TRS 979 62nd Report
TRS 979 62nd Report
TRS 979 62nd Report
W H O Te c h n i c a l R e p o r t S e r i e s
979
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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
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
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
WHO Expert Committee on Biological Standardization
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)
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WHO Expert Committee on Biological Standardization Sixty-second report
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)
WHO Technical Report Series, No. 979, 2013
3
For specific agenda items only – by telephone.
ix
WHO Expert Committee on Biological Standardization Sixty-second report
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
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WHO Expert Committee on Biological Standardization Sixty-second report
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
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.
WHO Technical Report Series, No. 979, 2013
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.
4
General
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
WHO Technical Report Series, No. 979, 2013
The Committee noted the developments in this area, and welcomed the
inclusion of MHLW as a new member of the BRN.
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.
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WHO Expert Committee on Biological Standardization Sixty-second report
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
8
General
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.
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WHO Expert Committee on Biological Standardization Sixty-second report
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,
WHO Technical Report Series, No. 979, 2013
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.
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.
After making suitable amendments, the Committee recommended that
the Recommendations be adopted and appended to its report (Annex 3).
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
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
International Recommendations, Guidelines and other matters
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.
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
International Recommendations, Guidelines and other matters
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.
After making suitable amendments, the Committee recommended that
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.
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.
23
5. International reference materials – blood
products and related substances
All reference materials established at the meeting are listed in Annex 8.
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.
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
International reference materials – blood products and related substances
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.
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WHO Expert Committee on Biological Standardization Sixty-second report
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
6. International reference materials –
in vitro diagnostic device reagents
6.1 WHO International Standards and Reference
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.
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.
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.
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
International reference materials – in vitro diagnostic device reagents
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.
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
International reference materials – in vitro diagnostic device reagents
40
7. International reference materials –
biotherapeutics other than blood products
All reference materials established at the meeting are listed in Annex 8.
44
8. International reference materials – antibiotics
All reference materials established at the meeting are listed in Annex 8.
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
World Health Organization
20 avenue Appia
1211 Geneva 27
Switzerland
1
Abbreviated in the following pages to “TRS”.
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WHO Expert Committee on Biological Standardization Sixty-second report
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)
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
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Annex 1
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)
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WHO Expert Committee on Biological Standardization Sixty-second report
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);
Amendment 2003, TRS 926 (2004)
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)
WHO Technical Report Series No. 979, 2013
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
(live, attenuated) 81
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
(live, attenuated) 87
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
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WHO Expert Committee on Biological Standardization Sixty-second report
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.
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WHO Expert Committee on Biological Standardization Sixty-second report
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
WHO Technical Report Series No. 979, 2013
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).
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.
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WHO Expert Committee on Biological Standardization Sixty-second report
(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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WHO Expert Committee on Biological Standardization Sixty-second report
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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Annex 2
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.
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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Annex 2
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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Annex 2
in vivo guinea-pig test for the detection of mycobacteria after suitable validation
and agreement from the NRA (33).
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).
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.
have been discarded for any reason by the end of the test period.
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.4.2.5 Storage
Monovalent virus harvest pools should be stored at a temperature that ensures
stability.
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
WHO Technical Report Series No. 979, 2013
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.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.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:
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.
Table A2.1
Nonclinical evaluation of dengue vaccines
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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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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.
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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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.
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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.
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
WHO Technical Report Series No. 979, 2013
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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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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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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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.
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).
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.
and of any associated decisions taken, both for information and to ensure that
the appropriate procedures have been followed.
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
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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.
Figure A2.1
Typical steps in an environmental risk assessment
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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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.
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
WHO Technical Report Series No. 979, 2013
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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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,
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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
Report Series, No. 927), Annex 1.
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.
American Journal of Tropical Medicine and Hygiene, 2003, 69:48–60.
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.
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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
Organization, 2008 (WHO/IVB/08.12; http://whqlibdoc.who.int/hq/2008/WHO_IVB_08.12_eng.
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57. Guidelines for plaque-reduction neutralization testing of human antibodies to dengue viruses.
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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.
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deliberate release into the environment of genetically modified organisms and repealing Council
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61. Commission Decision 2002/623/EC of 24 July 2002 establishing guidance notes supplementing
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in ChimeriVax-dengue 4 does not enhance infection of Aedes aegypti mosquitoes. Journal of
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67. Monath TP et al. Recombination and flavivirus vaccines: a commentary. Vaccine, 2005, 23:2956–
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68. Seligman SJ, Gould EA. Safety concerns with regard to live attenuated flavivirus vaccines.
