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Preparation, Characterization and Establishment of A WHO International Biological Reference Preparation

This document discusses the process for establishing an international biological reference standard for Chagas disease. It describes how source material is selected, tested for safety and suitability, and pooled. A collaborative study is conducted globally to evaluate the candidate standard in different assays. Stability testing is also done by accelerating degradation. If approved, the standard can improve standardization of Chagas disease diagnostic testing worldwide.

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jyothi
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19 views

Preparation, Characterization and Establishment of A WHO International Biological Reference Preparation

This document discusses the process for establishing an international biological reference standard for Chagas disease. It describes how source material is selected, tested for safety and suitability, and pooled. A collaborative study is conducted globally to evaluate the candidate standard in different assays. Stability testing is also done by accelerating degradation. If approved, the standard can improve standardization of Chagas disease diagnostic testing worldwide.

Uploaded by

jyothi
Copyright
© © All Rights Reserved
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
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Preparation, characterization and

establishment of a WHO International


Biological Reference Preparation
Dr Sjoerd Rijpkema
Division of Bacteriology
NIBSC
How are International Standards
prepared?
 Experts select material that is suitable to
serve as a standard for diagnostic assays
 Filled and freeze dried
 Long term stability
 Suitability for use is established in a
collaborative study
 Statistical analysis of raw assay data
 Arbitrary unitage is assigned
Reference panel for Chagas disease
 Chagas disease is a global threat to public
health
 Antibody panel
o Suitable for all diagnostic tests
o Specific for Tc I and Tc II
o Cross-reactive antibodies
o Antibody titre range
Source material
 Represent geographic origins of disease
 Informed consent/local ethical approval
 Serum/Plasma/Recalcified plasma
 Pooled samples or individual sample
 Certified free of HIV1/2, HepA/B/C
 Certified free of T. cruzi parasites
 Source material ‘fit for purpose’ pre & post freeze
drying
 Pilot study FD conditions
 ‘fit for purpose’ post freeze drying
 1000 – 5000 ampoules
Aim of the collaborative study

 Establish suitability of the candidate standard in a


range of assays including the ‘gold’ standard
 Collect international data set
 Statistical analysis of raw data
 Assign a unitage
 Evaluate differences in assay methods
 Evaluate inter-laboratory differences (GCV)
Collaborative study design

 Create project team


 Select international participants
o Represent reference and specialist
diagnostic laboratories
 Select samples for analysis pack
 Select test or range of tests
o Samples tested as part of routine
o No re-imbursement for testing
Project Team

 Internal
o Project leader
o Statistician
o Formulation scientist
o Fill & freeze dry/packaging & distribution
 External
o Expert(s)
o Specialist diagnostic laboratories
o Participants of the collaborative study
Collaborative study (1)

 Participants
o Global distribution
 Duplicate sample packs distributed
o Blinded samples
o Candidate standard duplicated
o Positive + Negative control samples
 Tests
o Selection agreed in the CS design
o Carried out in duplicate on at least two
separate days = 4 tests per sample
Sample pack for the collaborative
study of 05/122 & 05/132
Study NIBSC Sample RPR TPPA EIA
Code Code Ig IgM
HS HS 1st International 64 >1280 pos pos
Standard 24 IU/ml
G, E 05/132 Pooled candidate 8 1280 pos pos
standard
C 05/122 Pooled candidate neg 1280 pos neg
standard
F 97/682 Normal serum neg neg neg neg
A 05/142 Positive serum neg 1280 pos pos
B 83/571 Sera with high level neg neg neg neg
D 82/585 of cortisol neg neg neg neg
Collaborative study (2)

 Collate raw test data


 Calculate mean test value, CI and GCV
 Accelerated degradation studies
o Test stability of candidate standards stored at
+37oC to –20oC
 Draft report and propose unitage
 Final report endorsed by all members of CS
 Report send to WHO-ECBS for approval
 Publish study
Collection of raw data

 Titration methods: yield endpoint titre


o Each sample tested 4x on two separate days
o 8 data points/sample
 EIA Methods: produce a numerical value
o Each sample tested in 4 sequential dilutions
on two separate days
o 8 data points/sample
 In-house methods: standard procedures
 Commercially available tests: manufacturer’s
protocol
Stability of biological standards

 The stability of a biological standard is predicted


by an accelerated thermal degradation test.
 Samples of the standard are stored at elevated
temperatures at 4oC to 56oC.
 Storage time ranges from 6 months to 2 years.
 The activity of these samples is then calculated
relative to those samples stored at -20oC (T0).
The method

 Biological activity is assumed to degrade


exponentially with time. This is a “first-
order” process.
 An estimate of k(T0) is required to predict
the stability of the standard, but k(T) can
only be estimated for T>T0.
Arrhenius equation

 log{k(T)} is:
o Linear with 1/T
o log{k(T)} =  + /T
o Independent of time
Log { k(T) }

1/T
37oC 20oC 4oC -20oC
Accelerated degradation
 Predicts degradation process & rate
Samples removed Sample removed from
from storage site at -20oC 4oC 20oC 37oC 45oC 56oC
1 month √ √ √ √
2 months √ √ √ √
3 months √ √ √ √ (√)
6 months √ √ √ (√)
12 months √ √ √ √
Subsequent times √ √ √ √
Effect of accelerated degradation
on potency of 05/122 and 05/132
Storage Temp. TPPA endpoint Potency relative
for one year #1 #2 #3 #4 to -20oC
05/122 -20oC 640 640 640 640 -
+ 4oC 640 640 640 640 1.00
+20oC 640 640 640 640 1.00
+37oC 320 320 320 640 0.59
05/132 -20oC 5120 5120 5120 5120 -
+ 4oC 5120 5120 5120 5120 1.00
+20oC 5120 5120 5120 5120 1.00
+37oC 5120 5120 2560 2560 0.71
Timeline of a collaborative study
• MTA agreed 1 mo
• Source samples shipped +1 mo 2 mo
• Pilot study freeze dry +2 mo 4 mo
• Test FD samples +2 mo 6 mo
• Fill and freeze dry +2 mo 8 mo
• Ship sample packs +3 mo 11 mo
• Collect results +3 mo 14 mo
• Analyse data +3 mo 17 mo
• Submit report
• Approval by WHO ECBS 2011?
• Publication
Biological standards produced by
NIBSC meet WHO criteria

 ISO 9001 and ISO 13485


 All containers in the batch are identical
 Potency is unchanged after processing
 Stable – indefinite shelf life
 Low moisture content
 Oxygen level (<1%)
 Sterility testing: pre & post filling & freeze drying
 Fill weight variation control
 Full production records
References

 For a detailed description of the standard


specification see:
o WHO technical bulletin series 932. ISBN 92-4-120932-1
o http://www.who.int/biologicals/expert_committee/TRS932CVR%
20with%20full%20Texts.pdf
 For accelerated degradation see:
o Kirkwood TBL. Predicting the stability of biological standards and
products. Biometrics 1977; 33: 736-742.
o Shin J, Nam J. Validation of a computer software program for
statistical analysis of accelerated stability studies on biological
standards. Biologicals 2007; 35: 27-30.
Acknowledgements

 Peter Rigsby, Statistical Division


 Paul Matejtschuk, Centre for Biological
Reference Materials

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