Update on the Evaluation of the Behavior of Synthetic Materials and Biologics

1. Update on the Evaluation of the Behavior of Synthetic Materials and Biologics SoGAT XX Warsaw, 12 June 2007 Roberta M. Madej Roche Molecular Diagnostics Representing the ILC

2. Purpose • To evaluate the behavior of synthetic materials and biologics in three commercially available quantitative HCV NAT assays.

3. Design “Parallel-line” experiment with • Synthetic RNA from Siemens • Asuragen armored HCV RNA • PEI Reference (80,000 IU/ml) • HCV positive serum sample • HCV positive EDTA plasma sample • WHO Second International Standard for HCV (96/798, 100,000 IU/ml)** • ( Materials “pre-diluted where possible.) Using the following Diagnostic / Quantitative Assays • Bayer VERSANT HCV RNA 3.0 Assay (bDNA) /HCV RNA TMA QN Assay • Roche COBAS AmpliPrep / COBAS TaqMan HCV • Abbott RealTimeTM HCV Six dilution points of five different materials in three different assays.

4. Considerations • Materials • Asuragen and Siemens Materials • Donated by Asuragen and Siemens • Calibrated by independent means to copies/ml Phosphate std and OD readings • PEI material • Donated by M. Nübling • Calibrated to the WHO material std in IU/ml (80,000 IU/ml) • Plasma and Serum • Made and donated by Abbott • Initial testing by Abbott • Assays and logistics • Siemens assay is calibrated with the Siemens standard material already and reports in copies/ml • Abbott and Roche: calibrated against WHO standard: report in IU/ml • 5 copies/IU used throughout the study • PEI and Siemens material diluted at each site into the same base matrix. (PEI was also reconstituted at each site) • Asuragen, Plasma and Serum were diluted and tested BEFORE distribution to each site.

5. Testing Results: Asuragen Material

6. Testing Results: Siemens Material

7. Std Curves with Output (Ct and RLU): Synthetic Materials

8. Testing Results: PEI material

9. “Adjustment” with Synthetic: PEI

10. Testing Results: Plasma Material

11. Plasma material “adjusted” by either of the synthetics

12. Comparative Characteristics • Within Run cv’s 4-60% • Across Run cv’s: 8-46% • Across Platform cv’s: 19-117%

13. Summary Stats: All Methods

14. Summary Stats: All Methods

15. Variability Gage Charts Asuragen Siemens Plasma PEI

16. Summary • The synthetic materials behaved in a similar manner to the biologic materials in the three assays used. • Materials were recovered by the three assays used. • Parallel along the linear range • Similar variability • Relative quantification of each material was not necessarily consistent among the methods.

17. Biologic Standard Synthetic Is There a Role? Can we utilize the attributes of both types of materials to maximize the information? Establish Test Test Test Test

18. Issues to be addressed • Stability • Manufacturability • Commutability • Science is moving forward: • Whole length genome? • Cultured virus? • Subject to the same questions

19. Abbott: G. Schneider G. Gabriel Asuragen: J. Hedges E. Labourier C. Walker Peach PEI Micha Nübling Roche: R. Madej J. Saldanha Z. Wang G. Schilling C. Calado Siemens: S. Cruz A. Ocampo ILC (Industry Liaison Council) J. Reid (Chair) Abbott G. Schneider BioMerieux P. van de Weil R. Bally Bio-Rad M. Gonzales J. Jones A. Khellaf Chiron-Novartis N. Lelie Gen-Probe D. Kolk Roche P. Colucci R. Madej Siemens J. Turczyn Evaluation Study: Participants