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 o Suitable for all diagnostic tests o Specific for Tc I and Tc II o Cross-reactive antibodies o 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 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

7. 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

8. 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

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 o Test stability of candidate standards stored at +37 o C to –20 o C 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 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: manufacturers 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 4 o C to 56 o C. Storage time ranges from 6 months to 2 years. The activity of these samples is then calculated relative to those samples stored at -20 o C (T 0 ).

13. The method Biological activity is assumed to degrade exponentially with time. This is a first- order process. An estimate of k(T 0 ) is required to predict the stability of the standard, but k(T) can only be estimated for T>T 0.

14. 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 37 o C20 o C4oC4oC-20 o C

15. Accelerated degradation Predicts degradation process & rate Samples removed from storage site at Sample removed from -20 o C4oC4oC20 o C37 o C45 o C56 o C 1 month 2 months 3 months() 6 months() 12 months Subsequent times

16. Effect of accelerated degradation on potency of 05/122 and 05/132 Storage Temp. for one year TPPA endpointPotency relative to -20 o C #1#2#3#4 05/122-20 o C640 - + 4 o C640 1.00 +20 o C640 1.00 +37 o C320 6400.59 05/132 -20 o C5120 - + 4 o C5120 1.00 +20 o C5120 1.00 +37 o C5120 2560 0.71

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 mo11 mo Collect results+3 mo14 mo Analyse data+3 mo17 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: o WHO technical bulletin series 932. ISBN 92-4-120932-1 o http://www.who.int/biologicals/expert_committee/TRS932CVR% 20with%20full%20Texts.pdf 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.

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