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70. Pugachev KV et al. Construction and biological characterization of artificial recombinants
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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.
American Journal of Tropical Medicine and Hygiene, 2004, 71:811–821.
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
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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,
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WHO Technical Report Series No. 979, 2013
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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.
Expiry date:
Storage conditions:
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.
3.1.2 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.
Antibiotics
Nature and concentration of antibiotics or selecting
agent(s) used in production cell culture maintenance
medium:
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:
Genetic/phenotypic characterizations
Method:
Reference reagents:
Specification:
Dates of test (start, end):
Result:
Adventitious agents
Volume of virus seed samples for neutralization
and testing:
Batch number(s) of antisera/antiserum used for
neutralization of virus seeds:
Tests in nonhuman primates (either master or working seed lot) for neurovirulence
For details, please see Recommendations for yellow fever vaccine (51)
Method:
Specification:
Dates of test (start, end):
Result:
Tests in suckling mice (either master or working seed lot, where necessary)
for neurovirulence
(Detailed protocol should be developed)
Method:
Specification:
Dates of test (start, end):
Result:
Incubation conditions:
Method:
Specification:
Dates of test (start, end):
Ratio of cultures viable at end of test:
Result:
Identity test
Method:
Specification:
Dates of test (start, end):
Result:
Specification:
Date of test:
Result:
Result:
Test for pH
Method:
Specification:
Date of test:
Result:
Media:
Volume tested:
Temperatures of incubation:
Dates of test (start, end):
Result:
Tests in guinea-pigs
Date of inoculation:
Number of animals tested:
Volume and route of injection:
Observation period:
Specification:
Results (give details of deaths):
Residual moisture
Method:
Specification:
Date:
Result:
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6.2 Diluent
Name and composition of diluent:
Lot number:
Date of filling:
Type of diluent container:
Filling volume per container:
Maximum period of storage approved:
Storage temperature and period:
Name (typed)
Signature
Date
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.
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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.
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
WHO Technical Report Series, No. 979, Annex 2.
6
WHO Technical Report Series, No. 961, Annex 3.
7
WHO Technical Report Series, No. 822, Annex 1.
8
WHO Technical Report Series, No. 978, Annex 2.
9
Evaluation of summary protocol, independent laboratory testing, and/or specific procedures laid down in
defined document etc., as appropriate.
134
Annex 2
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
General considerations 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.5 Filling and containers 152
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
A.11 Stability, storage and expiry date 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
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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
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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.
General considerations
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).
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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).
WHO Technical Report Series No. 979, 2013
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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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
WHO Technical Report Series No. 979, 2013
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.
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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Annex 3
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.
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.
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.
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.
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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Annex 3
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.
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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WHO Expert Committee on Biological Standardization Sixty-second report
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.
The survival rate after freeze-drying is usually not less than 20%.
Since ATP is present in all living cells and is immediately destroyed when
the cell dies, ATP is a reliable marker for living cells.
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.
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.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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Annex 3
The label for the diluent should state “Reconstituting fluid for BCG
vaccine [proprietary name]”.
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.
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).
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).
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.
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.
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WHO Expert Committee on Biological Standardization Sixty-second report
References
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No. 745), Annex 2.
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.
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held at Pasteur Institute, Paris, France, 7 June 2005. Vaccine, 2006, 24:3874–3877.
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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.
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Quality of Medicines of the Council of Europe (EDQM), 2008 (01/2008:0163).
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Standardization. Fifty-fourth report. Geneva, World Health Organization, 2005 (WHO Technical
Report Series, No. 927), Annex 1.
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.
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Committee on the Use of Essential Drugs. Sixth report. Geneva, World Health Organization, 1995
(WHO Technical Report Series, No. 850), Annex 3.
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on Biological Standardization. Fifty-second report. Geneva, World Health Organization, 2004 (WHO
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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.
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Infectious Disease, 1999, 28:785–790.
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(WHO Technical Report Series, No. 552).
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Appendix 1
History and genealogy of BCG substrains
WHO Technical Report Series No. 979, 2013
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.
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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.
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.
on master and working seed lots are required on first submission only and whenever
a change has been introduced.
