating assay plates and determination
of the optimal antigen-coating concentration. For this purpose, the production
and standardization of a working-reference mouse serum is of utmost importance.
Calibration of the working-reference mouse serum in terms of the international
reference serum (97/642) may provide a suitable control and facilitate inter-
laboratory comparisons. Studies for reference serum standardization should

include an evaluation of the parallelism of the titration curves of reference and
test sera. Another component of the antibody detection system requiring careful
study is the anti-mouse-immunoglobulin-enzyme conjugate. This reagent should
be characterized in terms of isotype specificity and subclass reactivity, and a
suitable working dilution should be determined.
The reproducibility (intra-assay and inter-assay) of the assay for sera
containing different amounts of antibody and the limits of detection and
quantitation (LOD and LOQ, respectively) should be studied.
The development of criteria for acceptance of a vaccine lot subjected to
the immunogenicity test should take into account the following ELISA validity
criteria:
242
Annex 4
■ The average absorbance value for normal mouse serum should

be below a historically defined upper limit. Normal mouse serum
should be obtained from mice injected with diluent and housed
with vaccinated mice for the duration of the immunization period.
The absorbance of normal mouse serum should be measured in the
same ELISA as the sera of immunized mice.
■ The parameters of the curve relating absorbance to dilution for the
reference serum should be within historically defined upper and
lower limits.
■ A control serum with characteristics similar to the test sera and stored
in a separate location from the reference serum should be included
in every ELISA plate. The ratio of the ELISA units calculated for the
control serum to those for the reference serum should be within
historically defined upper and lower limits.
If the ELISA meets these validity criteria, the antibody values should be
calculated for mice immunized with the reference (or control) vaccine and the
test vaccine. Sera with ELISA unit values below the LOQ should be qualified
as belonging to non-responder mice. For the purpose of calculating geometric
mean antibody level, an arbitrary value (e.g. 1/2 LOQ) may be assigned to such
sera. Alternatively, the geometric mean antibody level could be calculated using
only those values above the LOQ, provided a limit is in place for the minimal
acceptable number of values to use for the calculation. If immunogenicity is
being expressed in terms of dose of vaccine, then the number of mice responding
to each antigen is used to calculate the ED50. If the ELISA validity criteria are not
met, the ELISA should be repeated.
After either a geometric mean or ED50 has been calculated for the reference
(or control) vaccine, the value should be compared with the criteria for sufficient
antibody response that were established when the assay was validated. If these
validity criteria are met, the results for the test vaccine should be evaluated as
described below. If the validity criteria for the reference vaccine are not met, the
ELISA should be repeated on all sera (from mice inoculated with both reference
and test vaccine). If the criteria are not met after a second ELISA, immunization
should be repeated.
To pass the immunogenicity test, the geometric mean antibody levels or
ED50 for mice immunized with test vaccine should meet the criteria that were
established when the assay was validated. Alternatively, immunogenicity of the
test vaccine can be expressed relative to the immunogenicity of the reference
vaccine. Acceptance criteria should be determined by performing multiple tests
on several lots (preferably three or more) that have shown acceptable performance
(i.e. efficacy, immunogenicity, or both) in clinical studies. If geometric mean
antibody levels, in absolute terms or relative to the geometric mean antibody
243
levels induced by the reference vaccine, are below the established limit, or if
the ED50 or ED50 ratio fails to meet the established limit, immunizations and
ELISAs should be repeated for those antigens that fail the test. After a second
test (if valid), the geometric mean antibody levels, geometric mean ratio, ED50
or ED50 ratio should be calculated, and results of the two tests may be combined
by appropriate statistical methods. The acceptance criteria to consider when
two tests are performed should be statistically adjusted. If the results of single
or double tests for all antigens in the vaccine satisfy their corresponding limits,
the vaccine passes the immunogenicity test. If any antigen does not satisfy its
adjusted limit after two assays, the vaccine fails the test.
The test – including the specifications, the method used to calculate
antibody response, and the treatment of non-responder mice in the calculation
of vaccine potency – should be approved by the NRA.
244
Annex 4
Appendix 5
Method for respiratory challenge
The respiratory challenge model is designed to demonstrate the protective effect
of immunizing mice with acellular pertussis vaccines or candidate antigens.
However, it is important to note that the activity of a vaccine or antigen in this
model is not an index of clinical efficacy. In general, it involves immunizing
mice, which have the capacity to give an adequate immune response to pertussis
vaccine, with pertussis vaccine at appropriate doses. Mice are then challenged
with live B. pertussis suspension. Two challenge routes/methods have been
reported, namely intranasal or aerosol administration of challenge. The response
to challenge is measured by dissecting out mouse lungs after a suitable time
and determining the number of bacterial colony-forming units (CFU). The
mouse protective effect of a test vaccine can be determined by comparison
of its responses with the responses of a vaccine of known clinical efficacy or
an appropriate Reference Preparation. The aerosol challenge method requires
specialized aerosol equipment and this may not be readily available in most
laboratories. The intranasal challenge method using a harmonized protocol
has been proved to be transferable between laboratories in international
collaborative studies. However, the current assay is not designed as a routine test
for determining vaccine potency. Nevertheless, by comparing with a reference
vaccine included in the assay, the respiratory challenge method may be useful
to assess the potential impact of changing the manufacturing process and/or
formulation; to evaluate new formulations; to investigate potential interactions
in combinations; to monitor stability of product and to assess lot consistency.
A number of designs are possible with variation in age of the mice, sampling
times, and so on. A brief outline of the procedure for a harmonized intranasal/
challenge method is given below.
Mice
Balb/c mice, three weeks old; 15 mice are to be ordered per vaccine group
(i.e. five mice at each time of sampling. Sampling time can be at two hours,
five days and eight days post-challenge; or alternatively on other days after the
validation study).
Immunization
First immunization:
Prepare vaccine doses: one vaccine dose = ¼ of a human dose (e.g.125 µl) per
mouse and per immunization. The mice are immunized using a 1 ml syringe.
245
The syringe is divided into 125 µl sections with a marker. The vaccine is injected
subcutaneously. When the vaccination is correct, a liquid-filled blister should be
visible under the skin.
The second immunization is carried out two weeks after the first
immunization, and as described above.
The challenge
All animals are challenged two weeks after the second immunization.
Preparation of challenge suspension:

The bacterial suspension of B. pertussis 18323 used for challenge is prepared
from an 18–24 hrs culture grown on Bordet–Gengou medium (alternatively,
charcoal agar plates containing blood may also be used). Colonies are picked
and resuspended in fresh Stainer–Scholte medium or in 1% casamino acids
solution. The opacity of the bacterial suspension is adjusted to OD650nm = 1,
which corresponds to 3×10 9 CFU/ml (this may vary according to the individual
laboratory) and further dilution is carried out to obtain a suspension containing
10 8 CFU/ml. Fifty µl of this suspension is used for infection of each mouse.
One aliquot of this suspension is serially diluted to 10 -4, 10 -5 and 10 -6,
and 100 µl of the latter two dilutions are plated on Bordet–Gengou plates, in
duplicate, in order to enumerate the CFU/ml content of the bacterial suspension
used to infect the mice.
Intranasal challenge:
The mice immunized by each vaccine under test should survive until challenge,
and each mouse should appear healthy prior to challenge.
All mice are anaesthetized before the challenge. A total of 50 µl of the
bacterial suspension is delivered in the nostril, or 25 µl into each nostril, with
an automatic 50 µl pipette (in some laboratories, the nose of the mouse is dried
with a paper towel before the challenge).
Sampling and CFU count

Five lungs from each group of mice are removed two hours, five and eight days
post-challenge following terminal anaesthesia and deposited into tubes containing
1 or 2 ml of saline or 1% casamino acids. The lungs are homogenized individually
and plated out under appropriate dilution on a Bordet–Gengou plate or charcoal
agar plate. Plates are incubated at 36–37 °C for 4–5 days.
Result
For one animal the lungs homogenate (in 1 ml) is normally diluted to 10 -1, 10 -2,
10 -3 and 10 -4, and up to 10 -6 may be needed for the control group.
246
Annex 4
For each point, the number of colonies on each plate is counted.

Mean of CFU/lungs:
Σ CFU on the 3 plates × dilution factor
m=
Σ volumes used
Note: dilution factor: 10 if the lungs were homogenized in 1 ml or 20 if the lungs
were homogenized in 2 ml.
Example:
For one animal the lungs homogenate (in 1 ml) was diluted 10 -2,
10 -3 and 10 -4:
For 10 -2: 500 colonies were counted
500 + 47 + 6
M= × 10 = 5.53 ×10 5 CFU/ml
0.01+ 0.001 + 0.0001
Log10 is calculated for each mouse and the arithmetic mean is calculated for each
vaccine group.
The curve is then traced: mean of log10 CFU/lungs versus day after infection.
Suggested validity criteria of the test

Several important criteria have to be met in order to validate the assay.
■ Respect of the protocol: there is no technical problem in assay
performance.
■ The number of bacteria used to challenge the mouse is not below 10 5.
■ The calculated log10 mean number of CFU/lung for the negative
control group at two hours after challenge should be above 5.4.
■ The calculated log10 mean number of CFU/lung for the positive
reference group at five days after challenge should be below 3.75.
■ A significant difference of at least 3.1 log CFU between the reference
group and the negative control group should be detected at five days
after challenge.
Both reference and test vaccines should be included in the assay. The
log10 CFU/lung for the test and reference vaccines can be used for quantitative
analysis, either for comparison of results for groups of mice treated with single
doses or, if suitable doses of each vaccine have been used, interpretation of
potency using a parallel line assay could be achievable.
247
Appendix 6
Summary protocol for production and testing of acellular
pertussis vaccine
1. Summary information on final lot
Lot no.:
Date of filling:
Date of manufacture:
Nature of final product (absorbed):
Volume of each recommended single human dose:
No. of doses per final container:
No. of final containers:
Expiry date:
2. Detailed information on manufacture and control

Strain
Identity of B. pertussis strains used in vaccine:
Serological types of strains:
Reference no. of seed lot:
Date(s) of reconstitution of ampoule(s) for manufacture:
Culture media for production

Name of the culture medium:
Control of bacterial purity

Result:
Date:
Control of antigen purification

Purification of PT:
Purification of FHA:
Purification of pertactin:
Purification of FIM 2/3:
Identification:
Volume:
248
Annex 4
Test on purified antigens

For purified antigens
Methods:
Purity:
Date:
For co-purified antigens

Purity of claimed antigens:
Proportion of each antigen claimed:
Methods:
Date:
Residual level of endotoxin

Methods:
Content:
Date:
Antigen content
Methods:
Content:
Date:
Sterility test:
Method:
Media:
Date of test (start, end):
Result:
Detoxification
Detoxifying reagent:
Detoxifying conditions:
Control of bulk
Identification:
Volume:
249
Test for antigen content

Methods:
Content:
Date:
Residual activity of pertussis toxin

1. HIST by temperature measurement
Date:
Strain of mice/Sex of mice:
No. of mice per dilution:
No. of mice dilutions injected:
Age range or weight range on day of immunization:
Immunization route/Immunization dose:
Challenge route/Challenge dose:
Interval between immunization and challenge:
Results (IU/SHD or HSU/SHD):
Calculation method:
Rectal temperature or dermal temperature:
Temperature
average variance
Reference dilution 1
Reference dilution n
Test vaccine
Negative
• Dilution must contain 1.09 or 2.18 IU (0.2 or 0.4 HSU)/SHD.
2. HIST by lethal end-point assay

Date:
Result:
250
Annex 4
No. of deaths/No. of animals inoculated
Reference dilution 1 /
Reference dilution n /
Test vaccine /
Negative /
Residual level of endotoxin

Methods:
Content:
Date:
Sterility test
Method:
Media:
Result:
Control of final bulk

Identification:
Volume:
Detoxifying agent
Methods:
Content:
Date:
Preservative content
Methods:
Content:
Date:
Adjuvant
Methods:
251
Content:
Date:
Sterility
Method:
Media:
Result:
Residual activity of pertussis toxin

Date:
Calculation method:
Temperature
average variance
Test vaccine
Negative
252
Annex 4

Date:
Result:
Test vaccine /
Negative /
Reversion to toxicity
Incubation period: start date end date temperature
Methods:

Date:
Calculation method:
253
Temperature
average variance
Test vaccine
Negative

Date:
Result:
Test vaccine /
Negative /
Immunological activity
1. MIT
Strain of mice:
No. of dilutions injected:
Identification of reference:
Date of bleeding:
Antibody titration:
254
Annex 4
Result for test vaccine:

GMT value of anti-PT:
GMT value of anti-FHA:
GMT value of anti-PRN:
GMT value of anti-Fims:
Result for reference vaccine:
GMT value of anti-PT:
GMT value of anti-FHA:
GMT value of anti-PRN:
GMT value of anti-Fims:
Or ratio of test vaccine to reference vaccine:
Ratio for anti-PT:
Ratio for anti-FHA:
Ratio for anti-PRN:
Ratio for anti-Fims:
Date:
2. MICA
Strain of mice:
No of dilutions injected:
Date of injection:
Identification of reference:
LD50 in challenge dose:
No. of colony-forming units in challenge dose:
Date of challenge:
Date of end of observation:
Results (IU/SHD):
Calculation method:
Dilution No. of survivors/ Median effective

No. of animals inoculated dose (ED50)
Reference:
Vaccine: ml
(IU/ml)
Test vaccine ml
Potency of test vaccine is IU per single human dose. Limits of 95%

confidence interval (in %) are: .
255
Final product
Identification:
Volume:
Identity test
Method of testing:
Result:
Date of test:
Sterility test
Method:
Media:
Result:
Test for adjuvant

Nature and concentration of adjuvant/SHD:
Method of testing:
Specification:
Result:
Date of test:
Test for preservative

Nature and concentration of preservative:
Method of testing:
Specification:
Result:
Date of test:
pH
Method of testing:
Specification:
Result:
Date of test:
Endotoxin test
Method of testing:
Specification:
256
Annex 4
Result:
Date of test:
Immunological activity
If the test was not performed on the final bulk, indicate this and report the data
obtained on the final product in the space provided for biological activity tests in
the “final bulk” section.
Innocuity test
Tests in mice
Date of start of test:
Date of end of test:
No. of animals tested:
Route of injection:
Observation period:
Tests in guinea-pigs
Date of start of test:
Date of end of test:
No. of animals tested:
Route of injection:
Observation period:
Inspection of final containers

Date of inspection:
Organoleptic characteristics:
Number of containers inspected:
Percentage of rejected containers:
3. Certification by the manufacturer
Name of head of production (typed)
Certification by person from the control laboratory of the manufacturing company

taking overall responsibility for the production and control of the vaccine
I certify that lot No. of acellular pertussis vaccine,

whose number appears on the label of the final containers, meets all national
257
requirements and satisfies Part A1 of the WHO Recommendations to assure the

quality, safety and efficacy of acellular pertussis vaccines (2013) 2 (if applicable).
Name (typed)
Signature
Date
4. Certification by the NRA

If the vaccine is to be exported, attach a certificate from the NRA as shown in
Appendix 7, a label from a final container and an instruction leaflet for users.
1
to assess.
2
258
Annex 4
Appendix 7
Model certificate for the release of acellular pertussis
vaccine by NRAs
This certificate is to be provided by the NRA of the country where the vaccine has
been manufactured, on request of the manufacturer.
Certificate no.
The following lot(s) of acellular pertussis vaccine produced by 1
in 2
whose numbers appear on the labels of the final containers,
complies with the relevant specification in the marketing authorization and
provisions for the release of biological products 3 and Part A4 of the WHO
Recommendations to assure the quality, safety and efficacy of acellular pertussis
vaccines (2013) 5 and comply with WHO good manufacturing practices for
pharmaceutical products: main principles; 6 Good manufacturing practices for
biological products; 7 and Guidelines for independent lot release of vaccines by
regulatory authorities.8

■ Name and address of manufacturer
■ Site(s) of manufacturing
1
2
Country of origin.
3
4
to assess.
5
6
7
8
9
defined document etc. as appropriate.
259
■ Trade name and common name of product

■ Marketing authorization number
■ Lot number(s) (including sub-lot numbers and packaging lot
numbers if necessary)
■ Type of container
■ Number of doses per container
■ Number of containers/lot size
■ Date of start of period of validity (e.g. manufacturing date) and/or
expiry date
■ Storage condition
■ Signature and function of the authorized person and authorized
agent to issue the certificate
■ Date of issue of certificate
■ Certificate number.
Name (typed)
Signature
Date
260
Annex 5
Generic protocol for the calibration of seasonal and
pandemic influenza antigen working reagents by WHO
essential regulatory laboratories
Abbreviations 262
1. Introduction 262
2. Essential regulatory laboratories (ERLs) 263
3. Reagents supplied to collaborating ERLs 263
4. Internal compliance testing and documentation 264
5. Antigen reagent supply to vaccine manufacturers 264
6. ERL calibration methodology 264
6.1 Procedure 264
6.2 Technical details 265
7. Assignment of calibrated potency value by the lead ERL 266
8. Calibration of secondary and replacement reagents 266
9. Review of this document 267
Appendix 1
Sample data sheet 268
Appendix 2
Supplementary data sheet 269
261
Abbreviations
CBER Center for Biologics Evaluation and Research
ERL Essential regulatory laboratory
GISRS WHO Global Influenza Surveillance and Response System
NIBSC National Institute for Biological Standards and Control
NIID National Institute for Infectious Disease
PLS Primary liquid standard
SDS–PAGE Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
SRD Single radial diffusion assay
SRID Single radial immunodiffusion assay
TGA Therapeutic Goods Administration
WHO World Health Organization
1. Introduction
Vaccination is the principal measure for preventing influenza and reducing
its impact.1 Since 1973, WHO has provided formal recommendations for the
composition of influenza vaccines on the basis of information provided by the
WHO Global Influenza Surveillance and Response System (GISRS).
WHO technical consultations are convened each year in February and
September to recommend the viruses for inclusion in influenza vaccines for the
northern and southern hemispheres, respectively.2 For countries in equatorial
regions, epidemiological considerations influence which recommendation
(northern or southern) individual national and regional authorities consider
more appropriate.
High-yield candidate vaccine viruses are developed by collaboration
between laboratories involved in developing reassortants and WHO collaborating
centres following the strain recommendations. Once developed, these candidate
reassortants are sent to WHO collaborating centres for characterization of their
antigenic and genetic properties before being released to interested institutions
on request. Reference reagents are subsequently developed and standardized by
ERLs in collaboration with vaccine manufacturers, and are made available to
manufacturers worldwide upon request.
1
See: http://www.who.int/influenza/vaccines/virus/en/, accessed 23 February 2013.
2
See: http://www.who.int/influenza/vaccines/virus/recommendations/en/, accessed 23 February 2013.
262
Annex 5
This document provides a description of the generic protocol for the

calibration of influenza antigen working reagents used by the four WHO ERLs.
It represents the consensus of the ERLs on the process of assigning a potency
value to a newly established influenza antigen reagent for use in potency testing
of inactivated influenza vaccines. An influenza antigen working (or reference)
reagent is a preparation of inactivated whole virus that has been freeze-dried and
calibrated as outlined in this document.
The calibration process involves the preparation of a primary liquid
standard (PLS) and a large batch of freeze-dried antigen by one of the ERLs.
The PLS is distributed to all other ERLs for independent calibration by
physicochemical means. Samples of the freeze-dried antigen are distributed to
the ERLs at the same time and are calibrated against the PLS using the single
radial immunodiffusion assay (SRID; also SRD).
2. ERLs
The ERLs are as follows:
■ Australia – Therapeutic Goods Administration (TGA)
■ Japan – National Institute for Infectious Disease (NIID)
■ United Kingdom – National Institute for Biological Standards and
Control (NIBSC)
■ USA – Center for Biologics Evaluation and Research (CBER).
The participation of all ERLs is assumed, as is current practice, with a
minimum of three contributing data for each calibration. The laboratories agree
a timeline for completion of all calibration tests, with an expectation that most
calibrations will be completed within 15 working days.
The lead ERL is the ERL that produces the freeze-dried antigen reference
reagent and sends out materials to the other ERLs for use in calibration (as
specified in section 4 below). The lead ERL will inform WHO in a timely manner
about the availability of a new reagent and progress of the calibration.
3. Reagents supplied to collaborating ERLs

