Yi-Chen Yang, Drug Biology Division Bureau of Food and Drug Analysis

1. Collaborative study for establishment of the first national standard for Parvovirus B19 DNA NAT assays Yi-Chen Yang, Drug Biology Division Bureau of Food and Drug Analysis Department of Health, Taiwan SoGAT XX June 2007

2. Background • NAT requirements in Taiwan ( 2002 ) • NAT test for Parvovirus B19 is suggested on plasma pool or mini-pool the cut-off limit of B19 DNA should be < 105 IU/mL • WHO International standard for B19 virus DNA (99/800) • European Pharmacopoeia Biological Reference Preparation (BRP) for B19 Virus DNA Testing of Plasma Pools by NAT • To facilitate the implementation of the policy in Taiwan • national standard and working reagent for human parvovirus B19 DNA NAT assays

3. National Standards for NAT • Reasons for Preparation • Difference genotypes among countries and regions (HBV, HCV) • Limited vials of international standards • One of BFDA’s task: supply of reference standards • Intended use • National standard : as a laboratory standard or reference material • Working reagent : as a run control for routine NAT assays • Blood/cell/tissure donor screening by NAT assays • Plasma pool screening by NAT assays • Testing for Class III IVD marketing approval • Quality control of IVD • Post marketing surveillance of IVD • Research However it is for the user to establish suitability of purpose

4. National Standards for NAT in Taiwan

5. Objective • To establish the B19 DNA national standard • 106 IU/mL, 0.5 mL/vial • around 1,000 vials • To prepare the B19 DNA working reagent • 104 IU/mL, 1 mL/vial • around 1,000 vials

6. Pooled cryosupernatant Dilute positive plasma to suitable concentration Calibrate the titers of candidates against the international standard by a collaborative study Stability study Flow Chart of NAT Standard and Working Reagent Preparation High titer positive plasma

7. Preparation of B19 DNA National Standard and Working Reagent • High titer positive plasma • Screening for other viruses • Anti-HIV 1/2, HBsAg, Anti-HCV by EIA: (-) • HAV RNA, HBV DNA, HCV RNA & HIV-1 RNA by NAT: (-) • Quantitative analysis of B19 DNA • DNA sequencing/ Nucleotide-nucleotide BLAST (NCBI) • Diluent : Pooled human cryosupernatant • Anti-HIV 1/2, HBsAg, Anti-HCV by EIA: All (-) • HAV RNA, HBV DNA, HCV RNA, HIV-1 RNA & B19 DNA by NAT : All (-) • Anti-B19 IgM, Anti-B19 IgG by EIA: All (-) • Check the titers of preparations in 3 different assay methods • LightCycler Parvovirus B19 Quantification Kit • RealArt Parvo B19 LC kit • In-house assay

8. International Collaborative Studyfor B19 DNA Standards • Participating Labs including: 10 Labs from 7 countries • Official Medicine Control Laboratories (OMCL) • NAT testing laboratory • Manufacturers of plasma products • Manufacturers of in vitro diagnostics • Each Lab received 3 vials of each sample, and 1 vial of WHO B19 DNA IS (code: 99/800) • Perform 3 independent assays for each sample • Calibrate the candidates against the IS

9. Data for the Collaborative Study National standard * in development

10. Mean 6.269 +2SD (6.627) - 2SD (5.911) • All data were within the mean ± 2 SD for national standard, showed that all laboratories are in good agreement with the results.

11. Results of the Collaborative Study National standard

12. Data for the Collaborative Study Working reagent * in development

13. Mean 4.309 +2SD (4.682) - 2SD (3.936) • All data were within the mean ± 2 SD for working reagent, showed that all laboratories are in good agreement with the results.

14. Results of the Collaborative Study Working reagent

15. B19 DNA National Standard and Working Reagent

16. Stability Study for B19 DNA Standards • Check the titers in 2 different assay methods • RealArt Parvo B19 LC kit • In-house assay • Performed 3 independent assays for each method

17. 6.269 p＞0.05, n.s. Results of the Stability Studies ( 25℃ ) 6.269 p＞0.05, n.s. 4.309 p＞0.05, n.s.

18. Results of the Stability Studies ( 4℃ ) 6.269 p＞0.05, n.s. 4.309 p＞0.05, n.s.

19. Results of the Stability Studies ( -20℃ ) 6.269 p＞0.05, n.s. 4.309 p＞0.05, n.s.

20. Results of the Stability Studies ( -80℃ ) 6.269 p＞0.05, n.s. 4.309 p＞0.05, n.s.

21. Summary • In this international collaborative study, a high level of agreement between results was obtained from different laboratories. • The first national standard and working reagent for B19 DNA NAT assays with an assigned potency of 1.9 × 106 IU/mL and 2.0 × 104 IU/mL, respectively, were established. • The national standard and working reagent were stable at 25℃ for 4 weeks, 4℃ for 8 weeks, -20℃ and -80℃ for at least 12 months.

22. Acknowledgements • thanks to all participants of the collaborative study group • Dr. M. Y. Yu CBER, USA • Dr. C. M. Nübling, Dr. M. Chudy PEI, Germany • Dr. S. Baylis NIBSC, UK • Dr. Y. Okada NIID, Japan • Dr. M. Gessner, Dr. A. Klotz Baxter AG, Austria • Dr. D. Johnstone CSL Bioplasma, Australia • Dr. R. Smith NGI, USA • Dr. T. Grewing QIAGEN, Germany • Dr. D. Sizmann, Dr. A. Schubert Roche, Germany • Dr. J. Saldanha Roche, USA • Dr. A. Heath NIBSC, UK Thank you for your attention

23. NAT requirement in Taiwan (Dec. 19 2002 ) to improve the safety of blood products • NAT tests on plasma pool are required: negative for HIV, HBV, HCV • Virus inactivation/removal steps for enveloped and non-enveloped viruses: two steps or one step ( shown to be reliably effective ) • For S/D treated blood products One additional step should be performed e.g. monoclonal purification or nanofiltration ( at least 4 log reduction of HAV ) or The plasma pool should be HAV NAT(-) before the manufacturing process • NAT test for Parvovirus B19 is suggested on plasma pool or mini-pool the cut-off limit of B19 DNA should be < 105 IU/ml