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20–25 °C
30–36 °C
Negative
control
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20–25 °C
30–36 °C
Negative
control
Date of freeze-drying:
Number of containers rejected during inspection:
Number of containers sampled:
Total number of containers (net):
Maximum period of storage approved:
Storage temperature and period:
Result:
Recommended reconstitution fluid:
Volume of reconstitution fluid per final container:
20–25 °C
30–36 °C
Negative
control
Specification:
Result:
Specification:
Result:
Details of working Reference Preparation:
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 3.
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Appendix 3
Model certificate for the release of BCG vaccine by NRAs
Lot release certificate
Certificate no.
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
WHO Technical Report Series, No. 979, Annex 3.
6
WHO Technical Report Series, No. 961, Annex 3.
7
WHO Technical Report Series, No. 822, Annex 1.
8
WHO Technical Report Series, No. 978, Annex 2.
9
Evaluation of summary protocol, independent laboratory testing, and/or specific procedures laid down in
defined document etc., as appropriate.
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■ 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
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Annex 4
Recommendations to assure the quality, safety and
efficacy of acellular pertussis vaccines
Replacement of Annex 2 of WHO Technical Report Series, No. 878
Introduction 189
Background 189
Scope 191
General considerations 192
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.4 Filling and containers 207
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
A.10 Stability, storage and expiry date 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
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Acknowledgements 228
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
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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
WHO Technical Report Series No. 979, 2013
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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General considerations
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
WHO Technical Report Series No. 979, 2013
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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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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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
WHO Technical Report Series No. 979, 2013
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.3.4.2.2 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
WHO Technical Report Series No. 979, 2013
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.
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.
containing genetically detoxified PT, this test may not be necessary as agreed
with the NRA.
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.
Other assays
The development of alternative assays to MIT and MICA is encouraged.
WHO Technical Report Series No. 979, 2013
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.
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.
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.6 Records
The recommendations given in Good manufacturing practices for biological
products (22) should apply.
A model of a suitable summary protocol to be used for pertussis vaccines
is given in Appendix 6.
208
Annex 4
A.7 Samples
The recommendations given in Good manufacturing practices for biological
products (22) should apply.
A.8 Labelling
The recommendations given in Good manufacturing practices for biological
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.
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.
the design, conduct, analysis and evaluation of nonclinical studies are available in
the WHO Guidelines on nonclinical evaluation of vaccines (37).
Table A4.1
Summary of published studies evaluating the ability of purified pertussis antigens to
protect in mouse challenge models
214
Annex 4
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.
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).
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).
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WHO Expert Committee on Biological Standardization Sixty-second report
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).
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.
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.
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
WHO Technical Report Series No. 979, 2013
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
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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
WHO Technical Report Series No. 979, 2013
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.
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
WHO Technical Report Series No. 979, 2013
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
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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
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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-
WHO Technical Report Series No. 979, 2013
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.
WHO Technical Report Series No. 979, 2013
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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.
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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.
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
WHO Technical Report Series No. 979, 2013
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.
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Annex 4
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.
Appendix 6
Summary protocol for production and testing of acellular
pertussis vaccine
1. Summary information on final lot
Name and address of manufacturer:
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:
Identification:
Volume:
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Annex 4
Antigen content
Methods:
Content:
Date:
Sterility test:
Tests for bacteria and fungi
Method:
Media:
Number of containers tested:
Volume of inoculum per container:
Volume of medium per container:
Temperatures of incubation:
Date of test (start, end):
Result:
Detoxification
Detoxifying reagent:
Detoxifying conditions:
Control of bulk
Identification:
Volume:
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Temperature
average variance
Reference dilution 1
Reference dilution 2
Reference dilution n
Test vaccine
Negative
WHO Technical Report Series No. 979, 2013
Reference dilution 1 /
Reference dilution 2 /
Reference dilution n /
Test vaccine /
Negative /
Sterility test
Tests for bacteria and fungi
Method:
Media:
Number of containers tested:
Volume of inoculum per container:
Volume of medium per container:
Temperatures of incubation:
Date of test (start, end):
Result:
Detoxifying agent
Methods:
Content:
Date:
Preservative content
Methods:
Content:
Date:
Adjuvant
Methods:
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Content:
Date:
Sterility
Tests for bacteria and fungi
Method:
Media:
Number of containers tested:
Volume of inoculum per container:
Volume of medium per container:
Temperatures of incubation:
Date of test (start, end):
Result:
Temperature
WHO Technical Report Series No. 979, 2013
average variance
Reference dilution 1
Reference dilution 2
Reference dilution n
Test vaccine
Negative
252
Annex 4
Reference dilution 1 /
Reference dilution 2 /
Reference dilution n /
Test vaccine /
Negative /
Reversion to toxicity
Incubation period: start date end date temperature
Methods:
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Temperature
average variance