For each antigen reagent to be calibrated, the following is to be supplied:
■ at least 30 ampoules of freeze-dried antigen;
■ 10 vials of antiserum (if more than one lot is shipped, 10 vials of
each lot);
■ a batch, preferably with two aliquots, of a whole virus preparation
(i.e. the “PLS”) consisting for example of an in-house live or
inactivated preparation or liquid pre-freeze-dried antigen.
263
The PLS will be characterized by all ERLs with respect to protein

content and the proportion of haemagglutinin by physicochemical means to
independently determine the haemagglutinin content in micrograms. The PLS
serves as the standard against which the secondary freeze-dried antigen reagent
is calibrated. The PLS is supplied with a protein concentration estimated by the
lead ERL.
4. Internal compliance testing and documentation

The lead ERL performs the tests on the supplied materials prior to distribution,
provides test data upon request and supplies interim documentation (e.g.
instructions for use). The tests performed by the lead ERL are as follows.
■ A protein estimation on the PLS, preferably confirmed by using
more than one method with a confirmatory value of ±20% of the
estimated value.
■ An estimated range of working dilutions of the specific antiserum.
■ When possible, SRID assays of the PLS and the freeze-dried antigen
using the specific antiserum to confirm their antigenic identity, the
estimated potency value of the freeze-dried antigen and a qualitative
assessment of SRID zones. If specific antiserum becomes available
only after distribution of the PLS and freeze-dried antigen, SRID
with a cross-reactive antiserum is performed if possible.
5. Antigen reagent supply to vaccine manufacturers

Upon request, vaccine manufacturers are to be provided with freeze-dried antigen
reference reagents as soon as they are available and prior to final calibration.
These may be supplied with interim estimated values for use in SRID potency
assays. Manufacturers’ data (e.g. comparison of SRID values with manufacturers’

in-house methods for preliminary yield analysis of monovalent bulks) can be
supplied to ERLs for review.
6. ERL calibration methodology

6.1 Procedure
■ Calibration of the PLS: the haemagglutinin content of the
PLS is determined by physicochemical methods. Total protein
is determined by nitrogen analysis and/or Lowry assay. The
percentage haemagglutinin is determined by sodium dodecyl
264
Annex 5
sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using

band densitometry, and the haemagglutinin content of the PLS is
expressed in micrograms.
■ Preparation of strain-specific antiserum reagents: the virus is
digested with bromelain to remove haemagglutinin from virus
particles. The haemagglutinin missing the transmembrane portion
is purified and sheep are immunized multiple times to generate
hyperimmune antiserum which is tested for suitability in SRID
assays in terms of potency and the quality of the SRID zones
produced. The serum is not calibrated but a suitable concentration
range for its use is suggested. The reference antiserum is aliquoted
and distributed with reference antigen.
■ Preparation of reference antigen: a reference antigen is prepared
on an industrial scale, and is aliquoted and lyophilized. The
haemagglutinin content of the reference antigen is assigned by
SRID against the PLS using the specific antiserum. The ERLs assign
potencies to the PLS and reference antigen independently and the
lead ERL collates the results and proposes a final consensus value
for agreement by the ERLs. Reagents are distributed as soon as they
become available. This process takes several weeks. Note: specific
antigen standards are required for each candidate vaccine virus
strain. Specific antisera are required for each recommended vaccine
virus – i.e. antisera are prepared for groups of antigenically similar
(“like”) viruses, and are cross-reactive between candidate vaccine
viruses covered by a particular recommended virus.
6.2 Technical details

■ PLS protein estimation: this should be performed by a recognized
assay (e.g. Lowry or total nitrogen determination), according to the
local ERL standard operating procedure or methodology. Assays
should include an appropriate protein control (ideally a large batch
of common protein standard shared between the ERLs).
■ PLS PAGE assay: the PLS should be treated as appropriate prior
to the PAGE analysis (e.g. reduction or deglycosylation). Assays
should be performed according to the local ERL standard operating
procedure or methodology, with a minimum of two independent
assays, preferably performed by different analysts. Protein bands
should be visualized using Coomassie blue-based staining.
265
■ PAGE band analysis: analysis of PAGE gels should be performed

according to the local ERL standard operating procedure or
methodology, recording any parameters that vary from their usual
procedures. General guidance for confirmation of accuracy of PAGE
band analysis:
– the ratio haemagglutinin 1: haemagglutinin 2 is approximately 3:2
– the haemagglutinin content should be between 20% and 50% of
total protein
– the analysis is to be repeated if there is > 20% variation between
replicates.
■ SRID assay: the assay is based on diffusion of virus antigen
(e.g. detergent-disrupted virus or vaccine) through an agarose
gel containing haemagglutinin-specific antiserum. The square
of the diameter of the precipitin ring is proportional to the
antigen concentration and a standard curve is used to quantify
haemagglutinin in vaccine samples. Preferably, three to six assays
should be performed involving more than one operator. Assays
should be performed using the local ERL standard operating
procedure or methodology. Freeze-dried antigen should be analysed
using the PLS as standard antigen on plates containing the appropriate
antiserum. The final potency value of the freeze-dried antigen is
derived using the mean potency values of all assays.
■ Complete the ERL data sheet: a sample data sheet is attached to this
protocol (Appendix 1). The data sheet is to be sent to the lead ERL.
Supplementary data may also be included (Appendix 2).
7. Assignment of calibrated potency value by the lead ERL

Data generated by the ERLs (see Appendix 1 and Appendix 2) are collected
by the lead ERL and compiled for the final potency value agreement and
confirmation. Manufacturers’ data may also be considered, if available. Before
the assigned value is made public (e.g. through the ERL web site, WHO web site
or Instructions for Use), the final data sheet and proposed calibration value are
sent to all participating ERLs for comment and/or approval. The lead ERL has
final authority in assigning a potency value.
8. Calibration of secondary and replacement reagents

The antigen reagent that is developed first for a given candidate vaccine virus
is calibrated according to the process outlined in sections 2–7 above. When
266
Annex 5
another antigen reagent for the same candidate vaccine virus is required, either as
a replacement to replenish stocks or as an alternative reagent provided by another
ERL, it is calibrated against the first antigen reagent to ensure equivalence
of reagents and to ease the switch from one lot of reagent to a new one. The
process for calibration (cross-calibration) of these types of secondary reagents is
abbreviated: calibration uses the antigen reagent that was the first to be developed
as the relevant calibrant, and not the PLS. In all cases, calibration is performed
using the SRID assay. The exact process for cross-calibration varies between
ERLs, and the involvement of more than one laboratory is encouraged – either
within the same institution or by using external laboratories (e.g. other ERLs, or
national control laboratories with proven experience in influenza vaccine potency
testing). If this is not feasible, more than one operator within the calibrating ERL
laboratory will need to be engaged in the calibration process.
9. Review of this document

The ERLs will review this document periodically – at least once a year – to ensure
that it reflects best practice within ERLs and any updated methodology that may
be implemented in the future. Review may take place through electronic means
or during meetings between ERL representatives. Any updates of this document
will be posted on the GISRS web site.3
3
See: http://www.who.int/influenza/gisrs_laboratory/en/, accessed 23 February 2013.
267
Appendix 1
Sample data sheet
Date:
Reference:
Primary liquid standard ([ERL]): [virus name] ([reassortant]) (Lot no. [yyy])
Protein concentration No. of deaths/No. of Haemagglutinin (HA)

(µg/ml) animals inoculated content (µgHA/ml)
Antiserum:
Anti-[virus name] sheep antiserum (Lot no. [zzz], [ERL])
x µl/ml agarose
Sample:
Lot no. [yyy], [ERL]
Tube no. Haemagglutinin (HA) content (µgHA/vial)

Mean
Standard deviation of the mean (SD)
Coefficient of variation (CV %)
268
Annex 5
Appendix 2
Supplementary data sheet
Calibration of [virus name] reference antigen lot [yyy] with antiserum lot [zzz]
Collaborative study summary results
TGA NIID CBER NIBSC Mean [n]

mean
Lowry total
SD [n] []
protein (µg/ml)
CV (%)
mean
Primary
Haemagglutinin
liquid SD [n] []
(% total protein)
standard
CV (%)
mean
Haemagglutinin
SD [n] []
(µg/ml)
CV (%)
mean
Reference Haemagglutinin
SD [n] []
antigen (µg/ml)
CV (%)
CBER: Center for Biologics Evaluation and Research; CV: coefficient of variation; NIBSC: National Institute
for Biological Standards and Control; NIID: National Institute for Infectious Disease; SD: standard deviation;
TGA: National Institute for Infectious Disease.
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Guidelines for thromboplastins and plasma used to control
oral anticoagulant therapy with vitamin K antagonists
1. Introduction 273
2. Definitions 275
3. International Reference Preparations of thromboplastins 276
4. Preparation of thromboplastins 279
5. Tests on thromboplastins 280
5.1 Response to coumarin-induced coagulation defect 280
5.2 Content of haemoglobin and serum 280
5.3 Opacity and sediment volume 280
5.4 Containers 280
5.5 Stability 281
6. Calibration of prothrombin-time systems 281
6.1 Calibration of International Reference Preparations 282
6.2 Calibration of secondary standards 282
6.3 Calibration of individual batches of thromboplastins 283
7. Calibration procedure 283
7.1 Procedure 1: Calibration of a secondary standard using individual fresh plasma
or blood samples 284
7.2 Procedure 2: Calibration of individual batches of thromboplastin 287
7.3 Procedure 3: Local system calibration using certified plasmas 289
8. The use of calibrated thromboplastins in clinical practice 297
Authors 297
References 298
Appendix 1
Criteria which may assist clinical laboratories in the choice of a reagent 304
Appendix 2
Example of the use of the suggested method for reporting the data for the calibration
of any system or a secondary standard of thromboplastin against an International
Standard preparation 305
271
Appendix 3
Example of the use of the suggested method for reporting the data for the
calibration of individual batches of thromboplastin 314
272
Annex 6
1. Introduction
Oral anticoagulant drugs derived from 4-hydroxycoumarin (and sometimes from
indandiones) are widely used in the treatment and prophylaxis of thrombotic
disorders. Coumarin drugs inhibit the biosynthesis of vitamin K-dependent
coagulation factors by the liver. For each patient, the dose of these drugs must
be adjusted periodically to ensure that an adequate, but not excessive, degree of
anticoagulation is achieved. The adjustments are made on the basis of the results
of the prothrombin-time or a similar test on the patient’s blood. The test, which
requires reagents called thromboplastins, is controlled by the use of calibrated
thromboplastins and plasmas.
Various types of thromboplastin are prepared commercially and, in order
to be able to interpret the results of the prothrombin-time test, it is essential that
each reagent is correctly calibrated. This will ensure that the results of tests with
different products and batches are reproducible and can be compared. A procedure
for the calibration of thromboplastins using a logarithmic plot of prothrombin
times (PTs) has been developed (1) and was described in the report of the forty-
eighth meeting of the WHO Expert Committee on Biological Standardization
(2). With this procedure, the definition of a calibration parameter called the
International Sensitivity Index (ISI) became feasible. It is possible to express
prothrombin-time results on a common scale, i.e. the International Normalized
Ratio (INR), if the ISI of the thromboplastin used is known.
Many routine laboratories use automated coagulometers for detection
of the clotting end-point. There is now substantial evidence that coagulometers
can have unpredictable and marked effects on the ISI of thromboplastins (3–6).
Because of these effects, some manufacturers provide a “system ISI” for a
particular thromboplastin/coagulometer combination. However, this procedure
appears to have limitations since variations in the system ISI with the same reagent
and coagulometer at different centres have been demonstrated in collaborative
studies (7, 8).
In general, the calibration of a given thromboplastin is more precise if
performed against an International Reference Preparation1 of similar composition
and from the same species (9–11). A system of coexisting International Reference
Preparations has been established in which each of these materials is related to the
first primary International Reference Preparation – the First WHO International
1
International reference materials established by the WHO Expert Committee on Biological Standardization
have been denoted, variously, as International Reference Preparations, International Reference Reagents
and International Standards. These Guidelines refer to all thromboplastin reference materials established
by the WHO Expert Committee, independent of the nomenclature. International reference materials so
established are by definition “primary” Reference Preparations, secondary Reference Preparations being
calibrated in relation to them.
273
Reference Preparation of thromboplastin (human, combined), coded 67/40 (see

Figure A6.1). Two International Reference Preparations of thromboplastin are
currently available from the relevant WHO International Laboratory for Biological
Standards: the Fourth WHO International Standard for thromboplastin (rabbit,
plain) (coded RBT/05) (12) and the Fourth WHO International Standard
for thromboplastin (human, recombinant, plain) (coded rTF/09) (13). Other
International Reference Preparations have been discontinued. The development
of these preparations is described in section 3.
In theory, the ISI/INR system should ensure that the ISI value calculated
for a given reagent is independent of the species from which the International
Reference Preparation is derived, because all have been directly or indirectly
calibrated against the First WHO International Reference Preparation of
thromboplastin (human, combined) (coded 67/40). However, this is not always
the case; several observations have demonstrated that reagents calibrated against
the Second WHO International Reference Preparation of thromboplastin (human,
plain) – a material coded BCT/253 (the predecessor of rTF/95) (14) – provide
lower INR values than those calibrated against RBT/79 (the predecessor of
RBT/90) or OBT/79 (9, 11, 15). The extent of these differences in INR is not
usually large enough to cause serious concerns from a practical point of view.
The discrepancy is due to calibration errors that persist because the different
International Reference Preparations were not checked against each other in
the original studies. A new procedure has now been agreed upon: international
thromboplastin Reference Preparations, whatever their origin and composition,
will be calibrated against all existing International Reference Preparations in
order to ensure consistency of results between different routes of calibration (16).
It is recommended that the International Reference Preparation of the
same species or composition should be used for calibration of secondary standards,
e.g. working standards, by manufacturers and national reference laboratories.
Thus, plain rabbit thromboplastins should be calibrated against RBT/05 and
plain human thromboplastins against the human recombinant material rTF/09.
It has been demonstrated that bovine or rabbit combined thromboplastins can be

calibrated with acceptable precision against RBT/05 (17). Thus, it is recommended
that bovine or rabbit combined thromboplastins should be calibrated against
RBT/05.
The calibration of prothrombin-time systems is not easy. Furthermore,
there is considerable variation in results from different laboratories performing
the same procedures, as shown by published multicentre calibration studies
(9–14, 18–20). In these studies, interlaboratory variation in ISI, expressed as a
coefficient of variation, ranged from approximately 1.7% to 8.1%.
The preparation, certification, and use of deep-frozen or lyophilized
plasmas for ISI calibration and INR determination has been described as an
important adjunct to fresh-plasma ISI calibration (21). The purpose of these
274
Annex 6
Guidelines, which replace the Requirements published in the forty-eighth

report of the WHO Expert Committee on Biological Standardization (2), now
discontinued, is to take account of the above-mentioned observations and to
describe in detail the technical methods currently in use. Modifications to the
methodology may give comparable results, but must be validated against the
methodology described in these Guidelines.
How to use these Guidelines

Both manufacturers and clinical laboratories should be informed of the definitions
used for the control of oral anticoagulant therapy with vitamin K-antagonists
(section 2). Manufacturers of thromboplastins and certified plasmas should be
informed of the current Guidelines. These Guidelines contain information for
manufacturers of thromboplastins on the methods for calibration of their reagents
against International Standards and for calibration of consecutive lots of each
type of reagent (section 6). Calibration with International Standards is generally
not to be performed by clinical laboratories.
These Guidelines contain information for clinical laboratories on the
methods for simplified local-system calibration as described in section 7.3. The
use of certified plasmas in clinical laboratories is described in section 7.3.4.
Appendix 1 contains criteria for clinical laboratories that may assist with the
choice of a commercial thromboplastin reagent or a set of certified plasmas.
2. Definitions
International Normalized Ratio (INR): for a given plasma or whole
blood specimen from a patient on long-term oral anticoagulant therapy, a value
calculated from the prothrombin-time ratio using a prothrombin-time system
with a valid ISI according to the formula INR = (PT/MNPT) ISI.
International Sensitivity Index (ISI): a quantitative measure, in terms of
the First WHO International Reference Preparation of thromboplastin (human,
combined), coded 67/40, of the responsiveness of a prothrombin-time system to
the defect induced by oral anticoagulants (see Appendix 2).
Mean normal prothrombin time (MNPT): the geometric mean of the
PTs of the healthy adult population. For practical purposes, the geometric mean
of the PT calculated from at least 20 fresh samples from healthy individuals,
including both sexes, is a reliable approximation of MNPT. It is recommended
that individual samples should be collected and tested over at least three working
days in order to include inter-assay variation. It is also recommended that each
laboratory should determine MNPT using its own prothrombin-time system.
Pooled normal plasma (either deep-frozen or freeze-dried) may be suitable if
the clotting time obtained is related to the MNPT value and its storage stability
is acceptable.
275
Prothrombin time (PT) (tissue-factor-induced coagulation time): the

clotting time of a plasma (or whole blood) sample in the presence of a preparation
of thromboplastin and the appropriate amount of calcium ions. The time is
reported in seconds (22).
Prothrombin-time ratio (tissue-factor-induced coagulation relative
time): the PT obtained with a test plasma or whole blood divided by the MNPT,
all times having been determined using the same prothrombin-time system.
Prothrombin-time system: a procedure by which the PT is determined
using a specific thromboplastin reagent and a particular method, which may be
manual, e.g. a tilt-tube method, or involve the use of an instrument that records
the coagulation end-point automatically. The method should be described and
the description should include all procedures and equipment used, e.g. the
pipettes and test-tubes.
Thromboplastin: a reagent containing tissue factor and coagulant
phospholipids. Many commercial thromboplastins are crude extracts prepared
from mammalian tissues, in which tissue factor is only a minor component
on a weight basis, and which also contain phospholipids. A preparation of a
thromboplastin consisting of a tissue extract alone, either with or without added
calcium chloride, is termed “plain”. When the preparation contains adsorbed
bovine plasma as a source of additional factor V and fibrinogen it is termed
“combined”. Thromboplastins may also be grouped into types, according to the
tissue source from which they are derived, e.g. human, bovine, rabbit brain or
lung, or human placenta. The tissue-factor component of recombinant human
thromboplastin reagents is produced in Escherichia coli or insect cells by
recombinant DNA techniques and then lipidated in vitro.
Tissue factor: an integral transmembrane protein functioning as a
cofactor enhancing the proteolytic activity of factor VIIa towards factor X and
factor IX in the blood. Tissue factor needs to be associated with coagulant
phospholipids for the full expression of its cofactor function.
3. International Reference Preparations

of thromboplastins
International Reference Preparations, International Standards and International
Reference Reagents are intended to serve throughout the world as sources of
defined biological activity quantitatively expressed in International Units or in
terms of a suitable property or characteristic defining the biological activity. These
preparations are used to calibrate secondary standards, which include regional,
national and manufacturers’ working standards. Normally, working standards
are used for routine calibration of individual batches of thromboplastin, and
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Annex 6
working standards should have been calibrated with the appropriate International
Reference Preparation. If secondary standards are developed using procedures
that involve multiple calibration steps, there is a risk that unnecessary variability
and discontinuity will occur in relation to the primary International Reference
Preparation because of cumulative serial calibration errors.
Current prothrombin-time systems are based on the use of three different
species of thromboplastin reagents: human, bovine and rabbit. Originally, the
standardization of these thromboplastin reagents likewise involved three different
Reference Preparations, one for each of the three species of plain thromboplastin
reagents in use (Figure A6.1).
The First WHO International Reference Preparation of thromboplastin
(human, combined) (coded 67/40), was established by the WHO Expert
Committee on Biological Standardization in 1976 (23). It was a freeze-dried
preparation, filled in sealed glass ampoules, and contained a human brain extract
to which adsorbed bovine plasma had been added to optimize the content of
non-vitamin-K-dependent coagulation factors (i.e. factor V and fibrinogen). Its
ISI value was set at 1.0 by definition. In 1983, this preparation was discontinued
and replaced by the Second WHO International Reference Preparation of
thromboplastin (human, plain) (coded BCT/253), a human brain extract with
no added coagulation factors and an assigned ISI value of 1.1 (24). When stocks
of BCT/253 became exhausted, a new preparation of human recombinant
thromboplastin (coded rTF/95) was established in 1996 as the Third WHO
International Standard for thromboplastin (human, recombinant, plain) with an
assigned ISI value of 0.94 (18, 25). When stocks of rTF/95 became exhausted,
a new preparation of human recombinant thromboplastin (coded rTF/09) was
established in 2009 as the Fourth WHO International Standard for thromboplastin
(human, recombinant, plain), calibrated against rTF/95 and RBT/05, with an
assigned ISI value of 1.082 (13).
The First WHO International Reference Preparation of thromboplastin
(bovine, combined) (coded 68/434) was established by the WHO Expert
Committee on Biological Standardization in 1978 (26). It was calibrated using
the First WHO International Reference Preparation of thromboplastin (human,
combined) (67/40). Another material, also calibrated against 67/40, was established
as the Second WHO International Reference Preparation of thromboplastin
(bovine, combined) (coded OBT/79), in 1983 with an assigned ISI of 1.0 (27).
This material (OBT/79), which was derived from bovine brain and combined
with factor V and fibrinogen, was used to calibrate thromboplastin materials of
bovine origin and combined thromboplastins of whatever origin. When stocks
of OBT/79 became exhausted in 2004, it was not replaced by a new International
Reference Preparation of bovine origin.
277
Figure A6.1
WHO International Reference Preparations, International Reference Reagents and
International Standards for thromboplastins, and their calibration relationships
IRP = International Reference Preparation; IRR = International Reference Reagent; IS = International Standard.
a
Now discontinued.
For the calibration of thromboplastins of rabbit origin, the First WHO

