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Preparation, characterization and establishment of a WHO International
Biological Reference PreparationDr Sjoerd Rijpkema
Division of BacteriologyNIBSC
How are International Standards How are International Standards prepared?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 oSuitable for all diagnostic testsoSpecific for Tc I and Tc IIoCross-reactive antibodiesoAntibody 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 participantsoRepresent reference and specialist diagnostic
laboratories Select samples for analysis pack Select test or range of testsoSamples tested as part of routine oNo re-imbursement for testing
Project Team
InternaloProject leader oStatisticianoFormulation scientist oFill & freeze dry/packaging & distribution
External oExpert(s) oSpecialist diagnostic laboratoriesoParticipants of the collaborative study
Collaborative study (1)
Participants oGlobal distribution
Duplicate sample packs distributedoBlinded samplesoCandidate standard duplicatedoPositive + Negative control samples
Tests oSelection agreed in the CS designoCarried 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 Code
NIBSC Code
Sample RPR TPPA EIA Ig IgM
HS HS 1st International Standard 24 IU/ml
64 >1280 pos pos
G, E 05/132 Pooled candidate standard
8 1280 pos pos
C 05/122 Pooled candidate standard
neg 1280 pos neg
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
of cortisol
neg neg neg neg D 82/585 neg neg neg neg
Collaborative study (2)
Collate raw test data Calculate mean test value, CI and GCV Accelerated degradation studiesoTest 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 titreoEach sample tested 4x on two separate dayso8 data points/sample
EIA Methods: produce a numerical valueoEach sample tested in 4 sequential dilutions on two
separate days o8 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/To log{k(T)} = + /T o Independent of time
Log
{ k(
T) }
1 / T37oC 20oC 4oC -20oC
Accelerated degradation Predicts degradation process & rate
Samples removed from storage site at
Sample removed from-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. for one year
TPPA endpoint Potency relative to -20oC#1 #2 #3 #4
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.
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