Reference dilution 1
Reference dilution 2
Reference dilution n
Test vaccine
Negative
Reference dilution 1 /
Reference dilution 2 /
Reference dilution n /
WHO Technical Report Series No. 979, 2013
Test vaccine /
Negative /
Immunological activity
1. MIT
Strain of mice:
No. of mice per dilution:
No. of dilutions injected:
Volume and route of injection:
Identification of reference:
Date of bleeding:
Antibody titration:
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2. MICA
Strain of mice:
No. of mice per dilution:
No of dilutions injected:
Volume and route of injection:
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:
Test vaccine ml
Final product
Identification:
Volume:
Identity test
Method of testing:
Result:
Date of test:
Sterility test
Tests for bacteria and fungi
Method:
Media:
Number of containers tested:
Volume of inoculum per container:
Volume of medium per container:
Temperatures of incubation:
Date of test (start, end):
Result:
Specification:
Result:
Date of test:
pH
Method of testing:
Specification:
Result:
Date of test:
Endotoxin test
Method of testing:
Specification:
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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:
Volume and route of injection:
Observation period:
Results (give details of deaths):
Tests in guinea-pigs
Date of start of test:
Date of end of test:
No. of animals tested:
Route of injection:
Volume and route of injection:
Observation period:
Results (give details of deaths):
Name (typed)
Signature
Date
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 4.
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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.
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
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
WHO Technical Report Series, No. 979, Annex 4.
6
WHO Technical Report Series, No. 961, Annex 3.
7
WHO Technical Report Series, No. 822, Annex 1.
8
WHO Technical Report Series, No. 978, Annex 2.
9
Evaluation of summary protocol, independent laboratory testing, and/or specific procedures laid down in
defined document etc. as appropriate.
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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
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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
WHO Technical Report Series No. 979, 2013
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.
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Annex 5
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.
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.
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.
3
See: http://www.who.int/influenza/gisrs_laboratory/en/, accessed 23 February 2013.
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Appendix 1
Sample data sheet
Date:
Reference:
Primary liquid standard ([ERL]): [virus name] ([reassortant]) (Lot no. [yyy])
Antiserum:
Anti-[virus name] sheep antiserum (Lot no. [zzz], [ERL])
x µl/ml agarose
Sample:
Lot no. [yyy], [ERL]
Mean
268
Annex 5
Appendix 2
Supplementary data sheet
Calibration of [virus name] reference antigen lot [yyy] with antiserum lot [zzz]
Collaborative study summary results
269
Annex 6
Guidelines for thromboplastins and plasma used to control
oral anticoagulant therapy with vitamin K antagonists
Replacement of Annex 3 of WHO Technical Report Series, No. 889
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
Acknowledgements 298
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
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Appendix 3
Example of the use of the suggested method for reporting the data for the
calibration of individual batches of thromboplastin 314
WHO Technical Report Series No. 979, 2013
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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.
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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.
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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.
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Figure A6.1
WHO International Reference Preparations, International Reference Reagents and
International Standards for thromboplastins, and their calibration relationships
WHO Technical Report Series No. 979, 2013
IRP = International Reference Preparation; IRR = International Reference Reagent; IS = International Standard.
a
Now discontinued.
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
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5. Tests on thromboplastins
Each batch of thromboplastin should satisfy the following criteria.
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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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).
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).
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.
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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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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
Reference Preparation.
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
WHO Technical Report Series No. 979, 2013
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).
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.
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.
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.
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.
may seek help from expert laboratories. The validation process should go through
the following steps.
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.
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.
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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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
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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.
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
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Table 1 continued
Date Plasma rTF/95 Secondary standard
23 February 2009 Patient 5 35.6 21.7
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Table 1 continued
Date Plasma rTF/95 Secondary standard
26 February 2009 Normal 9 13.1 10.9
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
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Table 1 continued
Date Plasma rTF/95 Secondary standard
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
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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.
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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.
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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)
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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,
Gralnick HR, eds. Thromboplastin calibration and oral anticoagulant control. Boston, Martinus
Nijhoff, 1984: 87–108.
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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
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
WHO Technical Report Series No. 979, 2013
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)
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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
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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
WHO Technical Report Series No. 979, 2013
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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
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318
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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.
