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Preparation, characterization and establishment of a WHO International Biological Reference Preparation

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

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Preparation, characterization and establishment of a WHO International Biological Reference Preparation

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  1. Preparation, characterization and establishment of a WHO International Biological Reference Preparation Dr Sjoerd Rijpkema Division of Bacteriology NIBSC

  2. 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

  3. Reference panel for Chagas disease • Chagas disease is a global threat to public health • Antibody panel • Suitable for all diagnostic tests • Specific for Tc I and Tc II • Cross-reactive antibodies • Antibody titre range

  4. 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

  5. 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)

  6. Collaborative study design • Create project team • Select international participants • Represent reference and specialist diagnostic laboratories • Select samples for analysis pack • Select test or range of tests • Samples tested as part of routine • No re-imbursement for testing

  7. Project Team • Internal • Project leader • Statistician • Formulation scientist • Fill & freeze dry/packaging & distribution • External • Expert(s) • Specialist diagnostic laboratories • Participants of the collaborative study

  8. Collaborative study (1) • Participants • Global distribution • Duplicate sample packs distributed • Blinded samples • Candidate standard duplicated • Positive + Negative control samples • Tests • Selection agreed in the CS design • Carried out in duplicate on at least two separate days = 4 tests per sample

  9. Sample pack for the collaborative study of 05/122 & 05/132

  10. Collaborative study (2) • Collate raw test data • Calculate mean test value, CI and GCV • Accelerated degradation studies • 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

  11. Collection of raw data • Titration methods: yield endpoint titre • Each sample tested 4x on two separate days • 8 data points/sample • EIA Methods: produce a numerical value • Each sample tested in 4 sequential dilutions on two separate days • 8 data points/sample • In-house methods: standard procedures • Commercially available tests: manufacturer’s protocol

  12. 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).

  13. 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.

  14. Arrhenius equation Log { k(T) } 1 / T 37oC 20oC 4oC -20oC • log{k(T)} is: • Linear with 1/T • log{k(T)} =  + /T • Independent of time

  15. Accelerated degradation • Predicts degradation process & rate

  16. Effect of accelerated degradation on potency of 05/122 and 05/132

  17. 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

  18. 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

  19. References • For a detailed description of the standard specification see: • WHO technical bulletin series 932. ISBN 92-4-120932-1 • http://www.who.int/biologicals/expert_committee/TRS932CVR%20with%20full%20Texts.pdf • For accelerated degradation see: • Kirkwood TBL. Predicting the stability of biological standards and products. Biometrics 1977; 33: 736-742. • 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.

  20. Acknowledgements • Peter Rigsby, Statistical Division • Paul Matejtschuk, Centre for Biological Reference Materials

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