1. Roche Molecular Diagnostics
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 PEI material
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: %

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

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

19. Evaluation Study: Participants
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