International Reference Preparation of thromboplastin (rabbit, plain) (coded
70/178), was established in 1978. This material was calibrated against the First
WHO International Reference Preparation of thromboplastin (human, combined)
(coded 67/40), in an international collaborative study which also included the First
WHO International Reference Preparation of thromboplastin (bovine, combined)
(26). When stocks of 70/178 became exhausted, the Second WHO International
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Annex 6
Reference Preparation of thromboplastin (rabbit, plain) (coded RBT/79), was

established in 1982 with an ISI value of 1.4; this was also calibrated against 67/40
(27). The Third WHO International Reference Reagent for thromboplastin (rabbit,
plain) (coded RBT/90), obtained from rabbit brain with no added factors, was
calibrated against each of the three species of thromboplastins and established
by the WHO Expert Committee on Biological Standardization in 1995 with an
ISI of 1.0 (28). When stocks of RBT/90 were exhausted, a new preparation of
rabbit brain thromboplastin (coded RBT/05) was established as the Fourth WHO
International Standard for thromboplastin (rabbit, plain), calibrated against rTF/95
and OBT/79, with an assigned ISI value of 1.15 (12). This material should be used
for the calibration of rabbit thromboplastins as well as bovine thromboplastins.
The widespread use of these International Reference Preparations for
calibrating secondary standards reflects the value placed on them by the scientific
community responsible for the control of thromboplastins. An independent
control of a manufacturer’s ISI assignments by a national reference laboratory is
also recommended. National control authorities should consider designating an
expert laboratory in their country for testing thromboplastin reagents and plasmas
used by clinical laboratories to control oral anticoagulant therapy to ensure that
they are in accordance with guidelines published by WHO.
The international reference materials for thromboplastins are in the
custody of the National Institute for Biological Standards and Control, Potters
Bar, England. Samples of these materials are distributed to national reference
laboratories or national control laboratories for biological products and, upon
payment of handling charges, to other organizations such as manufacturers,
universities, research institutes and hospital laboratories. The principle that WHO
International Reference Preparations are distributed free of charge to national
control authorities for the purpose of the calibration of national standards has
been adhered to since the establishment of international biological standardization
activities (29).
4. Preparation of thromboplastins
The method of preparation of thromboplastins should be such that there is
consistency from batch to batch and that the preparations are suitable for use in
the control of oral anticoagulant treatment. The thromboplastins should comply
with the specifications outlined in section 5.
Every attempt should be made to use the least contaminated source
material possible and to use a manufacturing procedure that prevents further
contamination and the growth of organisms during manufacture. Thromboplastins
of animal origin should be prepared only from healthy animals. Thromboplastins
prepared from bovine brain should be derived only from cattle from countries
279
that have not reported indigenous cases of bovine spongiform encephalopathy

(BSE) and which have a compulsory BSE notification system, compulsory clinical
and laboratory verification of suspected cases and a surveillance programme in
place (30).
Human brain tissue should not be used because of the risk of transmission
of Creutzfeldt–Jakob disease. Thromboplastins derived from human placenta
should be prepared from donors in whom there is no evidence of systemic
microbiological infection or localized infection and who have been shown to be
free from hepatitis B surface antigen, antibodies to human immunodeficiency
viruses (HIV-1 and HIV-2) and antibodies to hepatitis C virus.
5. Tests on thromboplastins
Each batch of thromboplastin should satisfy the following criteria.
5.1 Response to coumarin‑induced coagulation defect

The response to the coumarin-induced coagulation defect should be measured
by the PT obtained using normal and coumarin plasmas. Thromboplastins with
a manual ISI between 0.9 and 1.7 are acceptable. However, ISIs towards the lower
end of this scale are desirable, since some studies have shown that interlaboratory
variation in ISI is greater for high than for low ISI systems (20). It has been
suggested that the INR is less accurate when PT is determined with insensitive
thromboplastins that have high ISI values (31).
5.2 Content of haemoglobin and serum

To prevent contamination of the product with (activated) clotting factors, the
thromboplastin preparation should be free from serum and show no detectable
haemoglobin.
5.3 Opacity and sediment volume

The method of manufacture, particularly the method of breaking up the
tissue, has a marked effect on the activity, opacity and sediment volume of the
thromboplastin. The opacity of preparations intended for use in photoelectric
instruments should be suitably low, as specified by the manufacturer.
5.4 Containers
International Reference Preparations for thromboplastins are freeze-dried in
sealed glass ampoules (32), but secondary standards may be freeze-dried in
ampoules or vials.
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Annex 6
5.5 Stability
The method of manufacture should be such that the thromboplastin preparations
are stable for at least one year. All reagents eventually lose activity when stored
at elevated temperatures, and stability should be checked by an accelerated
degradation test (33, 34).
Accelerated degradation studies are considered to be only an indicative
rather than an absolute guide to the stability of thromboplastins maintained at the
storage temperatures recommended by the manufacturer. Lyophilized standard
thromboplastins are routinely stored at low temperatures to maintain their
stability. A small part of the standard material may be stored at an even lower
temperature (“ultra-low temperature stock”). Under the assumption that the rate
of degradation is different under the two storage conditions, a comparison of
the results of samples of stock kept under the routine storage conditions with
those of the ultra-low-temperature stock can be used to assess the stability status
of the standard material (35). The stability of the thromboplastins must also be
determined for the conditions under which they are stored, i.e. in a real-time
stability study (36, 37).
6. Calibration of prothrombin-time systems

Four types of calibration should be distinguished:
a. calibration of International Reference Preparations;
b. calibration of secondary standards, e.g. national Reference
Preparations and manufacturers’ working standards;
c. calibration of manufacturers’ commercial preparations against the
corresponding working standard (“lot-to-lot” calibration);
d. local-system calibration.
In general, the results of calibrations are used by laboratories other than
the calibrating laboratories. The clinical laboratories should therefore be aware
of the interlaboratory variation in ISI values for the thromboplastin reagent.
Type (d) calibration involves the use of deep-frozen or freeze-dried plasmas with
assigned INR or prothrombin-time values which are described below. Type (a)
and (b) calibrations should be carried out with a large number of fresh plasma or
whole blood samples. Several studies suggest that, under certain circumstances,
fresh plasmas for type (c) calibrations can be reliably replaced by frozen, freeze-
dried, pooled plasma or plasmas artificially depleted of vitamin K-dependent
coagulation factors (38–40). Manufacturers should validate this procedure by
means of fresh plasmas.
Prothrombin-time systems should be calibrated in terms of the appropriate
International Reference Preparation of thromboplastin, and the response to the
281
coumarin-induced coagulation defect should be defined by the ISI obtained in

the calibration procedure. Supplies of International Reference Preparations are
limited, and it is not possible to use these materials in routine tests to calibrate
each batch of the many thromboplastins produced by different manufacturers.
Calibration of individual batches of thromboplastin should be carried out by
comparison with a secondary standard, which should be a batch of the same
or a similar thromboplastin calibrated against the appropriate International
The basis of the thromboplastin calibration model is necessarily an
empirical one. While there is good evidence that the calibration relationship
defined in a double-logarithmic plot of PTs is usually linear, and that the same
line represents data points for both patients and healthy subjects, the possibility
of departure from these assumptions cannot be ruled out. Statistical methods
to test deviations from the above-mentioned assumption have been described
(41, 42). In the case of marked deviation, the assignment of an ISI would not
be meaningful. For practical purposes, the assignment of an ISI is acceptable
if INRs calculated with the ISI derived from the overall regression line (i.e. for
patients plus healthy subjects) do not differ by more than 10%, in the INR range
2–4.5, from INRs calculated with the equation describing the regression line for
patients only (see Appendix 2). A difference of 10% is considered as a critical
difference according to the Scientific and Standardization Committee of the
International Society on Thrombosis and Haemostasis (SSC/ISTH) Guidelines
on preparation, certification, and use of certified plasmas for ISI calibration and
INR determination (21).
6.1 Calibration of International Reference Preparations

The calibration of the International Reference Preparations for thromboplastins,
and their future replacements, should be carried out as part of international
multicentre collaborative studies using fresh coumarin, normal plasma and manual
techniques. In recent studies for the calibration of replacement International
Standards, approximately 20 centres participated. These centres were located in
North and South America, Asia, and Europe (12, 13). Each collaborative study
for replacement of an International Reference Preparation should include the
testing of all existing International Reference Preparations. The ISI assigned to
the replacement material should be the mean of the ISIs obtained by calibration
with all existing International Reference Preparations (16).
6.2 Calibration of secondary standards

Secondary standards of human origin should be calibrated against the
current International Standard, i.e. the Fourth WHO International Standard
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Annex 6
for thromboplastin (human, recombinant, plain) (coded rTF/09); plain

thromboplastins of rabbit brain and rabbit lung should be calibrated against the
Fourth WHO International Standard for thromboplastin (rabbit, plain) (coded
RBT/05). Thromboplastins of bovine or rabbit brain combined with adsorbed
bovine plasma should also be calibrated against RBT/05.
In view of the interlaboratory variation observed in multicentre
calibration studies, it is recommended that calibration of national reference
materials or manufacturer’s working standards should be performed by at least
two laboratories. The ISI assigned to the national reference material or the
manufacturer’s working standard should be the mean of ISIs obtained by the
individual laboratories.
6.3 Calibration of individual batches of thromboplastins

The precision of calibration is greatest when similar materials and methods are
compared. For this reason, a national Reference Preparation or manufacturer’s
working standard used for the calibration of individual batches of thromboplastin
should be a thromboplastin with similar characteristics to the batches being
calibrated (i.e. it should be derived from the same tissue of the same species,
using a similar manufacturing process). Batch-to-batch calibration should be
performed by the manufacturer before release of the reagent, and consistency of
ISI values should be shown. Manufacturers should state the applicable end-point
detection systems including any relevant coagulometer lines alongside any stated
ISI values.
7. Calibration procedure
The calibration procedure entails the determination of a series of PTs, using
normal and abnormal plasmas or whole blood samples, with both the reference
and the test thromboplastin. The tests are performed using either fresh samples
from individual subjects (procedure 1) or freeze-dried or frozen plasmas
(procedures 2 and 3). Abnormal plasmas for procedure 1 are obtained from
patients undergoing long-term oral anticoagulant treatment. Freeze-dried or
frozen plasmas for procedure 2 may be pooled plasmas from healthy subjects
and from patients undergoing long-term anticoagulant treatment.
Procedure 1 is recommended for the calibration of secondary standards
or any other prothrombin-time system against the appropriate International
Reference Preparation and for the calibration of whole-blood coagulometers.
Procedure 1 can also be used for the calibration of individual batches of
thromboplastin against the corresponding secondary standard (i.e. lot-to-lot-
calibration), but may be replaced by procedure 2 if the same results are obtained.
283
The precision of the calibration relationship depends on the number of

plasmas and on a balanced distribution of normal and abnormal plasmas over
the “therapeutic” range of INR values. The recommended number of abnormal
plasmas is three times the number of normal plasmas.
7.1 Procedure 1: Calibration of a secondary standard

using individual fresh plasma or blood samples
This procedure consists of a set of tests using freshly opened or reconstituted
thromboplastins and a number of different individual samples of fresh plasma
or whole blood. The procedure should be repeated on at least five separate
occasions using fresh reagents on each occasion (see section 7.1.4). The procedure
need not be repeated on consecutive days but should be completed as soon as
possible. The tests in any one laboratory on any one day should be performed by
the same person.
7.1.1 Blood samples

Blood samples from healthy subjects and patients who have been on oral
anticoagulants for at least 6 weeks should be selected. Samples from patients
treated with heparin should not be used. Patients’ samples with INR values in the
range 1.5–4.5 should be selected.
Blood should be obtained by venipuncture, avoiding haemolysis and
contamination with tissue fluids. It should be drawn either with a plastic syringe
and transferred to a plastic tube, or with other non-contact activation equipment.
Nine volumes of blood should be decalcified with one volume of 109 mmol/l
trisodium citrate solution (22). A mixture of trisodium citrate and citric acid is
also acceptable if the total citrate plus citric acid concentration is 109 mmol/l
and the pH is no lower than 5. The same procedure and materials should be used
for all the samples in a given calibration.
The lot number of tubes used for blood collection should be noted, as
there may be lot-to-lot variation. If tubes are made of glass, they must be properly
siliconized internally and the pH of the trisodium citrate plus citric acid solution
must be in the range 5–6 (43). The sample should be centrifuged as soon as it
is received but, in any case, no later than 2 hours after blood collection. The
centrifugation should render the plasma poor in platelets (i.e. at least 2500 g
for 10 minutes at a controlled room temperature, or at a speed and for a time
that allow a platelet count of the platelet-poor plasma lower than 10 × 10 9/l).
The plasma should be taken off the red-cell layer with a plastic pipette, stored
undisturbed in a narrow, stoppered, non-contact tube at room temperature and
tested within 5 hours after blood collection.
Some techniques or instruments require the use of non-citrated capillary
blood (44). Capillary blood can be obtained by finger or heel puncture. The
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Annex 6
capillary blood should be obtained without squeezing of the finger or heel and
tested immediately with the technique or instrument to be calibrated. Venous
blood should be obtained from the same subjects (healthy subjects and patients)
within 5 minutes of taking the capillary sample, for preparation of citrated
plasma as described above and testing with the most appropriate International
7.1.2 Reference thromboplastins

The appropriate International Reference Preparation of thromboplastin (human
or rabbit) should be reconstituted as instructed and the contents of the ampoules
transferred to a container in sufficient volume for all tests to be performed in a
single calibration session. Specific instructions for use should be supplied by the
custodian of these materials.
7.1.3 Prothrombin‑time test

The prothrombin-time test is performed either by mixing equal volumes of
citrated plasma, thromboplastin and calcium chloride solution (25 mmol/l), or by
adding a volume of plasma to the required volume of thromboplastin premixed
with calcium, and therefore available as a single reagent. The time (in seconds)
taken for the mixture to clot when maintained at a temperature of between
36.5 °C and 37.5 °C is recorded. Test instructions for commercial thromboplastins
should be provided by the manufacturers.
The coagulation end-point for International Reference Preparations
of thromboplastin must be detected by a manual (tilt-tube) technique because
the manual technique has been used for the establishment of the ISI for the
International Reference Preparations and International Standards. The angle and
speed of tilting the test-tube must be standardized (through 90 °C three times
every 5 seconds) to control glass activation and minimize cooling (45).
The coagulation end-point for other thromboplastins (e.g. commercial
preparations) may be detected with the aid of an automatic or semi-automatic
end-point recorder. The same technique should be used throughout the series of
tests with a given thromboplastin.
Each laboratory should have a system for internal quality control. Records
should be maintained of the lot number of all reagents and disposable equipment
used. Periodic checks of the temperature of incubation baths or heating blocks
and of the volumes of pipettes or pumps should be made and recorded.
An example of results obtained with procedure 1 is provided in
Appendix 2.
7.1.4 Statistical evaluation

The suggested procedure for calculation of the ISI is given in Appendix 2.
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Before the final orthogonal regression line for the ISI is calculated, it
is important to detect outliers and any samples beyond the therapeutic range.
Outliers may result from technical or clerical errors and may strongly influence
the estimated ISI. Outliers may be detected as points with a perpendicular distance
greater than 3 residual standard deviations from the preliminary orthogonal
regression line calculated with all data included (46). It is suggested that outliers
be detected and removed in a single step. In the next step any patient samples
beyond the therapeutic range (INR < 1.5 or INR > 4.5) should be removed. In
this procedure it is important to assess each patient’s INR as the mean INR
determined with the International Standard and with the system being calibrated
using the ISI obtained after the removal of outliers. Using the INR determined
solely with the International Standard could introduce a bias in the orthogonal
regression line and should be avoided (47).
It is not necessary to replace the removed outliers and non-therapeutic
patient samples with new samples, provided that the number of patient samples
remaining is at least 55. In any case, the within-laboratory coefficient of variation
of the slope of the orthogonal regression line for normal samples + patient
samples should be 3% or less. The number of normal samples should be at least
20 for the calculation of the MNPT. After removal of outliers and non-therapeutic
patient samples, the adequacy of the ISI model should be assessed. The ISI model
is deemed adequate if the deviation D of the INR calculated with the ISI model
from the INR calculated with the International Standard is not greater than 10%
(see equation 19 in Appendix 2). If the deviation of the INR calculated with the
ISI model is greater than 10%, it is advisable to use a different model according
to Tomenson (42).
The sequence of steps in the statistical evaluation is as follows.
1. Calculate preliminary orthogonal regression line (20 normal samples +
60 patient samples).
2. Detect outliers defined as points with a perpendicular distance
greater than 3 residual standard deviations from the preliminary line.

