1. SynTura: A New Ribonuclease Resistant Viral RNA Control Material Ralf Schönbrunner, Ph.D. SoGAT XXI Brussels, Belgium May 28, 2009

2. SynTura Project The SynTura project was created to provide RNA Internal Controls and Quantification Standards which behave like the target they intend to control Synthetic & Natural = SynTura

3. SynTura Project Objectives Develop technology platform which can provide control material close to real analyte HCV was used as model system Also functional as QS and IC

4. The Best Theoretical Alternative for Real HCV Similar virus to HCV Easy to culture Grows to high titers Non infectious to humans Detectable by HCV assays

5. CONFIDENTIAL Viruses Related to HCV Flaviviridae Flavivirus Yellow fever virus, West Nile virus Dengue virus St. Louis encephalitis virus Tick-borne encephalitis virus Japanese encephalitis virus Pestivirus Classical swine fever virus (CSFV) Border disease virus (BDV) Bovine Viral Diarrhea Virus 1 & 2 (BVDV) Hepacivirus HCV

6. CONFIDENTIAL BVDV, HCV and MS2 BVDVHCVMS2 Mammalian virus E. Coli Bacteriophage Flaviviridae Leviviridae Genome 12 kbGenome 9.5 kbGenome 3.57 kB Enveloped (lipid bilayer) Protein coat Detergent sensitive Detergent resistant

7. HCV & BVDV Genome Comparison BVDV genome is similar to HCV but can be easily cultured and manipulated Target Region of NAT HCV assays

8. Flaviviridae Translation HCV & BVDV

9. 5’UTR Secondary Structure Critical 5’UTR secondary structure –Transcripts often do not have correct secondary structure –Might give misleading results for certain HCV genotypes Is the Flaviviridae 5’-3’ Ring formation important?

10. 5’ & 3’ NTR Strategies SynTura

11. Position of HCV 5’NTR in BVDV 5’HCV NTR NFAR

12. Generation of a BVDV-Hybrid BVDV Plasmid (BVDV in blue) Insert desired Sequence (red) Make RNA Transcripts Transfect Cell Line Grow transfected Cell in culture Viral Particles Infected Cells produce Virus DNARNA

13. Development Timeline

14. SynTura HCV Growth Curve

15. Inactivation of BVDV Inactivated with ß-Propiolactone Protein-modifying agent –reacts with amides of Lys or Arg Commonly used for BVDV vaccines Alternative: Heat – 90% loss

16. Initial Titer Determination

17. Effect of Inactivation on TMA Results with ProCleix Ultrio TMA Assay No impact of ß-PL inactivation on TMA

18. Sequence Stability 3 passages 5 clones partially sequenced –No mutations found in 5 clones 7 passages 5 clones partially sequenced –1 clone no mutation –2 clones had 1 point mutation in BVDV sequence –2 clones had 1 point mutation in HCV insert Rate of mutation in insert similar to virus

19. SynTura Thermal Stability

20. 37ºC Accelerated Stability

21. SynTura HCV as Calibrator

22. OptiQuant-S HCV RNA Quantification Panel Based on SynTura Technology 100, 500, 5e3, 5e4, 5e5, 5e6 & 2.5e7 IU/mL Plasma Matrix

23. Multicenter Study

24. Roche CAP/CTM HCV IVD

25. Comparison of two Abbott realTime HCV ASR Assays

26. Key Results SynTura HCV titer 2.0E+08 IU/mL Concentrated to 1.5eE+10 IU/mL Stable over 7 passages –Insert remains intact –No mutation after 3 passages –Only 1 point mutation after 7 passages

27. Summary SynTura has properties very similar to HCV and probably other enveloped mammalian viruses SynTura technology allows integration of defined RNA sequences SynTura technology can be used for RNA assays as: –QS and IC –Positive Control –Calibrator

28. Thank You Martin Luther Universität Halle/Saale –Sven Behrens –Martina Behrens Acrometrix –Mona Shahbazian –Jerry Boonyaratanakornkit –Reina Karunaratne Steve Young, Jessie Kilgore –Tricore Hanna Rennert, John Sipley, - NYPH, Cornell Medical Center Melody Hung-Fan, Kara Lee, -Contra Costa Public Health Lab Linda Sabatini, Lech Mazur, -ACL Central Lab Maura Pieretti, Carolyn Dowell, -BayCare Lab Ted Schutzbank- Covance