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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
WHO Technical Report Series No. 979, 2013
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
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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:
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.
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Table continued
Main criteria related Rating* Indicators related to the
to the element main criteria
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.
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Table continued
Main criteria related Rating* Indicators related to the
to the element main criteria
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.
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WHO Expert Committee on Biological Standardization Sixty-second report
Table continued
Main criteria related Rating* Indicators related to the
to the element main criteria
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.
WHO Technical Report Series No. 979, 2013
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Table continued
Main criteria related Rating* Indicators related to the
to the element main criteria
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.
* R = required; S = suggested.
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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.
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Table continued
Main criteria related Rating* Indicators related to the
to the function main criteria
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
registration and/or application
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.
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Table continued
Main criteria related Rating* Indicators related to the
to the function main criteria
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.
WHO Technical Report Series No. 979, 2013
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).
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Table continued
Main criteria related Rating* Indicators related to the
to the function main criteria
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
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Table continued
Main criteria related Rating* Indicators related to the
to the function main criteria
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.
R 4.2.4 Advice for applicants is
available on the content,
format, requirements
(depending on the activities)
and procedures to follow in
order to submit a required
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Table continued
Main criteria related Rating* Indicators related to the
to the function main criteria
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
S 4.3.2 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.
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.
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Table continued
Main criteria related Rating* Indicators related to the
to the function main criteria
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.
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Table continued
Main criteria related Rating* Indicators related to the
to the function main criteria
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.
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Table continued
Main criteria related Rating* Indicators related to the
to the function main criteria
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
WHO Technical Report Series No. 979, 2013
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Table continued
Main criteria related Rating* Indicators related to the
to the function main criteria
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.
S 5.6.3 Written guidelines for
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.
S 5.7.2 Written guidelines for
assessment exist based
on the type of change
(e.g. significant, notifiable,
administrative).
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Table continued
Main criteria related Rating* Indicators related to the
to the function main criteria
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).
S 5.8.2 Written procedures for
selection, management, and
use of external experts are in
place.
* R = required; S = suggested.
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Main criteria related Rating* Indicators related to the
to the function main criteria
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).
R 6.2.5 Advice for applicants is
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.
S 6.2.8 Written procedures for
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.
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Table continued
Main criteria related Rating* Indicators related to the
to the function main criteria
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).
S 6.3.5 Written guidelines for
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
changes and documentation
required.
S 6.4.3 Written guidelines for
assessment are available
based on the type of change.
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Table continued
Main criteria related Rating* Indicators related to the
to the function main criteria
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.
* R = required; S = suggested.
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Main criteria related Rating* Indicators related to the
to the function main criteria
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.
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Main criteria related Rating* Indicators related to the
to the function main criteria
Main Indi‑
criteria cator
S 7.2.5 Written guidelines for
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.
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Main criteria related Rating* Indicators related to the
to the function main criteria
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
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Main criteria related Rating* Indicators related to the
to the function main criteria
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.
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Main criteria related Rating* Indicators related to the
to the function main criteria
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
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Table continued
Main criteria related Rating* Indicators related to the
to the function main criteria
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.
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Table continued
Main criteria related Rating* Indicators related to the
to the function main criteria
Main Indi‑
criteria cator
R 10.2.6 Procedures for
communication with the
product manufacturer are
defined.
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Main criteria related Rating* Indicators related to the
to the function main criteria
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.
* R = required; S = suggested.
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Table continued
Main criteria related Rating* Indicators related to the
to the function main criteria
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.
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Main criteria related Rating* Indicators related to the
to the function main criteria
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.
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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.
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Main criteria related Rating* Indicators related to the
to the function main criteria
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.
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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).
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Main criteria related Rating* Indicators related to the
to the function main criteria
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.
* R = required; S = suggested.
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Main criteria related Rating* Indicators related to the
to the function main criteria
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.
* R = required; S = suggested.
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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
Organization, 2011 (WHO Technical Report Series, No. 961), Annex 3.
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
Organization, 2007 (WHO Technical Report Series, No. 941), Annex 4.
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,
World Health Organization, 1994 (WHO Technical Report Series, No. 840), Annex 2.
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,
2006 (WHO Technical Report Series, No. 937), Annex 4.
WHO good manufacturing practices for sterile pharmaceutical products. In: WHO Expert Committee on
Specifications for Pharmaceutical Preparations. Forty-fifth report. Geneva, World Health Organization,
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WHO Technical Report Series No. 979, 2013
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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
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