3. Remove outliers in one step and recalculate the orthogonal regression
line (normal samples + patient samples) and ISI.
4. Calculate each patient’s INR using the PT determined with the
International Standard.
5. Calculate each patient’s INR using the PT determined with the system
being calibrated and the ISI from step 3.
6. Calculate each patient’s mean INR from steps 4 and 5.
7. Remove patients with mean INR < 1.5 or mean INR > 4.5.
8. Recalculate the orthogonal regression line (normal samples +
patient samples) and ISI.
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To assess the adequacy of the ISI model, calculate the deviation D of the
INR determined with the ISI model from the true INR for INR = 2.0 and for
INR = 4.5. If D < 10%, the ISI model is deemed to be adequate. If D > 10%, use
Tomenson's formula for INR calculation (see Appendix 2).
7.2 Procedure 2: Calibration of individual

batches of thromboplastin
Calibration of individual batches of thromboplastin may be carried out with
pooled normal plasmas and pooled coumarin plasmas or plasmas artificially
depleted of vitamin K-dependent coagulation factors (38, 39). The number of
plasma pools required for precise calibration is, in general, much smaller than the
number of fresh individual plasma samples required for procedure 1. The scatter
of data points about the regression line is relatively small because the batch to be
calibrated is very similar to the working Reference Preparation and/or because
the biological variation caused by individual samples is reduced by the pooling
of plasmas. It has been reported that lot-to-lot calibration of bovine and rabbit
thromboplastins could be performed with as few as three plasma pools (38, 39),
but the accuracy of such a simplified procedure may depend on the quality of the
pooled plasmas and the thromboplastin being calibrated. It is recommended that
any procedure using pooled or artificially depleted plasmas be validated against
the fresh plasma procedure (procedure 1).
7.2.1 Pooled plasma

7.2.1.1 Properties of pooled normal plasma
Plasma should be obtained from healthy adults and should comply with the
appropriate section of the Requirements for the collection, processing and quality
control of blood, blood components and plasma derivatives (48). The normal
plasmas for pooling should be obtained from at least 20 different donors with
an approximately equal number of males and females. Nine volumes of blood
should be decalcified with one volume of 109 mmol/l trisodium citrate solution.
The packed-cell volume-fraction should be between 0.35 and 0.45.
The final preparation should be platelet-poor plasma, which has been
freeze-dried or frozen (at –40 °C or below) in suitable containers. The stability
of deep-frozen plasma should be monitored regularly by testing the PT. Thawing
of deep-frozen plasma should be done in a water bath at 37 °C for a fixed time
depending on the volume in each container. After reconstitution or thawing,
the pH should not be lower than 7.3 and should not exceed 7.9, and the plasma
should not show any shortening or prolongation of clotting times for at least
2 hours when held at ambient temperature (49). The stability of freeze-dried
normal plasma should be checked by accelerated degradation tests. Such plasma
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should not show a prolongation of PT of over 5% after storage for 4 weeks at

37 °C. The factor V content should be between 60% (or 60 IU/dl) and 140% (or
140 IU/dl) of the average content of fresh normal plasma (50).
7.2.1.2 Properties of pooled coumarin plasma

Pooled coumarin plasma is obtained from patients who have been on oral
anticoagulant therapy for at least 6 weeks. Coumarin plasmas for pooling should
be obtained from at least 20 different donors.
Plasma should not be obtained from donors with a history of jaundice
or from those with plasma-lipid abnormalities. The collection of plasma, the
properties of the final preparation and the stability of the freeze-dried pools are
the same as described above for pooled normal plasma.
The INR of the pooled plasma should be stated, as should the
thromboplastins used for its assignment. It should be noted that the INR value
of a freeze-dried plasma usually depends on the thromboplastin used for its
assignment (51–53). At least two different plasma pools, having an INR between
1.5 and 4.5 and with a difference of at least 1.0 in their INRs, in combination with
one normal plasma pool are necessary for the calibration procedure.
The factor V content, opacity and citrate concentration for blood
decalcification should comply with the requirements for normal plasma (see
above).
7.2.1.3 Freedom from infectious agents

The plasma should be shown to be free from hepatitis B surface antigen, antibodies
to human immunodeficiency viruses (HIV-1 and HIV-2) and antibodies to
hepatitis C virus.
7.2.2 The test

The test should be carried out using the same procedure as described for
procedure 1 (see section 7.1.3). An example of the protocol for the recording of
the results is given in Appendix 3. The procedure should be repeated on at least
four separate occasions (40), with fresh reagents used on each occasion. At least
three plasma pools should be used to permit the testing of linearity.
Freeze-dried plasma pools should be reconstituted at least 15 minutes
before the actual test. Plasma that has been frozen and subsequently thawed,
or reconstituted freeze-dried plasma, should not be centrifuged, and unused
reconstituted or thawed material should be discarded after 2 hours.
7.2.3 Statistical evaluation

An orthogonal regression line should be calculated on the basis of the ln PT
value of the pooled plasmas. Individual determinations should be entered
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when multiple determinations for each plasma pool are available. Ln PT for
the working reference thromboplastin system is plotted on the vertical axis and
ln PT for the test batch of thromboplastin on the horizontal axis. Any samples
with a perpendicular distance greater than 3 residual standard deviations from
the regression line should be removed. After removal of such samples, the final
orthogonal regression line is calculated.
To define the ISI of a batch of thromboplastin, a sufficient number of
tests should be carried out to obtain a within-laboratory coefficient of variation
for the slope of the orthogonal regression line of 3% or less. The recommended
procedure for calculation of the ISI is given in Appendix 3.
7.3 Procedure 3: Local system calibration using certified plasmas

Laboratories may calibrate their own local system (i.e. instrument/thromboplastin
combination) using certified plasmas supplied by manufacturers or reference
laboratories. A certified plasma is a deep-frozen or lyophilized plasma with an
assigned PT or INR value. Two procedures using certified plasmas have been
described and are summarized below.
Local test system ISI calibration – this procedure is a modification of the WHO
method for ISI determination. In a set of plasmas, each plasma is assigned a
manual PT value by the manufacturer or reference centre using an International
Standard for thromboplastin. In the local laboratory, the PTs of each plasma are
measured with the local instrument/reagent combination, and the two sets of
PTs are plotted on a log/log plot. The slope of the orthogonal regression line
is used to determine the local ISI (see Appendix 2), which can then be used
for subsequent determination of INRs from the local PTs and MNPT (7, 8,
54–56). An underlying assumption of the WHO orthogonal regression model
is that a single line describes the relationship between log(PT) of abnormal and
normal plasmas. If there is a significant deviation of the two calibration lines
(i.e. abnormal-only and normal/abnormal combined), a correction according to
Tomenson should be applied (42, 57).
“Direct” INR determination – this procedure involves assignment of INR values
to a set of plasmas with the manual method and an international thromboplastin
standard by the manufacturer or reference centre. The PTs of these plasmas
are measured locally using the local instrument/reagent combination, and the
local test system PTs are plotted against the reference INRs on a log/log plot. An
orthogonal regression line is calculated, and the INRs of patients’ plasmas can be
interpolated directly from local PTs using this line, without the need for ISI or
MNPT determination. Although many studies of direct INR determination were
performed with linear rather than orthogonal regression, the latter is preferable
from a theoretical point of view (see section 7.3.2.5) (53, 58–66).
289
A number of studies have shown that use of either of these procedures can
considerably reduce inter-laboratory imprecision in INR determination (8, 59–61,
67, 68). For example, in one study the mean deviation of 95 local systems from
the “true” INR was +14.4% with the manufacturers’ ISI, but was reduced to
+1.04% with the local ISI (8). In another study, the inter-laboratory coefficient
of variation of the INR was reduced from 12% with the manufacturers’ ISI to 6%
using direct INR determination with a certified plasma procedure (59).
It should be recognized that there are a number of different ways in
which plasmas can be prepared and certified. The following sections describe
the various methods of preparation, certification and use, and their advantages
and disadvantages.
7.3.1 Preparation of certified plasmas

7.3.1.1 Type of plasma – AVK (from patients on anti-vitamin K therapy) or
artificially depleted of prothrombin complex factors (ART)
The intention is for certified plasmas to be as similar as possible to fresh
plasmas from patients, thus on theoretical grounds, AVK plasmas might be
preferred, although for practical reasons these have to be pooled, rather than
individual, donations. In some studies where the two types of plasmas have been
compared, AVK plasmas give closer agreement with fresh plasmas and better
inter-laboratory agreement than artificially depleted plasmas (51, 69). Artificially
depleted plasmas have several advantages over plasmas from patients on oral
anticoagulants, including availability of larger volumes, wider selection of PT
values across the therapeutic interval, and the possibly reduced risk of virus
transmission (70). It can be argued that larger volumes of AVK plasmas could be
obtained by pooling donations from patients on anti-vitamin K therapy, but this
procedure would make a spectrum of INR values more difficult to obtain because
of averaging of individual INRs in such a pool.
The European Concerted Action on Anticoagulation (ECAA) prepared
depleted plasmas using artificial depletion of normal human plasma by selective

adsorption of vitamin K-dependent clotting factors with barium sulfate to
provide a range of values which spanned the therapeutic interval (54). The ECAA
found that there is a small difference between the results obtained with ECAA
lyophilized artificially depleted plasmas and lyophilized AVK plasmas in ISI value
assignment, but both of these lyophilized plasmas differed by a similar amount
from a conventional fresh plasma ISI calibration (54). The mean calibration
slopes with both types of lyophilized plasma were generally higher than with
fresh AVK plasmas but the differences were not great in clinical terms. It should
be noted that the ECAA study was performed with one combination of a human
brain International Reference Preparation and recombinant thromboplastin and
the manual technique, and that the conclusions may not be applicable to all other
reagent/instrument combinations.
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The reliability of artificially depleted plasmas and AVK plasmas depends

on the method of preparation and certification.
7.3.1.2 Method of preparation – frozen or freeze-dried

Although lyophilization seems a simple solution to the difficulties associated
with storage and shipment of certified plasmas, there are problems associated
with lyophilized materials.
Studies have shown that the INR of fresh plasmas is largely unchanged
on freezing, whereas freeze-drying may change the INR significantly, depending
on the method of freeze-drying and the thromboplastin/instrument combination
used (50, 71–73). Buffering of plasmas shortly after blood collection can reduce
but not eliminate changes after freeze-drying. The magnitude of the changes is
not the same for all reagents or instruments. The measured INR of lyophilized
certified plasmas depends on the thromboplastin reagent and instrument used.
The use of RBT/90 presented problems relating to its poor end-point particularly
with lyophilized plasmas giving long PTs.
The widespread use of frozen plasmas presents logistical difficulties
due to their potential instability, although in some countries frozen certified
plasmas have been used with success regarding the reduction of interlaboratory
imprecision (63, 67).
Freeze-dried plasmas represent the most practical option for general
laboratories and their use has been associated with reduced interlaboratory
imprecision in several studies.
7.3.1.3 Citrate concentration

It is well known that citrate concentration can affect the PT, especially of high
INR plasmas (74, 75). Furthermore, citrate concentration has a variable effect
on the ISI, but the magnitude of the effect is not the same for all reagents and
instruments (75–78). The recommended citrate concentration for the collection
of blood (1 volume of citrate solution + 9 volumes of blood) for PT is 0.109 mol/l
(3.2%), although concentrations in the range 0.105–0.11 mol/l can be accepted
(77), and the citrate concentration of certified plasmas should be as close as
possible to that in fresh plasma collected in the above anticoagulant (70). Citrate
concentrations of 0.129 mol/l (3.8%) should not be used for PT tests.
7.3.1.4 Number of plasmas

The number of plasmas depends on the purpose for which they are used.
Local test system ISI calibration – according to the present Guidelines, to define
the ISI of a working thromboplastin, the number of tests carried out should be
sufficient to obtain a within-laboratory coefficient of variation (CV) for the slope
291
of the orthogonal regression line of 3% or less (see section 7.1.4). In an ECAA

study of lyophilized artificially depleted and individual AVK plasmas, it has been
shown that the requirement for 60 lyophilized abnormal samples for a full WHO
calibration can be reduced to 20 if combined with results from seven lyophilized
normal plasmas; reductions below this number were associated with decreased
precision of the calibration line and hence increased variability of the INR (79).
However, the use of pooled AVK plasmas may reduce the scatter of individual
plasmas about the line (80), and with pooled plasmas and repeat testing it is
possible that a lower number could be used, e.g. acceptable precision has been
achieved with six pooled AVK plasmas containing at least 50 patient samples
in each pool and two pooled normal plasmas if these were analysed on at least
3 days (40).
“Direct” INR determination – for “direct” INR determination a smaller number
of pooled plasmas can be used. Studies have shown improved inter-laboratory
variability with as few as six (63), five (53, 60, 61, 65), four (66), three (58),
or two (59, 81) plasmas, but considering that one of the plasmas should be a
normal plasma and that at least three plasmas are required to define a line, a set
of one normal and at least three abnormal plasmas is recommended. One study
documented the within-laboratory imprecision of the slope of a calibration line
(one normal + three abnormal plasmas): the CV ranged from 0.1% to 4.6% (64).
The number of donations in each pool should be at least 10, but higher numbers
are preferable to ensure normal levels of factor V.
For both procedures it is important that the abnormal plasmas be chosen to cover
the range of 1.5–4.5 INR. The fibrinogen and factor V content should be between
60% and 140% of the average content of fresh normal plasma (50).
7.3.2 Certification (value assignment) of plasmas

Manufacturers or suppliers are responsible for certification, i.e. value assignment

to the plasmas.
7.3.2.1 Thromboplastins for certification

WHO standard or European reference thromboplastins should be used directly
if possible. Assuming that the certified plasmas are intended for use with the
various types and species of thromboplastin, the two types of WHO standard
preparations should be used (human and rabbit). If insufficient supplies of
WHO or European standards are available, national or secondary standards
can be used provided they have been calibrated against the appropriate WHO
or European thromboplastin standards in a multicentre study. If the plasmas
are intended for use with only one type of thromboplastin (e.g. human), the
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appropriate thromboplastin standard preparation should be used. Several studies

have shown that the INR value for some lyophilized plasmas obtained with the
previous rabbit standard thromboplastin (RBT/90) was greater than the INR
obtained with the human and bovine standard preparations (11, 51, 69, 82),
especially for artificially depleted plasmas (52). Certified INRs for lyophilized
artificially depleted plasmas determined with the ECAA rabbit plain reference
thromboplastin were approximately 15% greater than those determined with the
recombinant human International Standard and approximately 30% greater than
those determined with the bovine combined International Standard (65).
For use with one manufacturer’s thromboplastin reagent only, certification
with the manufacturer’s calibrated reagent is acceptable; such “reagent-specific”
value assignments have been shown to be reliable in recent collaborative
studies (53, 64). The manufacturer’s thromboplastin reagent used for reagent-
specific certification of plasmas should be calibrated by at least two independent
laboratories using the original WHO procedure (see section 7.1).
Although thromboplastin standards should be used for the assignment
of values, the certified plasmas should be tested for suitability with a variety of
commercial thromboplastins before release for general use (see section 7.3.3).
7.3.2.2 Number of laboratories

It is recommended that three to five laboratories should be involved in the
certification process for each set of plasmas. An individual laboratory’s mean
value should differ by no more than ± 10% of the overall mean (in terms of INR)
obtained with a given thromboplastin reagent. If the difference is greater than
10%, the divergent individual laboratory’s value should not be used.
7.3.2.3 Manual technique or instruments

The manual tilt-tube method must be used for International Standard preparations,
as described in section 7.1.3. Once certified, the plasmas should be tested for their
suitability with various reagent/instrument combinations. Where certification of
plasmas is done with one manufacturer’s reagent only, an instrument may be
used. In this case the reagent/instrument combination must have been calibrated
using the original WHO procedure (see section 7.1).
7.3.2.4 Single or multiple values

For the local test system ISI calibration, the actual values of the PTs of the certified
plasmas will differ according to the species of the standard thromboplastin used,
and therefore PT values must be independently certified for the different species.
For the direct INR determination approach, the INR values of the plasmas should
theoretically be the same whichever reference thromboplastin reagent is used.
293
In practice, differences in INRs obtained using different thromboplastins have

been observed with some freeze-dried plasmas; results should not be averaged
into a single INR if the INRs obtained with individual standard reagents differ
by more than 10% from the mean. Large discrepancies between INRs obtained
with different thromboplastins may indicate that the plasmas are unsuitable for
use with thromboplastins of all types. It should be noted that the manufacturer
or supplier of the certified plasmas should clearly specify the set of reagent/
instrument combinations with which their materials may be reliably used (83)
(see section 7.3.3).
7.3.2.5 Orthogonal regression

Orthogonal regression is used if each coordinate is subject to independent
random error of constant variance (41, 84), e.g. PT measurements with two
different reagents recorded by the same instrument or operator. Linear regression
is used when one of the values is fixed, i.e. essentially without error. The use of
certified plasmas does not conform completely to either of these models, but
it is important to recognize that apparently “fixed” values of these plasmas are
themselves subject to error. Therefore, orthogonal regression should be used for
both procedures, i.e. local system ISI calibration and direct INR determination.
The equations for orthogonal regression are given in Appendix 2.
7.3.2.6 International reference plasmas

At present there are no established international reference plasmas. Work has
begun on the development of reference plasmas for “direct” INR assignment (58,
82). These could then be used for direct certification of batches of commercial
plasmas, in the same way as for coagulation factor assays. One difficulty, as
mentioned above, is that of preparing lyophilized plasmas with the same
properties as fresh plasmas, and it may be that frozen plasmas have to be used.
Furthermore, for long-term use, the stability of such reference plasmas would
need to be carefully checked. In the meantime, commercial plasmas will continue
to have their values assigned as described above.
It should be noted that the validity of lyophilized certified plasmas may
be limited to certain combinations of thromboplastins and coagulometers, and
may not be generalizable to all other reagent/instrument combinations.
7.3.3 Validation of certified plasmas

Each set or batch of certified plasmas intended for either local test system ISI
calibration or direct INR determination must be validated before release (21).
The validation should be the responsibility of the manufacturer or supplier who
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may seek help from expert laboratories. The validation process should go through
the following steps.
1. Ten or more fresh plasmas from patients on long-term oral

anticoagulants are selected to represent the full therapeutic range of
anticoagulation.
2. The INR of these fresh plasmas is determined with an appropriate
International Standard for thromboplastin, and the mean value
(INRR ) is calculated.
3. The INR of the same fresh plasmas is also determined with a variety
of commercial reagent/instrument combinations following the
certified plasma procedure (either ISI calibration or direct INR
determination). The mean value (INRC) is calculated.
4. Finally, paired INR values obtained with the International Standard
and with the local system are compared to assess their agreement
using Bland and Altman’s procedure (85).
If the relative difference between the mean values INRR and INRC, i.e.
2(INRR – INRC)/(INRR + INRC), is 0.1 or less, the set of certified plasmas is
considered acceptable and may be released for local ISI calibration or direct INR
determination. New batches of the same type of preparation should be validated
according to the above procedure.
Studies on simplified local calibration with certified plasmas have been
published (65, 81), but the value of the studies is limited if the sets of plasmas have
not been validated with fresh plasmas from patients as described in this section.
7.3.4 Use of certified plasmas in clinical laboratories

7.3.4.1 Quality assessment
An important use of certified plasmas is to perform internal or external quality
assessment, i.e. to determine whether or not corrective action is necessary (83,
86). For quality assessment, a set of three to five certified plasmas with INRs in
the range 1.5–4.5 would be required. The INRs of the certified plasmas should
be calculated from local PTs and routine ISI, and compared with the certified
INR values. If the differences between routine INR and certified INR are greater
than 15%, local ISI calibration or direct INR correction should be performed.
In addition, the manufacturer of the reagent and certified plasmas should be
informed about the discrepant results. Quality assessment with certified plasmas
should be performed regularly at intervals of no more than 6 months and should
be repeated whenever there is a change in reagent batch or instrument (e.g.
servicing, modification, or new instrument). It should be realized that errors
295
caused by local pre-analytical factors (e.g. divergent citrate concentration or

contamination of citrate with divalent cations) cannot be corrected by certified
plasma procedures (87).
7.3.4.2 Method for local ISI determination

PTs should be measured in quadruplicate in the same working session, with the
local instrument/reagent combination for the full set of normal and abnormal
plasmas. Duplicate PT measurements are permitted if the imprecision of the PT
system is not greater than 2% CV. It is recommended to repeat the measurements
over three sessions or on 3 days to control for day-to-day variation. Mean local
PTs should be plotted on the horizontal axis against the certified PT values on
the vertical axis (log scales). Tomenson’s test should be performed to test the
hypothesis that the mean log(PT) of the certified normal plasmas lies on the
same line as the log(PT) of the certified abnormal plasmas (42, 56, 57). If the
hypothesis is not confirmed, Tomenson’s correction formula should be applied
(42, 56, 57, Appendix 2). Like-to-like comparison should be used wherever
possible, i.e. if the local reagent is a human thromboplastin, the certified
values should be those determined with a human Reference Reagent. If the
INR difference between the routine ISI and the local ISI calibration procedure
is greater than 10%, the calibration should be repeated. If the discrepancy
persists, the manufacturer or supplier of the local thromboplastin reagent and
coagulometer and certified plasmas should be informed. After consultation with
the manufacturer of the certified plasmas and, if possible, an expert laboratory,
the clinical laboratory should decide which materials and methods should be
used for local ISI calibration.
7.3.4.3 Method for direct INR measurements

