1. An Overall View of Standardization May 26, 2004 Indira Hewlett, Ph.D. CBER/FDA

2. NAT and blood screening u NAT assays are used to screen blood and plasma donations for multiple viruses u NAT can reduce viral burden in plasma for further manufacturing u NAT can reduce virus transmission by blood through early detection of viremic window period donations

3. Standardization of NAT u Multiple, different NAT technologies: PCR, TMA, bDNA u Multiple, genetically diverse viral subtypes u Different pooling algorithms and pool sizes for minipool NAT : up to 512 for Source Plasma and 6-24 for Whole blood u Varied sensitivity, specificity and reproducibility of NAT

4. Standardization of NAT-con’t u Need analytical standards –Tools for quality control and quality assurance of NAT –Aid in licensing of investigational NAT and for post-market surveillance through lot release testing –Establish and monitor LOD and analytical sensitivity of NAT –Assure global standardization of NAT assays and reporting e.g. copies, IU/ml –Monitor laboratory proficiency

5. Quality Assurance of NAT (QA) u Assay validation – sensitivity, specificity, precision –clinical specificity and sensitivity u Quality control testing of components, final test kit, instrumentation u Acceptance criteria, specifications u Good manufacturing and good laboratory practices

6. QA Issues - I u Standardize assays using reference reagents and to compare values between laboratories u Ensure quality control of components and final test kit using panels and reference standards u Monitor operator proficiency using proficiency panels and training programs

7. QA Issues -II u Sample preparation including collection, storage and extraction u Manufacturing consistency of primers, probes and enzymes u Performance of controls, calibrators and quantitation standards u Specimen and kit stability u Instrument and software validation

8. QA: Controls and Quantitation Standards u Potency of specimen controls u Purity, identity and potency of synthetic oligo based internal controls or quantitation standards (e.g. transcripts) u Stability conditions of controls u Low and medium copy number kit controls, at least one close to LLOD u Quantitation standard close to LLOQ u Acceptance criteria, specifications

9. Current status of NAT u Donor screening NAT assays licensed for HIV-1 and HCV u Investigational NAT for West Nile virus (WNV) since July 2003 u HBV NAT in IND phase u In-process quality control NAT for Parvo virus B19, Hepatitis A virus (HAV)

10. Current status of CBER NAT standards u HIV-1, HBV and HCV NAT panels currently available for lot release of licensed NAT u HIV-1 subtype panel in final stages of formulation u HIV-2 panel in initial stages of testing in-house u WNV NAT panel in final stages of testing

11. CBER HIV-1 RNA Panel HIV-1 subtype B panel has 10 members –Cultured patient isolate, heat inactivated and diluted with defibrinated Ab-ve plasma –Gag, pol and env regions sequenced –Virus dilutions tested by 15 labs in collaborative study –8 positives: 10, 50, 100, 500, 2500, 5000, 2.5 x 10 4, and 2.5 x 10 5 copies/mL and 2 negatives –Current HIV-1 standard is 100 IU/ml for the pool test and 10,000 IU/ml for the original donation

12. 12 NIBSC/SoGAT Collaborative Study Calibration of HIV-1 Working reagents Candidate Log IU/mL Preparation QC105 (NRL, Australia)4.04 B5 (CBER, USA)2.21 B10 (CBER, USA)3.82 Pelispy (CLB, The Netherlands)4.43 PWS-1 (NIBSC, UK)3.56 PWS-3 (NIBSC, UK)2.72 IRC (Utrecht, The Netherlands)4.27 Ref: Davis et al (2003) J Virol Methods 107:37-44

13. CBER HIV-1 RNA Panel -con’t u HIV-1 subtype panel –7 subtypes of HIV-1 group M: A, B, C, D, E, F, G ; group N, and group O »Pilot-scale prototype panels were tested in collaborative study involving 5 NAT manufacturers at various dilutions »Data analyzed at FDA and consensus values assigned to viral stocks »Full-scale final panel currently under formulation

14. CBER HIV-1 RNA Panel -con’t Information of virus isolates IsolateIDPhenotypeSource A A2 C C2 D D2 E E2 F F2 G1 G2 N O1 O2 O3 O4 O5 B B2 DJ/258/91 UG/029/92 SE/364/90 192431 UG/035/92 UG/021/92 TH/022/92 TH/1465/95 BZ/162/90 BZ/126/89 G3/Nigeria HH8793 NA a NA NA NA NA NA 10207 9697 NSI SI NSI SI NSI SI NSI SI NSI SI NK b NK NSI NK NSI WRAMC NIBSC WRAMC CDC NIBSC WRAMC NIBSC Sinnousi Spain German US a NA= not available b NK= not known

