1. Establishment of the 1st WHO International Standard for Detection of Antibodies to Hepatitis B Virus Core Antigen (anti-HBc) SoGAT XXI 29 May 2009 Dr. Heiner Scheiblauer Paul-Ehrlich-Institut, Langen/Germany PEI-IVD

2. 2 Why Anti-HBc?  Anti-HBc are the first antibodies after Hepatitis B virus infection and persist trough out life  Anti-HBc can detect a persistent HBV viraemia, that is not easy detectable by HBsAg or HBV DNA  Anti-HBc can detect HBV infections with HBV escape mutants or genotypes which are not detectable by certain HBsAg or HBV DNA test kits  Anti-HBc screening can prevent transfusion-transmitted HBV infections  Anti-HBc has a regulatory impact: requirement for obligatory screening in some countries worldwide

3. 3 WHO Anti-HBc Standard Demand  Estimation of the analytical sensitivity for anti-HBc tests  Calibration of anti-HBc test systems by manufacturers  Accuracy and reproducibility verification in batch testing  Current national PEI anti-HBc standard (no 82, serum, 100 PEI U/ml) has been used widely  Sensitivity of assays and anti-HBc content of samples expressed as PEI-U/ml  PEI anti-HBc standard stocks declined, replacement preparation needed

4. 4 WHO Anti-HBc Collaborative Study Samples Tested Candidate Material

5. 5 WHO Anti-HBc Collaborative Study Study design  Analytical sensitivity of Samples A (NIBSC 95/522) and B (PEI 82) at the intercept with the assay´s cut-off (endpoint titer, detection limit in U/ml)  Potency of NIBSC 95/522 relative to PEI 82 (U/ml)  GMV ratio of the detection limit  Parallel line model  Correlation between analytical sensitivity for NIBSC 95/522 and anti- HBc detection in Samples C (PEI 108166) and D (CBER Panel #11)  Variability in results between assay methods (repeatability), and between laboratories (reproducibility)  Geometric coefficient of variation (GCV%)  Statistical significance of differences between methods and laboratories  Stability

6. 6 WHO Anti-HBc Collaborative Study Participants 1.Hemocentro de Sao Paulo, Brazil 2.CBER, USA 3.INTS, France 4.Abbott Laboratories, USA 5.KFDA, Seoul, Republic of Korea 6.American Red Cross, Gaithersburg, USA 7.National Institute of Infectious Diseases, Tokyo, Japan 8.NIBSC, Hertsfordshire, UK 9.AFSSAPS, France 10.National Institute for the Control of Pharmaceutical and Biological Products Beijing Zhongyuan, China 11.NRL, Australia 12.VQC Laboratory Sanquin-CLB Diagnostics, Netherlands 13.Paul-Ehrlich-Institut, Germany

7. 7 WHO Anti-HBc Collaborative Study Test kits 1.ADVIA Centaur HBcT 2.Architect Anti-HBc 3.AxSYM Core 4.Bioelisa anti-HBc 5.Corzyme 6.Elecsys Anti-HBc 7.Enzygnost Anti-HBc monoclonal 8.Genedia Anti-HBc ELISA Plus 9.Hepanostika anti-HBc Uniform 10.Immulite 2000 anti-HBc 11.Immuncomb II HBc IgG 12.IMx Core 13.Anti-HBc EIA Lumipuls 14.Presto HBc Ab-N 15.Monolisa anti-HBc Plus 16.Murex anti-HBc 17.Ortho anti-HBc 18.PRISM HBCore 19.ST AIA-Pack HBcAb 20.Architect Anti-HBc II (ex-US) / Architect Core (US)

8. 8 WHO Anti-HBc Collaborative Study Summary of test kits characteristics

9. 9 Collaborative Study - Suitability for variety of tests, mfcts, labs, geography  20 anti-HBc test kits included in the study from 15 different manufacturers and from 7 different countries  Tested in 10 different countries (Australia, Brazil, China, France, Japan, Germany, Korea, Netherlands, UK, USA)  Including the current available anti-HBc test technologies  Competitive test format (n=14)  Indirect test format (n=5)  Sandwich test format (n=1)  Reductant pre-treatment (e.g. DTT) with (n=5) or without (n=15)  Manually conducted (n=9)  Automated (n=10)  Rapid assay (n=1)  All anti-HBc test kits coated with recombinant HBc antigen

10. 10 WHO Anti-HBc Collaborative Study Potency of NIBSC 95/522 relative to PEI 82 Ø Ø Dilution ranges for NIBSC 95/522 (sample A) was in the dynamic measuring range of assays Ø Ø Within an individual assay, Sample A and B (PEI 82) gave comparable dose responses

11. 11 WHO Anti-HBc Collaborative Study Endpoint titers Dilution equivalent to cut-off – NIBSC 95/522 Endpoint dilution 1/32 1/64 1/128 1/256 1/512 1/1024 Laboratory Assay 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 7 12 10 13 1 7 12 1 4 7 9 7 5 2 6 10 8 2 5 7 11 12 5 9 4 1 3 4 8 1

12. WHO Anti-HBc Collaborative Study Overall Potency  Overall potency was 49.8 U/ml (95%-CI 44.2 - 56.1 U/m)  Assay 1 had a potency of 22.8 U/ml (mean of 2 labs)  Assay 8 had a potency of 73.3 U/ml  Overall potency excluding assays 1 and 8 was 51.1 U/ml (95%-CI 45.3 - 57.5 U/ml)

13. 13 WHO Anti-HBc Collaborative Study Commutability Ø Ø Analytical sensitivity (low detection limits) in Sample A (NIBSC 95/522) correlated with positive score for anti-HBc in Samples C (PEI 108166) and D (CBER panel #11) Ø Ø One assay of the study did not follow this correlation

14. 14 WHO Anti-HBc Collaborative Study Stability   The proposed anti-HBc standard NIBSC 95/522 is likely to be highly stable when stored at the recommended temperature of -20°C

15. 15 1st International Anti-HBc Standard Conclusions  NIBSC 95/522 1 st International Standard (IS) for detection of anti-HBc  Assigned unitage of 50 IU per ampoule (ml)  The IU provides continuity with the currently used PEI units/ml  Analytical sensitivity for anti-HBc was effective for estimation of sensitivity performance  the lower the detection limit with the IS, the higher the positive score for anti-HBc concentration in other samples  The proposed International anti-HBc Standard was found suitable for  estimation of analytical sensitivity for anti-HBc detection  calibration of anti-HBc test kit sensitivity by manufacturers  to calibrate secondary standards  for quality control procedures, e.g. in batch release testing

16. 16 1st International Anti-HBc Standard Acknowledgements  NIBSC: Dr. Morag Ferguson  CBER: Dr. Robin Biswas  PEI: Dr. Peter Volkers Dr. Sigrid Nick Dr. Micha Nübling  WHO: Dr. Ana Padilla

17. 17 Thank you for your attention!