This method is simpler to use than the one described above as it does not require
local ISI and MNPT determinations. PTs should be measured in duplicate with the
local instrument/reagent combination for each certified plasma. To allow for day-
to-day variation, the measurements should be repeated on at least three different
days. Mean PTs should be plotted on the horizontal axis against the certified INR
values on the vertical axis (log scales), and an orthogonal regression line derived.
Manufacturers of certified plasmas should state for which thromboplastin brands
the certified values are valid and provide instructions on how to calculate the
calibration line. The INRs of patients’ plasmas should be calculated from the
measured PTs. If the INR difference between the routine ISI procedure and the
direct determination is greater than 10%, the certified plasma procedure should
be repeated. If the discrepancy persists, the manufacturer or supplier of the
local thromboplastin reagent and coagulometer and certified plasmas should
be informed. After consultation with the manufacturer of the certified plasmas
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and, if possible, an expert laboratory, the clinical laboratory should decide which
materials and methods should be used for direct INR measurement.
8. The use of calibrated thromboplastins

in clinical practice
It is possible to express prothrombin-time results on a common scale, i.e. the
International Normalized Ratio (INR), provided that the ISI of the thromboplastin
and the method used are known. The following formula is used:
INR = (PT/MNPT)ISI
where PT is the patient’s PT and MNPT is the mean normal PT determined with
the same thromboplastin and method. The use of the INR enables comparisons to
be made between results obtained using different thromboplastins and methods.
It is a misconception, however, that for an individual patient’s plasma the INR
will always be identical when determined with different thromboplastins and
methods (42, 88). Different thromboplastins vary greatly in their responsiveness
to individual vitamin K-dependent clotting factors, i.e. factors II, VII and X,
as well as to some factors that are not dependent on vitamin K, e.g. factor V.
Discrepancies between INRs determined with different thromboplastins arising
from these biological variations and from additional technical errors are therefore
not unexpected.
All medical staff and health auxiliaries involved in managing oral
anticoagulant treatment should be encouraged to use the INR system. It should
be appreciated, however, that this system can be accurate only in the INR range
explored by the calibration procedure, i.e. 1.5–4.5.
Manufacturers of commercial reagents should state on the package insert
the ISI of the relevant batch of thromboplastin together with the Reference
Preparation against which it has been determined and the instrument for which
it is valid.
Authors
The first draft of these Guidelines was prepared by: Dr V. Chantarangkul, Angelo
Bianchi Bonomi Hemophilia and Thrombosis Center, Department of Internal
Medicine, University and Foundation IRCCS Ca’ Granda Ospedale Maggiore,
Milan, Italy; Dr A. Tripodi, Angelo Bianchi Bonomi Hemophilia and Thrombosis
Center, Department of Internal Medicine, University and Foundation IRCCS
Ca’ Granda Ospedale Maggiore, Milan, Italy; and Dr A.M.H.P. van den Besselaar,
Department of Thrombosis and Haemostasis, Leiden University Medical Center,
Leiden, the Netherlands.
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Acknowledgements
The Subcommittee for Control of Anticoagulation of the Scientific and
Standardization Committee of the International Society on Thrombosis and
Haemostasis supported the revision of the Guidelines.
Secretariat – Dr A. Padilla, Blood Products and Related Biologicals
programme, Quality Assurance and Safety: Medicines, World Health Organization.
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302
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303
Appendix 1
Criteria which may assist clinical laboratories in the choice
of a reagent
The purpose of this appendix is to provide criteria that are useful for a clinical
laboratory to apply when choosing a thromboplastin reagent. These criteria relate
to the manufacturer of the reagent providing standard information to the user on
the following:
■ instrument-specific International Sensitivity Index (ISI) values for
the reagent;
■ which International Standard has been used for the ISI calibration;
■ whether the adequacy of the ISI model has been checked;
■ local-system calibration (if an instrument-specific ISI is not available).
The manufacturer’s provision of the following may assist with the choice
of a set of certified plasmas:
■ information on the International Standards and other thromboplastin
reagents that have been used for the certification and the validation
of the set of plasmas;
■ a statement that the set of certified plasmas has been validated;
■ a value of the relative mean International Normalized Ratio (INR)
difference obtained in the validation procedure (according to
section 7.3.3 of the main text);
■ a list of the thromboplastin reagent brands for which the set of
plasmas can be used (for both quality assessment and local-system

calibration);
■ instructions for calculation of local-system ISI or direct INR
measurement;
■ a spreadsheet for performing the calculations.
304
Annex 6
Appendix 2
Example of the use of the suggested method for
reporting the data for the calibration of any system or
a secondary standard of thromboplastin against an
International Standard preparation
Thromboplastins: 1. Recombinant human thromboplastin
secondary standard
2. Third WHO International Standard for
thromboplastin (human, recombinant, plain)
(rTF/95) with an established ISI = 0.94.
End-point recording: 1. Automated photoelectric coagulometer for

secondary standard
2. Manual (tilt-tube) technique for rTF/95.
The tests were conducted on five different days (Table 1). On each day, fresh
samples from four healthy subjects and 12 patients were tested (plasma samples
from healthy subjects are referred to as “normal”). On each day, different subjects
were selected. The automated coagulometer and manual determinations were
performed more or less simultaneously.
Table 1
Prothrombin times for the calibration of a secondary standard of recombinant human
thromboplastin
Date Plasma rTF/95 Secondary standard

23 February 2009 Normal 1 12.8 10.8
Normal 2 13.2 10.5
Patient 1 33.0 18.5
Patient 2 50.8 27.8
Patient 3 32.0 18.7
Patient 4 46.2 25.4
305
Table 1 continued
23 February 2009 Patient 5 35.6 21.7
Patient 6 59.8 34.4
Patient 7 45.8 24.9
Patient 8 31.5 18.6
Patient 9 40.4 22.1
Patient 10 49.8 28.2
Patient 11 56.4 27.0
Patient 12 47.8 24.4
Normal 3 14.9 11.6
Normal 4 14.0 10.6

Normal 6 12.8 11.2
Patient 13 37.4 21.2
Patient 14 29.4 17.7
Patient 15 37.2 21.8
Patient 16 49.4 25.9
Patient 17 35.4 20.0
Patient 18 58.8 32.4

Patient 19 41.6 23.9
Patient 20 35.2 19.3
Patient 21 44.7 25.5
Patient 22 47.4 58.1
Patient 23 38.8 21.3
Patient 24 45.0 25.2
Normal 7 13.3 11.0
Normal 8 14.8 11.3
306
Annex 6
Table 1 continued
Normal 10 13.0 10.9
Patient 25 42.0 22.1
Patient 26 31.0 17.7
Patient 27 39.3 20.4
Patient 28 59.0 31.0
Patient 29 35.3 19.4
Patient 30 48.3 25.2
Patient 31 46.5 23.5
Patient 32 52.0 26.4
Patient 33 42.3 22.6
Patient 34 45.7 23.1
Patient 35 50.7 26.3
Patient 36 46.3 22.5
Normal 11 13.2 11.0
Normal 12 13.4 11.1
27 February 2009 Normal 13 13.2 10.3
Normal 14 11.4 10.4
Patient 37 39.0 21.6
Patient 38 32.0 18.7
Patient 39 45.2 24.6
Patient 40 35.8 20.8
Patient 41 40.0 22.3
Patient 42 25.8 16.3
Patient 43 64.0 33.2
Patient 44 51.0 28.1
Patient 45 41.4 23.3
307
Table 1 continued
27 February 2009 Patient 46 38.4 20.9
Patient 47 48.4 25.9
Patient 48 33.0 19.0
Normal 15 12.9 11.2
Normal 16 13.8 11.1
2 March 2009 Normal 17 15.0 11.9
Normal 18 12.8 10.0
Patient 49 35.8 19.2
Patient 50 43.0 23.1
Patient 51 44.3 24.4
Patient 52 32.3 18.5
Patient 53 43.3 23.9
Patient 54 30.0 17.6
Patient 55 50.0 27.9
Patient 56 43.0 22.8
Patient 57 28.6 18.0
Patient 58 41.6 23.1
Patient 59 39.0 21.4
Patient 60 38.8 22.9
Normal 19 13.1 10.9

Normal 20 13.3 10.9
308
Annex 6
Calculations
The International Sensitivity Index of the secondary standard (ISIw) is obtained
by plotting the prothrombin times of the two thromboplastins on logarithmic
scales as shown in Figure A6.2, fitting a straight line of the form:
Y = A + BX (1)
and estimating the slope B. The recommended method involves estimation of a
linear structural relation (also called an “orthogonal regression equation”). With
this technique, the slope B can be estimated as follows.
Consider a set of N independent observations (xi , yi ), where i = 1, 2, 3,..., N; for
N paired tests, yi represents the natural logarithm of the measured prothrombin
time of the International Standard, and xi that of the secondary standard. Write
x0 , y0 , for the arithmetic means of the N values of xi , yi , respectively. Write Q1 , Q2 ,
for the sums of the squares of (x i – x0) and (yi – y0), respectively, and P for the
sum of their products (x i – x0) (yi – y0). These quantities are all that is necessary
for computing a and b, the least-squares estimators for the parameters A and B
of equation (1). Now define:
E = (Q2 – Q1)2 + 4P2 (2)
Then
b = (Q2 – Q1 + √E)/2P (3)
and
a = y0 – bx0 (4)
are the estimators that minimize the sum of the squares of the perpendicular
distances of the N points from the line represented by equation (1). The variance
of b is given by:
Var(b) = {(1 + b2)P + NbV}bV/P2 (5)
where V is defined as
V = (Q2 – bP)/(N – 2) (6)
The standard error of b (s b ) is the square root of Var(b). The coefficient of variation
of b is CV(b) = 100 × (s b /b).
If t is a deviate from the t-distribution, with (N – 2) degrees of freedom and
at a chosen probability, approximate confidence limits for B can be obtained by
setting an interval t × s b on either side of b.
The residual standard deviation is the square root of V. Outlying points should
be rejected if their vertical (i.e. perpendicular) distance from the calibration line
is greater than 3 × √V.
309
The ISI w for the secondary standard is calculated as follows:

ISIw = ISIIS × b (7)
where ISIIS is the ISI of the International Standard.
The prothrombin-time ratio for a given patient (i) with the secondary standard
can be estimated according to the equation
Rw,i = exp(xi – xn ) (8)
where xn is the mean natural logarithm of the prothrombin times of the normals.
Likewise, the prothrombin-time ratio with the International Standard can be
estimated according to the equation
RIS,i = exp(yi – yn) (9)
where yn is the mean natural logarithm of the prothrombin times of the normals.
If the same linear structural relation is valid for patients and normals it can be
shown that the calibration model implies a relationship between prothrombin-
time ratios of the form
RIS = (Rw)b (10)
where Rw is the prothrombin-time ratio obtained with the secondary standard,
and RIS is the prothrombin-time ratio for the International Standard. A similar
equation can be written for the prothrombin-time ratio of the First WHO
International Reference Preparation of thromboplastin (human, combined) coded
67/40:
R 67/40 = (RIS )ISI IS (11)
Equations (7), (10) and (11) are the base for calculation of the INR according to
the ISI calibration model:
INRw = (Rw)ISIw (12)

One of the underlying assumptions of the ISI calibration model is that a single
line describes the relationship between logarithms of prothrombin times of both
normal and patient plasmas. Thus the line describing the relationship between
logarithms of patient prothrombin times should ideally pass through the mean
of the logarithms of normal prothrombin times. In the case of marked deviation,
the assignment of an ISI would not be meaningful. The natural way to overcome
this problem is to introduce a scale parameter and use a model for prothrombin
ratios of the form
RIS = ed × (Rw )bʹ (13)
The above model is referred to as Tomenson’s (1).
310
Annex 6
Tomenson’s model leads to the following equation for calculation of the

corrected INRw,p :
INRIS = INRw,p = {ed × (Rw )bʹ }ISI IS (14)
Clearly equation (10) is a particular case of equation (13) but the generalized
model will also cope with data sets for which the line describing the relationship
between logarithms of patient prothrombin times does not pass through the
mean of the logarithms of normal prothrombin times. It can be shown that d in
equations (13) and (14) is estimated as
d = aʹ + bʹxn - yn (15)
where xn and yn are the mean natural logarithms of the prothrombin times of
normals determined with the secondary standard and the International Standard,
and aʹ and bʹ the intercept and slope of the “orthogonal regression line” calculated
using only the patient data.
Example
For the full set of data shown in Table 1, the various parameters were calculated
according to equations (3), (4), (5), (6), (7) and (15). The results are shown in
Table 2. The next step is to detect any outliers. In this example there was one data
pair (patient number 22) for which the distance to the line was greater than 3×√V.
This data pair was excluded. The parameters calculated for the remaining 79 data
pairs are shown in Table 2. The ISIw calculated for the remaining 79 data pairs
was 4.8% greater than the preliminary ISI calculated with the one outlier included.
Each patient’s INR can be calculated in two ways. The first is to calculate INR
from the PT measured with the International Standard:
INRIS,i = (RIS,i )ISI IS (16)
The second way of calculating each patient’s INR is by using the PT measured
with the secondary standard:
INRw,i = (Rw,i )ISIw (17)
Now it is possible to calculate the mean INRm,i for each patient’s sample:
INRm,i = (INRIS,i + INRw,i )/2 (18)
For example, the INR for patient number 43 is 4.32 with the International
Standard and 4.70 with the secondary standard. The mean INR is 4.51 which is
just at the limit of the therapeutic range. There are no other patients for whom the
mean INR is outside the 1.5–4.5 interval.
311
The relative difference D between the INR calculated according to equation (12)
and equation (14) is given by:
D = 100 × (exp(ISIw × ((((yn + (ln(INRIS)/ISIIS))–aʹ)/bʹ)-xn))–INRIS)/INRIS (19)
In this example the orthogonal regression line calculated for 59 patient data
pairs did not pass through the mean of the normal data pairs (see Figure 1).
The difference D calculated at INRIS = 2 is –11% and at INRIS = 4.5 it is 7.8%. It
is therefore important to consider the alternative calibration model according
to equation (13). By substituting the values from Table 2 in equation (14) the
following formula is obtained:
INRw,p = {e 0.2431 × (R w)1.2024} 0.94 (20)
Table 2
Parameters calculated for the calibration of a secondary standard (see Table 1)
20 normal samples + 20 normal samples + 59 patient

60 patient samples 59 patient samples samples
(full data set) (outlier excluded)
Intercept –0.7598 –0.9432 –0.0386
Slope 1.4216 1.4889 1.2024
CV of slope 3.3% 1.6% 3.4%
ISI IS 0.94 0.94 0.94
ISI w 1.336 1.400 –
√V 0.0865 0.0396 0.0322
d – – 0.2431
yn 2.602 2.602 2.602

xn 2.399 2.399 2.399
312
Annex 6
Figure 1
Log‑log plot of prothrombin times for determination of ISI
Reference
1. Tomenson JA. A statistician’s independent evaluation. In: van den Besselaar AMHP, Lewis SM,
Nijhoff, 1984: 87–108.
313
Appendix 3
Example of the use of the suggested method for reporting
the data for the calibration of individual batches of
thromboplastin
Thromboplastins: 1. Rabbit brain thromboplastin secondary
standard
2. New batch of rabbit brain thromboplastin
End-point recording: Automated photoelectric coagulometer
Pooled coumarin plasmas: lot 960606, 1–5 (deep-frozen)
Pooled normal plasma: lot 900423 (deep-frozen)
The International Sensitivity Index (ISI) and mean normal prothrombin time
(MNPT) of the rabbit brain thromboplastin secondary standard used with this
automated photoelectric coagulometer are 1.31 and 12.7 seconds, respectively.
The tests were conducted in four separate runs. For each run,
thromboplastins were freshly reconstituted and deep-frozen plasmas were freshly
thawed. Since the secondary standard and the new batch were both timed with
the same photoelectric coagulometer, the order in which the two reagents were
tested was alternated from one run to the next. This was done to avoid any bias
due to possible instability of the thromboplastins and pooled plasmas.
Calculation
The ISI of the new batch (ISIb) is calculated as ISIb = ISIw × b, where b is the
slope of the straight line fitted to a double-logarithmic plot of the prothrombin
times in Table 1, with the prothrombin times for the secondary standard and
the new batch being shown on the vertical and horizontal axes, respectively. The
formula for b is given by equation (3) in Appendix 2. The standard error of b is
obtained from equation (5) in Appendix 2. The coefficient of variation (%) of b
is 100 × (sb/b).
Example
For the data from Table 1, the calculated residual standard deviation is
0.02482. One pair of determinations for plasma lot no. 960606-5 (run no. 3)
314
Annex 6
has a perpendicular distance from the line greater than three residual standard
deviations. When this pair is excluded, the calculated value for b is 0.9538. The
ISI for the secondary standard is given as 1.31. Thus, the ISI for the new batch is
estimated as 1.31 × 0.9538 = 1.25. The standard error for b is calculated as 0.0130.
The coefficient of variation for b is 100 × (0.0130/0.9538) = 1.36%.
Table 1
Prothrombin times (PT) for the calibration of a new batch of rabbit thromboplastin
Secondary standard New batch

Run Plasma Order of testing PT Order of testing PT
no. lot no. (within‑run) (within‑run)
1 900423 1 14.0 7 15.1
960606-1 2 20.5 8 21.5
960606-2 3 29.1 9 31.5
960606-3 4 32.9 10 36.4
960606-4 5 36.2 11 41.0
960605-5 6 39.7 12 44.6

2 900423 7 14.1 1 15.4
960606-1 8 20.3 2 22.6
960606-2 9 29.5 3 31.2
960606-3 10 32.8 4 37.6
960606-4 11 37.3 5 40.8
960605-5 12 39.8 6 44.5

3 900423 1 14.0 7 15.0
960606-1 2 20.0 8 21.5
960606-2 3 28.1 9 32.1
960606-3 4 31.8 10 34.2
960606-4 5 35.9 11 40.7
960605-5 6 37.2 12 47.7
315
Table 1 continued
Secondary standard New batch
Run Plasma Order of testing PT Order of testing PT
no. lot no. (within‑run) (within‑run)
4 900423 7 13.9 1 15.0
960606-1 8 20.0 2 21.9
960606-2 9 27.9 3 30.9
960606-3 10 31.5 4 35.7
960606-4 11 34.6 5 39.2
960605-5 12 37.6 6 44.4
316
Annex 7
Assessment criteria for national blood regulatory systems
Abbreviations 319
Glossary 319
Introduction 321
Part A. Essential elements 324
1. National regulatory system 324
2. National regulatory authority 326
Part B. Core functions 330
3. Licensing and/or registration of blood establishments 330
4. Licensing and/or registration of manufacturers and distributors of plasma-derived
medicinal products 333
5. Approval of blood and blood components (product and/or process approval) 336
6. Approval of plasma-derived medicinal products 340
7. Regulatory oversight of associated substances and medical devices including
in vitro diagnostics 343
8. Access to a laboratory independent of manufacturers 345
9. Control of clinical trials 349
10. System for lot release of plasma-derived medicinal products 351
11. Regulatory inspections and enforcement activities 353
12. Vigilance systems 356
13. Ensuring traceability and record-keeping by manufacturers for all regulated
products 360
14. International cooperation 360
Authors and acknowledgements 362
Bibliography 364
317
The following assessment criteria for national blood regulatory

systems were adopted by the WHO Expert Committee on
Biological Standardization at its sixty-second meeting, held
in Geneva from 17–21 October 2011. This annex reflects the
collective views of the WHO Blood Regulators Network, and
was developed in response to a request from WHO and the
International Conference of Drug Regulatory Authorities for an
assessment tool to support NRA capacity-building in relation to
the regulation of blood and blood products. The tool is intended
to help WHO Member States identify gaps and priorities when
developing capacity-building programmes, and to support the
introduction of regulation of blood products. The establishment
of such regulation was recommended in the 2010 World Health
Assembly resolution WHA63.12 on the availability, quality and
safety of blood products.
318
Annex 7
Abbreviations
AE adverse event
AR adverse reaction
BRN WHO Blood Regulators Network
GCP good clinical practice
GDP good distribution practice
GMP good manufacturing practice
ICDRA International Conference of Drug Regulatory Authorities
NCL national control laboratory
NRA national regulatory authority
QMS quality management system
SOP standard operating procedure
SPC summary of product characteristics
Glossary
The WHO Expert Committee on Biological Standardization adopted the following
definitions for the purpose of this report.
Approval: a decision to authorize marketing of a drug by a national
regulatory authority. The mechanism by which a regulatory authority ensures
that there is compliance with regulatory requirements and standards that
assure quality, safety and efficacy for all blood products and/or processes and
establishments involved in collecting blood donations and/or manufacturing
blood products. A regulatory approval is generally a precondition for marketing
of a blood product.
Associated medical devices: all devices involved in donor testing and/or
manufacturing activities.
Associated substances and materials: all substances or materials involved
in manufacturing of blood products, including anticoagulants, additive solutions
and storage solutions. These materials are regulated as drugs in some jurisdictions.
Blood component:1 a constituent of blood (erythrocytes, leukocytes,
platelets, cryoprecipitate and plasma) that can be prepared by various separation
1
Stem cells may or may not be included in the scope of the regulatory activity of the competent authority
for blood and blood products. Similar criteria for safety, quality and efficacy should be met as for blood
and blood components.
319
methods and under such conditions can be used either directly for therapeutic
purposes or for further processing or manufacturing.
Blood establishment: any structure, facility or body that is responsible
for any aspect of the collection, testing, processing, storage, release and/or
distribution of human blood or blood components when intended for transfusion
or further industrial manufacturing.
Blood product: any therapeutic substance derived from human blood,
including whole blood, blood components and plasma-derived medicinal products.
Core function: a specific function through which the regulatory system
assures the quality, safety and efficacy of blood products.
Distributor: any facility that engages in distribution, including storage,
importation or exportation of blood products, which may include wholesalers.
Essential element: a basic characteristic of a regulatory system as a
whole (such as a legal basis for its activities, enforcement power, independence
of the regulator from the regulated parties, etc.), which is fundamentally related
to the system’s ability to effectively ensure the quality, safety and efficacy of
blood products.
Good clinical practice (GCP): a standard for the design, conduct,
performance, monitoring, auditing, recording, analysing and reporting of clinical
trials that provides assurance that the data and reported results are credible
and accurate, and that the rights, integrity and confidentiality of trial subjects
are protected.
Good distribution practice (GDP): the part of quality assurance that
ensures the quality of a pharmaceutical product is maintained by means of
adequate control of all activities that occur throughout the distribution process.
Good manufacturing practice (GMP): all elements in the established
practice that will collectively lead to final products or services that consistently
meet appropriate specifications and compliance with defined regulations.
Legislation: a legal instrument of government that defines laws governing
a particular subject matter, e.g. regulation of quality, safety and efficacy of

medicines. Laws define the roles, rights and obligations of all parties involved in
the subject matter in general terms (see also Regulations).
Licensing: authorization by the national regulatory authority for the
manufacture, importation, exportation or distribution of medical products.
Manufacturer: any natural or legal person (structure, facility or body)
with responsibility for any aspect of the following activities in relation to blood
products: collection, testing, processing, storage, packaging, labelling, release,
and/or distribution.
National regulatory authority (NRA): national regulatory authorities
(also called national medicines regulatory authorities) are legally established
bodies that promulgate and enforce medicines regulations.
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Annex 7
Plasma-derived medicinal product: any therapeutic product derived from

human plasma and produced by an industrial-scale manufacturing process that
pools multiple units. Also called plasma derivatives or plasma-derived products.
Quality management system (QMS): a management system that directs
and controls an organization with respect to quality, and that ensures that steps,
processes, procedures and policies related to quality activities are being followed.
Registration: a procedure under which information regarding the
identification, location(s) and scope of activities of all parties involved in
manufacturing or supplying a medicinal product and associated medical devices
and substances is submitted to the regulatory authority in order to comply with
administrative requirements before starting, continuing or amending relevant
activities.
Regulations: legislative instruments of government that provide more
prescriptive information regarding compliance with relevant legislation.
Regulations are specifically designed to provide the legal framework and details
necessary to achieve the administrative and technical goals of legislation.
Standard operating procedure (SOP): a prescriptive document that
outlines how an activity is carried out.
Sponsor: an individual, company, institution or organization that takes
responsibility for the initiation, management and/or financing of a drug submission
or clinical trial.
Vigilance: a mechanism of oversight involving an organized system
for gathering safety information. This term encompasses pharmacovigilance,
haemovigilance and materiovigilance.
Introduction
Blood transfusion is an indispensable, potentially life-saving medical intervention,
and blood products such as clotting factors and some immunoglobulins are
designated by WHO as essential medicines. However, the inherent risks of
blood and the complexity of providing adequate, timely and equitable access to
safe blood products require an organized national or regional blood regulatory
system. Within that system, a competent blood products regulatory authority
assures that appropriate standards are met for production of blood products and
monitoring of blood safety. Consequently, as a pillar for the establishment of
safe blood programmes globally, WHO has advocated for the establishment and
sustenance of strong national regulatory authorities (NRAs) both in developed
and developing countries.
In 2010, in resolution WHA63.12, the World Health Assembly expressed
its concern about the inequality of access to patients in different parts of the
world to blood products, particularly plasma-derived products, which leaves
321
many without needed transfusions and many of those with severe congenital
and acquired disorders without adequate plasma-derived treatments. In this
resolution, the World Health Assembly urged Member States:
to take all the necessary steps to update their national regulations on

donor assessment and deferral, the collection, testing, processing, storage,
transportation and use of blood products, and operation of regulatory
authorities in order to ensure that regulatory control in the area of quality
and safety of blood products across the entire transfusion chain meets
internationally recognized standards.
Purpose and application of the document

The purpose of this document is to provide a tool to assist capacity-building of
national regulatory authorities (NRAs) for the regulation of blood and blood
products. Ancillary to the existence of NRAs to regulate activities assuring the
provision of safe blood products, there is currently a need to develop criteria
defining best practices or attributes of national blood regulatory systems globally
for activities related to regulation of blood products. This document provides
a description of elements and functions which may support the creation of an
appropriate blood regulatory system where none exists so far, and which may
also be used as a tool to assess strengths and gaps of established systems. For
both developed and developing countries, an assessment tool that reflects
international best practices in blood regulation could serve to highlight strengths
of the NRA while identifying gaps or areas for future development. In addition,
adoption of global criteria by NRAs could promote international convergence of
regulations, which can have a beneficial impact on global safety and availability
of blood products.
To promote these objectives, this document identifies the essential
elements and core regulatory functions that should be present in an effective

NRA to assure the quality, safety and efficacy of blood and blood products, as
well as associated substances and medical devices including in vitro diagnostics.
Additionally, this document provides major criteria, indicators and associated
ratings for the essential elements and core functions that are intended to help
NRAs assess their performance in the regulation of blood and blood products
and prioritize efforts to address any gaps that are identified.
Scope of the document

To achieve the aim of an international best practice national blood regulatory
framework, a set of integrated general and specific regulatory functions have been
identified that are applicable to all aspects of blood product regulation, from the
322
Annex 7
collection of source material through to the quality control of the final product,
and covering not only blood products but also associated substances and medical
devices, including in vitro diagnostics. Section A of this document identifies
essential elements that are necessary to establish the legal basis, authority and
general characteristics of the NRA. Section B identifies specific core functions
of the NRA that are necessary for comprehensive oversight of blood products,
related substances and medical devices. It is recognized that the functions may
be interdependent and that in some countries the specific functions captured in
this document may not be within the scope of one national blood regulatory
authority but may be captured by other national authorities or other acceptable
mechanisms to achieve compliance with the assessment criteria. Some regulatory
functions may be applicable regardless of the intended use of the blood (e.g. for
transfusion purposes or for further manufacturing use). However, regulatory
structures should be designed in such a way as to avoid fragmentation and
uncoordinated delegation.
This document provides the main criteria and indicators for each essential
element and core function. The criteria and indicators provide a framework that
will identify areas for improvement to governments, particularly in developing
countries. A self-assessment or external assessment process using these criteria
could also serve as a useful means to highlight strengths of NRA programmes
for regulation of blood products while identifying gaps or areas for future
development. National authorities are encouraged to use the assessment criteria
as a roadmap towards evolving a best practice blood regulatory system.
It is recognized that many national blood regulatory systems will not be
able to meet all the criteria and indicators listed in this document. The criteria
and indicators are therefore organized into those considered as being required
(R) and thus necessary in order to be effective as a blood regulator, and those that
are considered as being desirable or suggested (S) to achieve a blood regulatory
system of international best practice.
It is also recognized that single required criteria may not formally be
fulfilled even by regulators with proven effectiveness, but that underlying relevant
safety issues can be met by other means. This offers the opportunity to compare
different ways of ensuring the safety of blood products and to highlight areas
where refinement of the assessment criteria may need to be considered.
With experience of their application, future versions of these assessment
criteria are expected to better accommodate effective alternatives, and to highlight
aspects, such as the prioritization of efforts, that may require additional guidance.
323
Part A. Essential elements

1. National regulatory system
Applicable to blood, blood components, plasma-derived medicinal products, associated
substances, and medical devices including in vitro diagnostics
Main criteria related Rating* Indicators related to the
to the element main criteria
Main Indi‑
criteria cator
1.1 A comprehensive legal R R 1.1.1 Provisions for the main
(statutory) basis exists regulatory functions can be
for establishment of identified and are up to date.
a regulatory system
R 1.1.2 The regulations or their
applicable to blood,
adaptations take into
blood components,
consideration developments
plasma-derived products,
in the field of blood and
associated substances,
related technologies.
and medical devices
including in vitro R 1.1.3 Regulations have been
diagnostics. established and are available;
they are intelligible to those
that need to comply with or
enforce them, and the means
of communication used are
adequate.
R 1.1.4 Legislation exists that defines
therapeutic products for
human use to be regulated,
and establishes standards of
quality, safety and efficacy for:
a. blood, blood components

and plasma-derived products;
b. associated substances and
medical devices including in
vitro diagnostics.
R 1.1.5 Legislation exists that
provides a legal basis for the
responsible NRA to perform
the essential functions.
R 1.1.6 Legislation enables the
appropriate institutions to
issue regulations.
324
Annex 7
Table continued
Main Indi‑
criteria cator
S 1.1.7 The development of
regulations includes the
opportunity for public
consultation.
1.2 The legislation assigns R R 1.2.1 The competent authorities
the enforcement of involved in the regulatory
regulations regarding the system for blood, blood
products covered in 1.1 to components, plasma-
one or more responsible derived products, associated
regulatory authorities. substances, and medical
devices including in vitro
diagnostics are clearly
identified and can be named
for each of the regulatory
functions.
R 1.2.2 The responsibilities, functions
and the organization of
each of these authorities are
clearly defined, in particular
as regards the scope of
the regulation (regulatory
functions) they have under
their control.
R 1.2.3 The activities of the various
authorities involved are
coordinated and supervised
by an administrative
mechanism.
* R = required; S = suggested.
325
2. National regulatory authority

Main Indi‑
criteria cator
2.1 There is independence R R 2.1.1 A clear division of roles
of the regulatory and responsibilities is
authority in decision- implemented between the
making. NRA, blood establishments,
manufacturers and
distributors, reflecting
independence of the
regulatory system.
R 2.1.2 Accountabilities for decision-
making are clear.
R 2.1.3 An internal policy on
potential conflicts of interest
for staff exists.
R 2.1.4 NRA management and
assessment activities
(including use of expert
committees) never include
representatives from
manufacturers or licence
holders.
R 2.1.5 A code of conduct for
regulatory staff exists.
S 2.1.6 Written procedures for

meetings with manufacturers,
distributors and other
sponsors exist.
2.2 The NRA has established S S 2.2.1 The NRA has an institutional
an institutional development plan, which is
development plan. implemented and updated.
326
Annex 7
Table continued
Main Indi‑
criteria cator
S 2.2.2 The development plan
includes: vision; strategic
objectives; timeline
and deadline for target
implementation; indicators;
functions and/or duties
of the NRA; ongoing staff
training plan; resources
needed; information and/
or communication strategy;
and a human resources
development plan.
S 2.2.3 Performance indicators are
established and used for
monitoring attainment of
objectives.
2.3 The NRA has adequate R R 2.3.1 An adequate number of
resources to carry out its trained staff and budgetary
functions properly and provisions exist for all
to enforce regulatory essential functions.
functions.
R 2.3.2 All staff members have
appropriate qualifications to
conduct regulatory activities
and are provided with
timely, relevant and regularly
updated training.
R 2.3.3 Tasks and responsibilities
of staff members are well
defined.
R 2.3.4 Mechanisms are in place to
ensure that those performing
regulatory functions have
sufficient and current
expertise in specialized areas.
327
Table continued
Main Indi‑
criteria cator
R 2.3.5 Policies and procedures exist
for recruitment and selection
of external experts and the
management of expert
advisory committees, including
potential conflict of interest.
R 2.3.6 An agreement between
the NRA and external
experts defining roles and
responsibilities is established.
S 2.3.7 The sources of funding of
the responsible authorities
performing regulatory
functions are defined.
S 2.3.8 Written criteria for selection
and recruitment of regulatory
staff are defined.
2.4 A QMS is in place. S S 2.4.1 A QMS is implemented by the
NRA for all its core functions
as specified below.
S 2.4.2 Budgetary provisions are
made for implementation and
maintenance of the QMS.
S 2.4.3 A qualified quality manager

is designated as responsible
for the implementation of the
QMS.
S 2.4.4 The documentation needed
to establish, implement and
maintain the QMS is defined
(quality manual, SOPs, etc.).
S 2.4.5 The QMS is based on
recognized international
standards.
328
Annex 7
Table continued
Main Indi‑
criteria cator
S 2.4.6 The QMS is certified or
accredited by external bodies.
S 2.4.7 An internal and external
audit and review system
exists as well as evidence that
corrective and preventive
actions are taken as a result of
monitoring and/or audits.
2.5 Transparency and R R 2.5.1 Legally-specified,
accountability is ensured. confidential and trade secret
information is available for
internal use and decision-
making. However, all other
information is publicly
available and kept up to date.
R 2.5.2 Listing of authorized products
and companies is made
available where needed.
R 2.5.3 Information on sanctions,
recalls and public health
warnings is publicly available.
S 2.5.4 Information on decisions is
available and easily accessible
to the public and includes
negative decisions in selected
cases (may vary depending
on national regulation).
S 2.5.5 An opportunity for
interaction between the NRA
and stakeholders is given.
329
Part B. Core functions

3. Licensing and/or registration of blood establishments
Applicable to blood and blood components including plasma for fractionation
to the function main criteria
Main Indi‑
criteria cator
3.1 Legislative authority R R 3.1.1 Legislation and/or regulations
exists to require exist that require a blood
registration and/or establishment that intends
licensing of blood to collect, test, process, store,
establishments, and for manufacture, distribute,
enforcement power. import or export blood
and blood components to
be authorized, accredited,
registered or licensed by the
designated NRA.
R 3.1.2 The NRA has the authority
to take regulatory action
(e.g. revoke or suspend the
licence) if the establishment
does not comply with
regulatory requirements.
3.2 A licensing and/or R R 3.2.1 Activities that are
registration system decentralized or delegated to
is established and other agencies or authorities
operational for blood follow the standards,
establishments. guidelines and procedures
as agreed by the NRA, and
a reporting mechanism is
established between the
responsible authorities.
R 3.2.2 Required registration and/
or licence applications are
assessed by the NRA based on
written guidelines.
R 3.2.3 A list of all licensed and/
or registered blood
establishments is maintained
and made available where
needed.
330
Annex 7
Table continued
Main Indi‑
criteria cator
R 3.2.4 Advice for applicants is
available on the content,
format, requirements and
procedures to follow in
order to submit a required
registration and/or application
for an establishment licence.
S 3.2.5 Facility documentation (e.g.
site master file, qualification
of a responsible person) is
submitted as part of a required
for an establishment licence
and is assessed to demonstrate
that the facility is suitable for
the activities to be performed
(e.g. blood collection, donor
screening, testing, storage, etc.).
S 3.2.6 Renewal periods for an
establishment licence and/or
registration are defined and
consistent with mechanisms
of surveillance.
3.3 Significant changes to R R 3.3.1 Changes are assessed based
an establishment licence on the type of change.
and/or registration are
S 3.3.2 Written guidelines for
submitted and assessed
applicants are available that
by the NRA prior to
define the types and scopes of
implementation.
changes and documentation
required.
3.4 Compliance with the R R 3.4.1 Compliance with applicable
principles of good principles of GMP is a
manufacturing practice condition for maintaining an
(GMP) is assessed as part establishment licence and/or
of the establishment registration and for approval
licensing and/or of significant changes.
registration process.
331
Table continued
Main Indi‑
criteria cator
R 3.4.2 National GMP and GDP
principles are published and
are consistent with or based
on recognized standards
for the manufacturing and
distribution of blood and
blood components.
R 3.4.3 Periodic inspections according
to GMP and GDP principles
are carried out for supervision
of blood establishments. For
inspections carried out abroad:
a. there is an agreement with
other NRAs for exchange of
inspection reports and/or
certificates, or
b. a list of reference countries
and/or agencies whose
certificates and decisions
are accepted exists, or site
inspections are carried out
abroad.
3.5 QMS requirements R R 3.5.1 The essential components for
are established for all a QMS are covered.
functions performed by
blood establishments.
3.6 Assessment of compliance R R 3.6.1 Compliance with national
with standards regarding standards is a condition for
donor selection maintaining an establishment
criteria and testing of licence.
donations is part of the
R 3.6.2 National standards are
establishment licensing
published and are consistent
and/or registration
with or based on recognized
process (alternatively this
standards for blood and
requirement can be met
blood components.
under core function 5).
332
Annex 7
Table continued
Main Indi‑
criteria cator
R 3.6.3 Inspections are carried out
for checking compliance with
these national standards.
R 3.6.4 Defined procedures
are in place for taking
action in instances of any
nonconformity.
* R = required; S = suggested
4. Licensing and/or registration of manufacturers and distributors of plasma‑

derived medicinal products
Applicable to plasma-derived medicinal products
Main Indi‑
criteria cator
4.1 Legislative authority exists R R 4.1.1 Legislation and/or
to require registration regulations exist that
and/or licensing of require manufacturers and
manufacturers and distributors of plasma-
distributors of plasma- derived products that intend
derived products, and for to manufacture, distribute,
enforcement power. import or export plasma-
derived products to be
registered and/or licensed by
the designated NRA.
R 4.1.2 The NRA has authority to
take regulatory action (e.g.
revoke or suspend the
licence) if the company does
not comply with regulatory
requirements.
333
Table continued
Main Indi‑
criteria cator
4.2 A licensing and/or R R 4.2.1 Activities that are
registration system decentralized or delegated to
is established and other agencies or authorities
operational for follow the standards,
manufacturers and guidelines and procedures
distributors of plasma- as agreed by the NRA, and
derived products. a reporting mechanism is
established between the
responsible authorities.
R 4.2.2 Required registration and/
or licence applications are
assessed by the NRA based on
written guidelines.
R 4.2.3 A list of all licensed and/or
registered manufacturers and
distributors is maintained
and made available where
needed.
available on the content,
format, requirements
(depending on the activities)
and procedures to follow in
order to submit a required

for an establishment licence.
S 4.2.5 Facility documentation (e.g.
site master file, key personnel,
qualification of a responsible
person) is submitted as part
of a required registration
and/or application for an
establishment licence and is
assessed to demonstrate that
the facility is suitable for the
activities to be performed.
334
Annex 7
Table continued
Main Indi‑
criteria cator
S 4.2.6 Renewal periods for an
establishment licence and/or
registration are defined and
consistent with mechanisms
of surveillance.
4.3 Significant changes to R R 4.3.1 Changes are assessed based
an establishment licence on the type of change.
and/or registration are
submitted and assessed
applicants are available that
by the NRA prior to
define the types and scopes of
implementation.
required.
4.4 Compliance with R R 4.4.1 Compliance with applicable
principles of GMP and principles of GMP and GDP is
GDP is assessed as part a condition for maintaining
of the establishment an establishment licence
licensing and/or and/or registration and
registration process. for approval of significant
changes.
R 4.4.2 National GMP and GDP
standards are published and
are consistent with or based
on recognized standards
for the manufacturing and
distribution of plasma-
derived products.
335
Table continued
Main Indi‑
criteria cator
R 4.4.3 Periodic inspections according
to GMP and GDP principles
are carried out for supervision
of manufacturers and
distributors of plasma-derived
products. For inspections
carried out abroad:
a. there is an agreement with
other NRAs for exchange of
inspection reports and/or
certificates, or
b. a list of reference countries
and/or agencies whose
certificates and decisions
are accepted exists, or
c. site inspections are carried
out abroad.
4.5 QMS requirements R R 4.5.1 The essential components for

are established for all a QMS are covered.
functions performed
by manufacturers and
distributors.
5. Approval of blood and blood components (product and/or process approval)

Applicable to blood and blood components including plasma for fractionation
Main Indi‑
criteria cator
5.1 Legal provisions exist R R 5.1.1 An approval system is
for a system to ensure required that includes any
quality, safety and imported products.
efficacy of blood and
blood components.
336
Annex 7
Table continued
Main Indi‑
criteria cator
R 5.1.2 The NRA has the authority to
issue an approval, to suspend
it and to withdraw it if the
product is considered unsafe
or does not comply with
regulatory requirements
5.2 A system for ensuring R R 5.2.1 The capability exists to perform
quality, safety and science-based risk assessments
efficacy of blood and and risk management.
blood components
R 5.2.2 Specifications related to
is established and
quality, safety and efficacy of
operational.
blood and blood components
are defined and under the
supervision of the NRA.
R 5.2.3 The critical standards for
product manufacturing are
legally binding and include
donor selection, laboratory
testing, component
preparation, storage,
issuance, tracking, tracing,
record-keeping, and safe
disposal of units not meeting
specifications for use in
transfusion.
S 5.2.4 Procedures to recognize
exceptions are clearly defined
(e.g. if collected by a medical
practitioner for a specific
therapeutic purpose).
S 5.2.5 Requirements and standards
are based on internationally
recognized standards.
R 5.2.6 Plasma for fractionation
meets internationally
recognized standards.
337
Table continued
Main Indi‑
criteria cator
5.3 Donor selection R R 5.3.1 Donor selection and deferral
and deferral criteria criteria (temporary and
are established as permanent deferrals) take
appropriate to the into account the health of
intended use of the the donor and the safety and
component. suitability of the donation
consistent with current science.
R 5.3.2 Mechanisms for regularly
reviewing and updating the
donor selection and deferral
criteria are in place and
take into consideration the
development of issues that
might have a negative impact
on the quality and safety of
blood and blood components,
e.g. epidemiological situation
or emerging diseases.
5.4 Transmissible disease R R 5.4.1 Mechanisms for regularly
testing requirements reviewing (e.g. by qualified
are established as experts in epidemiology)
appropriate to the and updating the testing
intended use of the requirements are in place.
component.
R 5.4.2 Epidemiological data
regarding the prevalence
and incidence of infectious

disease markers in blood
donors are available and
regularly updated.
5.5 Labelling requirements R R 5.5.1 Each blood component has
are established. a unique and clear identifier
and is fully traceable.
R 5.5.2 Original labelling and
significant amendments
are submitted to the NRA
and assessed prior to
implementation.
338
Annex 7
Table continued
Main Indi‑
criteria cator
S 5.5.3 Product labelling includes
information on the risks and
benefits of product use.
S 5.5.4 Requirements are based on
internationally recognized
standards.
5.6 An approval system R R 5.6.1 Assessment exists that
for blood and blood includes relevant aspects of
components is quality, safety and, where
operational. applicable, efficacy of blood
and blood components.
S 5.6.2 Guidelines for applicants exist
on the content, format and
procedures to follow in order
to submit an application for
approval.
assessment of applications
are implemented.
S 5.6.4 Appeal procedures are in
place.
S 5.6.5 An assessment report is
prepared and used as a
reference for decision-making.
5.7 There is a requirement S S 5.7.1 Written guidelines for
for manufacturing applicants are available
changes to be submitted that define the types and
and assessed by the NRA. scopes of changes and
documentation required.
assessment exist based
on the type of change
(e.g. significant, notifiable,
administrative).
339
Table continued
Main Indi‑
criteria cator
5.8 Appropriate assessment R R 5.8.1 Access to experts with relevant
expertise is available. qualifications and experience
(internal and/or external) is
assured for assessment of
blood and blood components
(preclinical, clinical and quality
data).
selection, management, and
use of external experts are in
place.
6. Approval of plasma‑derived medicinal products

Applicable to plasma-derived medicinal products
Main Indi‑
criteria cator
6.1 Legal provision for a R R 6.1.1 Marketing approval is required

marketing approval for plasma-derived products,
system exists to ensure including imported products.
the quality, safety and
efficacy of plasma-
issue marketing approval for

derived products.
plasma-derived products, to
suspend an approval, and to
withdraw it if the product is
considered unsafe or does
not comply with regulatory
requirements.
6.2 A marketing approval R R 6.2.1 The capability exists to
system for plasma- perform science-based
derived products risk assessments and risk
is established and management.
operational.
340
Annex 7
Table continued
Main Indi‑
criteria cator
R 6.2.2 There is a requirement for the
applicant to include a list of
all the blood establishments
that collected the plasma
used in the product.
R 6.2.3 Specifications related to the
quality and safety of plasma
for fractionation are defined
and under the supervision of
the NRA.
R 6.2.4 Selection, deferral and
transmissible disease testing
requirements for plasma
donors are established (see
criteria 5.3 and 5.4).
available on the content, format
and procedures to follow in
order to submit an application
for market authorization.
S 6.2.6 Appeal procedures are in place.
S 6.2.7 The NCL is involved in
assessment as appropriate.
selection, management, and
use of external experts are
available.
6.3 Assessment of R R 6.3.1 Assessment of quality, safety
applications for market and efficacy of plasma-derived
authorization is products is performed,
implemented. including assessment of the
effectiveness of measures used
by manufacturers to inactivate
and/or remove transmissible
pathogens.
341
Table continued
Main Indi‑
criteria cator
R 6.3.2 Procedures to recognize
exceptions are clearly
defined.
S 6.3.3 Assessment reports are
prepared and used as a
reference for decision-
making.
S 6.3.4 Written criteria exist for
recognition of the reports
and/or decisions of other
NRAs (if applicable).
assessment of applications
are available.
6.4 There is a requirement R R 6.4.1 Changes are assessed.
for changes to be
submitted and assessed S 6.4.2 Written guidelines for
by the NRA prior to applicants are available that
implementation. define the types and scopes of
required.
assessment are available
based on the type of change.
6.5 Appropriate assessment R R 6.5.1 Access to experts (internal

expertise exists. and/or external) for
assessment of plasma-derived
products (preclinical, clinical
and quality data) is assured,
and lists staff and/or experts
with relevant qualifications
and experience.
6.6 Clear and comprehensive R R 6.6.1 The product information
information on made available is approved.
authorized plasma-
derived products is
available.
342
Annex 7
Table continued
Main Indi‑
criteria cator
R 6.6.2 A summary of product
characteristics (SPC) or
equivalent information is
available for all plasma-
derived products.
S 6.6.3 SPC-like information is
regularly updated and
publicly available.
6.7 A list of authorized R R 6.7.1 A list of authorized products
products exists. is made available where
needed.
S 6.7.2 A list of authorized products
is publicly available.
7. Regulatory oversight of associated substances and medical devices including in

vitro diagnostics
Applicable to associated substances and medical devices including in vitro diagnostics
Main Indi‑
criteria cator
7.1 Legal provisions exist for R R 7.1.1 Premarket review and
regulatory oversight of approval is required for in vitro
the relevant associated diagnostics and screening test
substances and medical kits used for donor selection,
devices. testing of blood and blood
components for therapeutic
use, and/or for further
manufacturing of plasma-
derived products (e.g. tests for
donor haemoglobin, tests for
infectious disease markers).
343
Table continued
Main Indi‑
criteria cator
R 7.1.2 Premarket review and
approval is required for
medical devices involved in
the manufacture of blood
components (e.g. apheresis
machines).
R 7.1.3 Premarket review and
approval is required for
associated substances (e.g.
anticoagulants, additive
solutions).
R 7.1.4 The NRA has the enforcement
power to investigate and act
against marketed products
and involved companies
that do not comply with the
requirements.
7.2 Systems for premarket R R 7.2.1 The capability exists to perform
review and approval of science-based risk assessments
associated substances and risk management.
and relevant medical
R 7.2.2 Premarket review includes an
devices are established
assessment of quality, safety
and operational.
and effectiveness.
R 7.2.3 Advice for applicants on

content (data requirements),
format and procedures for
submitting an application
exists.
R 7.2.4 If decentralized, roles and
responsibilities of the
bodies involved are defined
and there is a mechanism
for information exchange
between the control authority
and the NRA.
344
Annex 7
Table continued
Main Indi‑
criteria cator
product assessments exist.
7.3 Appropriate assessment R R 7.3.1 Access to experts with relevant
expertise is available. qualifications and experience
(internal and/or external)
for assessment of blood and
blood components (preclinical,
clinical and quality data) is
established.

selection, management and
use of external experts are in
place.
8. Access to a laboratory independent of manufacturers

Main Indi‑
criteria cator
8.1 Access by the NRA to R R 8.1.1 Policy and operational
an NCL independent of agreements are in place for
the manufacturer(s) is use of any external control
established. laboratories.
R 8.1.2 Adequate testing plans,

testing procedures and
related documentation are
available.
R 8.1.3 Responsibilities for testing in

the pre-licensing and post-
licensure period are clearly
defined.
345
Table continued
Main Indi‑
criteria cator
S 8.1.4 The NCL is involved in
defining the specifications
and analytical methods
during assessment of
marketing authorizations.
8.2 Appropriate organization R R 8.2.1 Written testing procedures
and financial support and related documentation
from management ensure are in place.
the implementation
of adequate testing R 8.2.2 A re-test policy is established.
programmes (including R 8.2.3 A strategy for the introduction
documentation) using and validation of new or
appropriate equipment, improved tests exists.
and qualified and
experienced staff. R 8.2.4 Reporting and issuance to
the NRA of all critical results
including out-of-specifications
handling is implemented.
S 8.2.5 Document control is
established.
S 8.2.6 SOPs, test procedures,
sample handling and data
management are organized.
8.3 An externally accredited S S 8.3.1 A quality policy and quality
quality management manual exist.
system (QMS) is in place
in the laboratory. S 8.3.2 A qualified quality manager

is designated and a QMS is in
operation.
8.4 Equipment R R 8.4.1 Calibration and maintenance
documentation is in schedules are available.
place.
R 8.4.2 Validation protocols are
available.
S 8.4.3 Equipment selection
processes are documented
and unique equipment
identification (ID) is in place.
346
Annex 7
Table continued
Main Indi‑
criteria cator
S 8.4.4 Commissioning records (i.e.
installation and qualification)
are available.
S 8.4.5 Operation manuals and logs
exist.
8.5 Human resource R R 8.5.1 Qualified and experienced
management is staff members with defined
implemented. responsibilities and
competencies are available.
S 8.5.2 A staff training plan is
developed and implemented.
S 8.5.3 The impact of staff training is
monitored.
8.6 An audit and review S S 8.6.1 Comprehensive internal audit
system exists. and review systems are in place.
S 8.6.2 Documentation of actions
taken as a result of audits is
available.
S 8.6.3 The laboratory is audited by
external organizations.
8.7 A validation policy for R R 8.7.1 A validation programme
the introduction of tests for non-compendial tests is
is implemented. available.
R 8.7.2 Procedures exist for transfers
of validated methods (i.e.
between the manufacturer
and the regulator).
8.8 A general safety R R 8.8.1 Lists of hazardous substances
programme exists. are available.
R 8.8.2 Responsible staff members
are designated.
S 8.8.3 A full safety programme exists.
347
Table continued
Main Indi‑
criteria cator
8.9 A policy for use of R R 8.9.1 Access to a catalogue (list,
reference standards specifications and sources)
and reagents exists. and regular supply system
for standards and reference
materials is implemented.
R 8.9.2 Appropriate use of reference
materials is ensured.
R 8.9.3 Use of reagents of assured
quality (e.g. grades) is
ensured.
S 8.9.4 A system is in place to
establish and qualify
national reference standards
in International Units (IU).
8.10 Data trends are R R 8.10.1 Results of reference
monitored and materials are monitored.
analysed.
R 8.10.2 Results are compared with
those of the manufacturer.
S 8.10.3 Laboratory results are
monitored and trends are
assessed.
8.11 Participation in S S 8.11.1 Regular participation
international (monitored by date of last

proficiency schemes participation, scope,
and collaborative product(s), coordinating
studies is organized. institution).
8.12 Regulatory outcome R R 8.12.1 Compliance with authorized
of testing is analysed specifications is checked.
and used as a basis for
R 8.12.2 Results are compared with
decision-making.
those of the manufacturer.
R 8.12.3 Corrective action is initiated
in case of non-compliance.
348
Annex 7
9. Control of clinical trials

Main Indi‑
criteria cator
9.1 Applicable legal R R 9.1.1 An authorization system for
provision for the clinical trials is required.
regulation of biomedical
R 9.1.2 The scope and requirements
research in human
for regulation of clinical trials
subjects exists.
are defined.
R 9.1.3 The NRA has the enforcement
power for the authorization,
suspension and withdrawal of
clinical trials.
R 9.1.4 Legal provisions are in place
to assure an ethical oversight
of clinical trials.
R 9.1.5 Compliance with principles of
good clinical practice (GCP) is
mandatory.
9.2 A system for authorization R R 9.2.1 A system is established for
of clinical trials is clinical trial assessment and
operational. authorization.
R 9.2.2 An inspection system is
established to verify compliance
with the principles of GCP.
R 9.2.3 Expertise is available from
within or outside the NRA.
assessment of clinical trials
and changes are implemented.
S 9.2.5 Written guidelines and forms
on the data requirements,
the format and procedures
for submitting a clinical trial
application are available to
sponsors.
349
Table continued
Main Indi‑
criteria cator
S 9.2.6 Provision for scientific advice
(e.g. preclinical and clinical)
on the design of clinical
trials or issues related to the
submission of appropriate
data is in place.
S 9.2.7 There are written guidelines
for GCP.
9.3 Data requirements for R R 9.3.1 Production and quality control
clinical trial applications of the clinical candidate
are defined. material (e.g. product
characterization, laboratory
specimens) are included.
R 9.3.2 Provision for preclinical data
exists.
R 9.3.3 Assessment of the clinical
trial protocol with respect to
patient safety and informed
consent is performed.
9.4 Assurance of ethical R R 9.4.1 A system of independent
oversight exists. ethical review and approval
exists in accordance with the
principles of GCP.
S 9.4.2 Ethics committees (e.g. an

Institutional Review Board)
are formally defined, including
their composition.
S 9.4.3 The ethics committees
include members external to
the concerned institution.
S 9.4.4 The roles and duties of ethics
committees to oversee
clinical trials are outlined.
350
Annex 7
10. System for lot release of plasma‑derived medicinal products

Applicable to plasma-derived medicinal products and donor screening tests
Main Indi‑
criteria cator
10.1 Legal provisions for R R 10.1.1 The NRA has the authority to
official lot release issue lot release certificates
certification are in and the enforcement power
place. to suspend or revoke lot
release.
R 10.1.2 The NRA has the legal
authority to perform lot
release and/or have in place
a policy and criteria for
acceptance of lot release
performed by another NRA
(e.g. a lot release certificate
from the country of origin).
S 10.1.3 Written criteria for exemption
from lot release exist.
10.2 A lot release system R R 10.2.1 Lot release protocols and
is established and procedures are established
operational. and/or acceptance of lot
release performed by
another NRA is in place.
R 10.2.2 Lot release is based at a
minimum on review of
summary lot-specific data.
R 10.2.3 Qualified staff members
(i.e. staff with relevant
qualifications, training and
experience) are available to
perform lot release.
R 10.2.4 Testing policy and test
protocols including
acceptance criteria are
defined.
R 10.2.5 Records on lot release are
maintained.
351
Table continued
Main Indi‑
criteria cator
R 10.2.6 Procedures for
communication with the
product manufacturer are
defined.
R 10.2.7 Written procedures and

guidelines (including
templates of certificates),
checklists, and/or SOPs are
developed and used to
review summary lot protocols
and are implemented for the
lot release process.
S 10.2.8 Testing procedures are

externally accredited.
10.3 A quality management R R 10.3.1 The laboratory that
system for official lot performs lot release within
release is implemented. or for the NRA complies with
core function 8.
S 10.3.2 Appropriate data collection

and analysis (e.g. lot-to-lot
consistency, trend analysis)
is implemented.
S 10.3.3 Continual review and

scientific dialogue exist

with the manufacturers and
product review experts on
issues of quality test results.
10.4 Access to product- R R 10.4.1 Approved relevant
related documentation marketing authorization and
to guide particular updates are available.
areas of scrutiny in lot
release is possible. R 10.4.2 Access to complaints and
adverse event (AE) reports is
possible.
352
Annex 7
Table continued
Main Indi‑
criteria cator
R 10.4.3 Access to the manufacturer's
batch records is possible.
R 10.4.4 Access to inspection reports
is possible.
11. Regulatory inspections and enforcement activities

Main Indi‑
criteria cator
11.1 Legal provision exists R R 11.1.1 A mandate exists for
to inspect premises inspections by the NRA and
where regulated enforcement of compliance
activities are performed with principles of GMP, GDP
in order to assess and and other standards.
enforce compliance
with the applicable R 11.1.2 Applicable standards and
laws, regulations and practices are defined in legal
standards. provisions.
to take enforcement action
against the accountable
companies or persons that
are not in compliance.
to sample products,
manufacturing materials
and records if necessary.
to recall products.
R 11.1.6 Provisions exist for conflict of
interest and confidentiality.
353
Table continued
Main Indi‑
criteria cator
11.2 Inspection and R R 11.2.1 Established policies and
enforcement systems programmes exist for
are established and conducting inspections of
operational. all regulated activities.
R 11.2.2 An inspection plan exists
with adequate human
and financial resources for
conducting inspections at
appropriate intervals.
R 11.2.3 The NRA maintains files of
each inspection, including
the inspection report and
final decisions taken.
R 11.2.4 There is an established
process for appropriate
regulatory action to address
inspectional findings (e.g.
recall of products, amended
licences).
R 11.2.5 If the mechanism is
adopted, provisions exist
for acceptance of external
inspectorates according to
internationally recognized
standards.
11.3 Inspectors with R R 11.3.1 Inspectors have the

appropriate expertise appropriate expertise
and qualifications are and training to conduct
available. inspections of blood
establishments, and
of manufacturers and
distributors of plasma-
derived products.
R 11.3.2 Training of inspectors
includes specific aspects
related to the activities of
relevant establishments.
354
Annex 7
Table continued
Main Indi‑
criteria cator
S 11.3.3 Use of a team approach is
possible in order to include
specialized knowledge
and expertise in specific
products where needed.
11.4 A quality management R R 11.4.1 Written procedures exist
system is implemented for conducting inspections
that is consistent with (inspection manual) and
international principles following up on deficiencies
for pharmaceutical and and/or violations.
related inspectorates.
S 11.4.2 An established procedure
(e.g. periodic internal and
external audits) exists to
monitor the inspection
process.
S 11.4.3 Monitoring of timelines

and indicated actions is
implemented.
11.5 A recall system exists R R 11.5.1 Policy and procedures for
with mechanisms to a recall system including
ensure the proper product disposition exist.
disposition of blood,
blood components, R 11.5.2 The recall system is based
plasma-derived on defined action and
products, associated documented communication
substances, and medical to the appropriate level of
devices including in the distribution system.
vitro diagnostics. R 11.5.3 A feedback mechanism exists
to confirm that appropriate
action (including destruction
when necessary) has been
taken at all appropriate
levels.
R 11.5.4 Full lot traceability is in place.

355
12. Vigilance systems

Main Indi‑
criteria cator
12.1 Legal provisions for R R 12.1.1 The NRA has a legal
a national vigilance mandate and enforcement
system exist. power for mandatory
reporting elements of the
national vigilance system.
specify reporting of adverse
events (AEs) and adverse
reactions (ARs) within the
national vigilance system.
R 12.1.3 Authority exists to require
the marketing authorization
holder to perform a specific
study of safety and/or
effectiveness in the post-
marketing period.
12.2 National vigilance R R 12.2.1 Roles and responsibilities of
systems for the the key parties, the NRA, and
monitoring and surveillance staff involved
management of AE in AE and AR monitoring
and AR are established and management activities
and operational. are clearly defined and
documented.
R 12.2.2 Guidelines exist and are
published and accessible
(i.e. distributed or available
when needed) to all staff
involved in AE and AR
surveillance.
356
Annex 7
Table continued
Main Indi‑
criteria cator
S 12.2.3 Guidelines include the
following:
a. objectives of the system;
b. a list of AEs and ARs to be
reported;
c. case definitions for all AEs
and ARs to be reported;
d. information on how to
report AEs and ARs for all
blood, blood components,
plasma-derived products,
associated substances, and
medical devices including
in vitro diagnostics (i.e.
who should report; how,
where and when reports
should be sent);
e. the process for analysing
data and providing
feedback to relevant staff
and key parties;
f. the process for
investigating and
responding to serious AEs
and ARs (including who
should be in charge of the
investigation);
g. the process for informing
patients, parents, the
community and country
(where relevant) of the
findings of an investigation
and relevant actions.
S 12.2.4 A standardized reporting
form exists with
comprehensive information
to monitor AEs and ARs.
357
Table continued
Main Indi‑
criteria cator
S 12.2.5 A system is established for
providing periodic feedback
on AEs and ARs, including
summary and specific
investigation reports from the
national to all levels (including
health-facility level).
12.3 Guidance on AE S S 12.3.1 Guidelines and templates

and AR monitoring on AE and AR reporting and
and management monitoring are provided to
is provided to appropriate staff dealing
appropriate staff. with AE and AR.
12.4 There is demonstrated R R 12.4.1 The NRA is regularly

capacity to detect, informed of data relevant
investigate and take to the quality and safety of
action regarding blood products including:
significant AEs and ARs. a. blood transfusion safety;
b. transmissible disease
surveillance data;
c. device failures.
R 12.4.2 Manufacturers are required

to inform the NRA of
any new safety issues or
marketing and/or regulatory
decisions taken in other

countries.
R 12.4.3 Procedures for initiating

corrective and/or regulatory
action (e.g. recall) are
available.
358
Annex 7
Table continued
Main Indi‑
criteria cator
R 12.4.4 There is documented
capacity to investigate AEs
and ARs, for example:
a. routine reporting of AEs
and ARs according to
established guidelines
and/or SOPs;
b. a clear understanding and
adequate training among
key parties of respective
roles and responsibilities;
c. access to resources
(personnel, laboratory) to
conduct comprehensive
investigations.
R 12.4.5 Case investigations are timely
and complete, for example:
a. timelines are established
for prompt investigation
and preliminary reporting
related to serious adverse
reactions;
b. investigation is thorough
and findings are clearly
described.
S 12.4.6 There is a demonstrated
reporting system (active or
passive, sentinel or universal)
with satisfactory sensitivity,
for example:
a. annual number of reports;
b. reporting rate;
c. breakdown of reports by
types of AE, age group,
districts etc.
359
13. Ensuring traceability and record‑keeping by manufacturers for all regulated

products
Main Indi‑
criteria cator
13.1 The NRA ensures that R R 13.1.1 A requirement exists for
standards for traceability manufacturers to implement
and record-keeping are methods and maintain
in place for all aspects records that enable
of manufacturing and traceability, including:
distribution. a. for manufacturers
of blood products,
traceability from donor to
recipient and vice versa;
b. ensuring the integrity of
manufacturing records
and completeness of
distribution records.
R 13.1.2 Procedures for record-

keeping and retention
periods defined by the NRA
are available.
14. International cooperation


Main Indi‑
criteria cator
14.1 A national policy to S S 14.1.1 A national policy and/or
facilitate international strategy on international
cooperation and interactions exist, e.g.
harmonization is information sharing on
implemented. product approvals, safety
data and policy initiatives.
360
Annex 7
Table continued
Main Indi‑
criteria cator
S 14.1.2 Agreements exist between
the NRA and other
international organizations
and regulatory authorities.
S 14.1.3 The NRA participates in
international harmonization
initiatives and forums.
14.2 Sharing of risk R R 14.2.1 Ability is shown by the NRA
information with to participate in international
international risk management efforts
organizations and other when needed.
regulatory authorities is
S 14.2.2 The NRA has the ability to
implemented.
engage in international
risk assessment when
needed, e.g. access to
epidemiological data,
expertise in risk assessment.
S 14.2.3 The capacity or expertise to
access epidemiological data
and formally assess risks is
available.
S 14.2.4 Documented procedures
for the timely sharing of risk
information internationally
exist.
S 14.2.5 Records are kept of risk
information that has been
exchanged.
361
Authors and acknowledgements

The drafting group was formed of Members of the WHO Blood Regulators Network
(BRN) and the WHO Blood Products and Related Biologicals programme,
Quality Assurance and Safety: Medicines, World Health Organization:
Dr F. Agbanyo, Health Canada, Canada; Dr J. Epstein, United States Food
and Drug Administration, USA; Dr P. Ganz, Health Canada, Canada; Dr M.
Heiden, Paul-Ehrlich-Institute, Germany; Dr M. Jutzi, Swissmedic, Switzerland;
Dr G. Michaud, United States Food and Drug Administration, USA; Dr A. Padilla,
World Health Organization, Geneva, Switzerland; Dr I. Prosser, Therapeutic
Goods Administration, Australia; Dr I. Sainte-Marie, French Agency for the Safety
of Health Products (AFSSAPS), France; Dr C. Schärer, Swissmedic, Switzerland;
Professor R. Seitz, Paul-Ehrlich-Institute, Germany; Dr G. Smith, Therapeutic
Goods Administration, Australia; Dr P. Zorzi, AFSSAPS, France.
Existing WHO evaluation templates for vaccines and medicinal products
were consulted in developing this tool. The first consolidated draft was discussed
at the Blood and Blood Products Workshop of the 14th International Conference
of Drug Regulatory Authorities, Singapore, 2010, where it was supported for
consideration by WHO Member States. More than 90 national regulatory agencies
were represented at the Conference.
Through a global consultation process involving all WHO regions,
regulators were encouraged to contribute their self-assessments and comments
on the usefulness of the tool to help towards its finalization. Thanks are due to the
WHO regional offices for their support in this process.
Valuable inputs in the form of comments and self-assessment feedback
were received from the following national agencies (in alphabetical order by
country):
Blood Bank Directorate, Ministry of Public Health, Afghanistan;
Administración Nacional de Medicamentos, Alimentos y Tecnología Médica
(ANMAT), Argentina; Scientific Center of Drug and Medical Technologies

Expertise (SCDMTE), Armenia; European Commission, Directorate General for
Health and Consumer Affairs (SANCO), Belgium; Gerência Geral de Sangue,
outros Tecidos, Células e Órgãos, Agência Nacional de Vigilância Sanitária
(ANVISA), Brazil; Department of Drug Registration, State Food and Drug
Adminstration, China; Instituto Nacional de Salud (INS) and Instituto Nacional de
Vigilancia de Medicamentos y Alimentos (INVIMA), Ministerio de la Protección
Social, Colombia; Centro para el Control Estatal de la Calidad de los Medicamentos
(CECMED), Cuba; Danish Medicines Agency, Denmark; The Minister’s Technical
Office, Ministry of Health, Egypt; Laboratory Services Department, Food and
Drugs Board, Ghana; Central Drugs Standard Control Organization, Ministry
of Health and Family Welfare, India; National Agency of Drug and Food Control
362
Annex 7
(NADFC), Indonesia; Food and Drug Organization, Islamic Republic of Iran;

Division of Blood and Blood Products, Ministry of Health, Labour and Welfare,
Japan; National Blood Service, Ministry of Health, Latvia; Comisión Federal para
la Protección contra Riesgos Sanitarios (COFEPRIS), Mexico; Department of
Health and Social Affairs, Micronesia (Federated States of); Department of Drug
Administration (DDA), Ministry of Health and Population, Nepal; Medicines
Evaluation Board, the Netherlands; Centro Nacional de Diagnóstico y Referencia
(CNDR), Ministerio de Salud de Nicaragua (MINSA), Nicaragua; National Agency
for Food and Drug Administration and Control (NAFDAC), Nigeria; Programa
Nacional de Sangre, Ministerio de Salud Pública y Bienestar Social, Paraguay;
Centre for Product Registration, National Pharmaceutical Control Bureau,
Ministry of Health, Malaysia; Korea Food and Drug Administration (KFDA),
Republic of Korea; Saudi Food and Drug Authority, Saudi Arabia; Direction de
la Pharmacie et des Laboratoires, Ministère de la Santé, Senegal; Medicines and
Medical Devices Agency, Serbia; Department of Health, South Africa; National
Drug Quality Control Laboratory, Medicines and Poisons Board, Sudan; Medical
Products Agency, Sweden; Ministry of Health, Syrian Arab Republic; Institute of
Biological Products, Department of Medical Sciences, Ministry of Public Health,
Thailand; Sharjah Blood Transfusion and Research Centre, Ministry of Health,
United Arab Emirates.
All comments received were reviewed by the drafting group and a
final proposed version was submitted to the sixty-second Expert Committee
on Biological Standardization. Special thanks are due to the Members of the
Committee, who provided valuable advice and agreed to the adoption of this
document:
Dr J. Epstein, Center for Biologics Evaluation and Research, Food and
Drug Administration, USA; Dr E. Griffiths, Biologics and Genetic Therapies
Directorate, Health Canada, Canada; Mrs T. Jivapaisarnpong, Institute of Biological
Products, Department of Medical Sciences, Ministry of Public Health, Thailand;
Dr H. Klein, National Institutes of Health, USA; Dr P. Minor, National Institute
for Biological Standards and Control, England; Dr F.M. Moftah, National Blood
Transfusion Service, Ministry of Health, Egypt; Dr J. Petricciani, International
Association for Biologicals, USA; Dr L.S. Slamet, National Agency of Drug and
Food Control (NADFC), Indonesia; Dr P. Strengers, Sanquin Foundation, the
Netherlands; Professor H. Yin, Center for Drug Evaluation, State Food and Drug
Administration, China.
For further information contact:
Department of Essential Medicines and Health Products
363
Bibliography
14th International Conference of Drug Regulatory Authorities. WHO Drug Information, 2011, 25(1)
(http://www.who.int/medicines/publications/druginformation/en/index.html, accessed 11 March
2013):3–18.
Catalogue of the WHO International Biological Reference Preparations: blood products and related
substances. Geneva, World Health Organization (http://www.who.int/entity/bloodproducts/catalogue/
en/index.html, accessed 16 May 2013).
Good manufacturing practices: main principles for pharmaceutical products. In: WHO Expert
Committee on Specifications for Pharmaceutical Preparations. Forty-fifth report. Geneva, World Health
Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human
blood plasma products. In: WHO Expert Committee on Biological Standardization. Fifty-second report.
Geneva, World Health Organization, 2004 (WHO Technical Report Series, No. 924), Annex 4.
Recommendations for the production, control and regulation of human plasma for fractionation.
In: WHO Expert Committee on Biological Standardization. Fifty-sixth report. Geneva, World Health
Requirements for the collection, processing and quality control of blood, blood components and
plasma derivatives. In: WHO Expert Committee on Biological Standardization. Forty-third report. Geneva,
Resolution WHA63.12. Availability, safety and quality of blood products. In: Sixty-third World Health
Assembly, Geneva, 17–21 May 2010. Resolutions and decisions, annexes. Geneva, World Health
Organization, 2010 (WHA63/2010/REC/1; http://apps.who.int/gb/ebwha/pdf_files/WHA63-REC1/WHA63_
REC1-en.pdf, accessed 11 March 2013).
Supplementary guidelines on good manufacturing practices: validation. In: WHO Expert Committee
on Specifications for Pharmaceutical Preparations. Fortieth report. Geneva, World Health Organization,
WHO good manufacturing practices for sterile pharmaceutical products. In: WHO Expert Committee on
Specifications for Pharmaceutical Preparations. Forty-fifth report. Geneva, World Health Organization,
WHO guidelines on good manufacturing practices for blood establishments. In: WHO Expert
Committee on Specifications for Pharmaceutical Preparations. Forty-fifth report. Geneva, World Health
364
Annex 8
Biological substances: WHO International Standards and
Reference Reagents
A list of WHO International Standards and Reference Reagents for biological
substances was issued in WHO Technical Report Series, No. 897, 2000 (Annex 4)
with an updated version available at: http://www.who.int/biologicals. Copies of
the list may be obtained from appointed sales agents for WHO publications or
from WHO Press, World Health Organization, 1211 Geneva 27, Switzerland
(tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: [email protected]; web site:
http://www.who.int/bookorders).
At its meeting in October 2011, the Expert Committee made the changes
shown below to the previous list.
Vaccines and related substances; blood products and related substances;
biotherapeutics other than blood products; and in vitro diagnostic device reagents
are held and distributed by the International Laboratory for Biological Standards,
NIBSC, Potters Bar, Herts, England. Antibiotic Reference Preparations are held
by the European Department for the Quality of Medicines, Council of Europe,
7 allée Kastner, CS 30026 F-67081, Strasbourg, France.
Additions
Preparation Activity Status
Antibiotics
Dihydrostreptomycin 19 425 IU per vial Third WHO International
Standard
Biotherapeutics other than blood products
Transforming growth factor 19 000 IU per ampoule First WHO International
beta-3 Standard
Blood products and related substances
Anti-human neutrophil No assigned value; First WHO Reference
antigen-1a antibody however, a 1-in-4 Reagent
dilution should define
the minimum potency
specification for
anti-HNA-1a detection
365
Preparation Activity Status

Blood group genotyping No assigned values First WHO Reference
Reagents
Factor VIII/von Willebrand 1.03 IU per ampoule for Sixth WHO International
factor (VWF), plasma VWF propeptide Standard
Fibrinogen, plasma 2.7 mg per ampoule Third WHO International
Standard
In vitro diagnostic device reagents
Anti-Trypanosoma cruzi 1 IU per ml First WHO International
group I antibodies, human Standard
Anti-Trypanosoma cruzi 1 IU per ml First WHO International
group II antibodies, human Standard
Epstein–Barr virus, for 5 000 000 IU per ml First WHO International
NAT-based assays Standard
Hepatitis B virus DNA, for 850 000 IU per ml Third WHO International
Hepatitis B virus genotypes, No assigned values First WHO International
for HBsAg assays Reference Panel
Hepatitis C virus RNA, for 260 000 IU per ml. Fourth WHO International
Hepatitis E virus RNA, for 250 000 IU per ml First WHO International
HIV-1 RNA, for NAT-based 185 000 IU per ml Third WHO International
assays Standard
Vaccines and related substances
Anti-pneumococcal serotype 1, 8.5 µg/ml First WHO International
antibodies, human, serum serotype 3, 1.45 µg/ml Standard

serotype 4, 3.33 µg/ml
serotype 6A, 3.93 µg/ml
serotype 6B, 9.05 µg/ml
serotype 7F, 8.3 µg/ml
serotype 9V, 6.44 µg/ml
serotype 18C, 7.3 µg/ml
serotype 19A, 13.87 µg/ml
Meningococcal 1.192 mg per ampoule First WHO International
serogroup C polysaccharide Standard
366

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979

WHO Expert Committee on Biological Standardization

WHO Technical Report Series

WHO Expert Committee

© World Health Organization 2013

WHO Expert Committee on Biological Standardization

Representatives of other organizations

Dr E. Charton, Department of Biological Standardisation, OMCL Network and HealthCare,

Dr A. Hubbard, Division of Haematology, National Institute for Biological Standards and

Dr I. Knezevic, Team Leader, Essential Medicines and Pharmaceutical Policies, WHO,

Professor R. Seitz, Head, Division of Haematology and Transfusion Medicine, Paul-Ehrlich-

HCV hepatitis C virus

UTR untranslated region

The results of a survey undertaken by WHO showed that norms and

to increase the implementation of the standards adopted by the Committee

2.1.2 Vaccines and biological therapeutics: recent and

Another important activity is the development of WHO guidance on

2.1.3 Blood products and related in vitro diagnostics: recent

An often under-appreciated but essential contribution of NIBSC and

the 7th IABS International Symposium on Advances in Transfusion Safety in

2.2.2 Reports from international laboratories and WHO

Fourteen new and replacement reference materials from NIBSC were

WHO Collaborating Centre for Quality Assurance of Blood Products and in

Other activities included work on developing a process (“animal rule”) to

both of which were ongoing). Other ongoing projects of potential interest

2.3.2 Issues shared with the WHO Expert Committee on

Proposed Third WHO International Standard for bacterial endotoxin

WHO International Standard for endotoxin established in 1996 now needed

Proposed global legally binding instrument on mercury:

alternatives; educating dental professionals; and raising public awareness.

3.1 Vaccines and related substances

3.1.2 Recommendations to assure the quality,

3.1.3 Recommendations to assure the quality, safety

The Committee was informed that the current Requirements for

3.1.4 Assessing risk when a potential adventitious agent is found

emphasizing the importance of coordination and collaboration among WHO,

3.1.5 Calibration of seasonal and pandemic influenza antigen

3.1.6 Proposed new projects for developing or updating written standards

3.2 Blood products and related substances

The draft Guidelines incorporated references to the SSC/ISTH Guidelines

3.2.2 Assessment criteria for national blood regulatory systems

4.1 WHO International Standards and Reference

4.1.2 First WHO International Standard for

selected an enzyme-linked immunosorbent assay protocol for the quantification

4.1.3 Third WHO International Standard for

4.2 Proposed new projects – vaccines and related substances

Influenza is considered to be of considerable public health importance

4.3 Ongoing stability monitoring – vaccines

In addition, although stability samples were laid down at the time of

The Committee recommended that information on potential contamination

4.4 Progress reports – vaccines and related substances

4.4.2 Hepatitis B vaccine

5.1 WHO International Standards and Reference

5.1.2 Third WHO International Standard for fibrinogen (plasma)

(plasma) necessitates the preparation of a replacement standard. A collaborative

5.1.3 First WHO International Reference Reagent for

5.1.4 First WHO International Reference Reagents

5.2 Proposed new projects – blood products

5.2.2 Replacement of the Third WHO International Standard for plasmin

As stocks of the Third WHO International Standard for plasmin were