15. Data Summary HIV-1 RNA copy numbers of the subtype panel HIV-1 subtype Mean a Min Max RangeSDCV(%) A A2 C C2 D D2 E E2 F F2 G1 G2 N O1 O2 O3 O4 O5 6.90 b 5.52 8.38 8.37 7.29 8.85 7.51 8.63 7.46 9.17 6.95 8.88 8.37 7.19 7.17 8.15 8.09 7.93 6.49 4.40 7.20 7.70 5.88 8.57 7.11 8.38 7.00 8.30 5.58 8.28 7.40 6.00 5.92 6.32 6.61 6.36 7.65 6.41 9.15 8.32 9.46 8.23 9.40 8.18 10.08 8.18 9.65 8.95 8.26 9.46 8.92 8.93 1.16 2.01 1.95 1.45 2.44 0.89 1.12 1.02 1.18 1.78 2.60 1.37 1.55 2.26 2.34 3.14 2.31 2.57 0.52 0.75 0.63 1.01 0.37 0.50 0.43 0.54 0.66 1.07 0.63 0.85 1.13 1.18 1.63 1.28 1.38 7.6 13.5 8.9 7.6 13.9 4.2 6.7 5.0 7.2 15.3 7.1 10.1 15.8 16.4 20.0 15.9 17.4 a The mean is the results from 5 independent labs b Values expressed as log10

16. CBER HCV Panel Stock u A window-period plasma unit –Negative for anti-HCV, anti-HIV & HBsAg –No detectable HIV RNA, HBV DNA, TTV DNA and B19 DNA u HCV genotype 1b u Entire HCV sequence determined u 5 x 10 7 copies/mL by 5 laboratories

17. 17 CBER HCV RNA Panel u A 10-member HCV panel derived from the HCV stock diluted with anti-HCV negative, defibrinated pooled plasma, genotype 1b –8 positives with target levels of 5, 10, 50, 100, 500, 10 3, 10 4, and 10 5 copies/mL, 2 negatives Current HCV standard: 100 IU/ml and 5,000 IU/ml for the original donation (Ref: Yu et al, Hepatology 1998; 28:566A)

18. 18 HCV NAT Standard Sample IU/mL Genotype Anti-HCV International Std 100,000 1a Pos NIBSC 96/586 710 3 Pos CLB/Pelispy 1,000 1a Neg PEI Ref 5 (Germany) 25,000 1 Neg ISS 0498 (Italy) 1,700 1 Pos CBER member #1 250 1b Neg (1000 copies/mL) [Ref: Saldanha et al, Vox Sang 2000; 78 (4) 217-24]

19. Parvovirus B19 NAT as an In-Process Control u Require validation as an analytical test and approve it under relevant product’s license u Proposed limit: <10 4 IU of B19 DNA per mL in all manufacturing pools –B19 transmissions associated with S/D Treated Pooled Plasma in a phase 4 study in healthy donors »<10 4 GE/mL in non-transmitting lots –Viral neutralization by anti-B19 in pools –Viral clearance by manufacturing procedures

20. CBER B19 DNA Standard u Derived from a window-period plasma unit, ~10 12 GE/mL u Diluted with pooled, cryo-poor plasma negative for HBsAg, anti-HIV, anti-HCV, anti-B19, HIV RNA, HCV RNA, HBV DNA, B19 DNA, and HAV RNA –~10 6 IU/mL (1 mL/vial) stored at  -70 °C

21. 21 WHO/NIBSC Collaborative Study International Standard for B19 DNA Candidate Log GE/mL Log IU/mL Preparation Targeted Mean AA (NIBSC, FD)* 6 5.92 6 BB (NIBSC, FD) 65.82 CC (CBER, Liquid) 65.89 6 DD (CLB, Liquid) 7 - 87.7

22. CBER HAV RNA Standard (I) u Derived from a window-period plasma unit, ~10 6 copies/mL u Diluted with a pooled, cryo-poor plasma negative for anti-HAV, HBsAg, anti-HIV, anti-HCV, HIV RNA, HCV RNA, HBV DNA, B19 DNA and HAV RNA –ca. 10 4 copies/mL; consensus level determined by the WHO/NIBSC collaborative study

23. 23 WHO/NIBSC Collaborative Study International Standard for HAV RNA Candidate Log GE/mL Log IU/mL Preparation AA (NIBSC, FD)* 5.29 5 BB (NIBSC, FD) 5.07 CC (CLB, Liquid) 4.99 DD (ISS, Liquid) 5.40 EE (CBER, Liquid) 4.08 Ref: Saldanha et al, WHO ECBS Report, Feb 2003.

24. HBV NAT panel u CBER HBV DNA panel derived from a window period specimen genotype A, serotype adw2 u Panel members are 0, 10 and 100 copies/ml u Panel tested by 3 NAT manufacturers

25. WNV testing u The 2002 outbreak indicated transmission of WNV by blood transfusion u All reported cases of transmission by blood transfusion occurred during the acute, viremic phase u NAT is the most appropriate strategy to interdict infectious donations u NAT on pooled donations implemented first since platforms for HIV/HCV NAT are already licensed u Need for standards to monitor sensitivity of WNV NAT assays

26. Need for Viral Titer Standardization u Lack of consensus for viral titer –Viral titer defined in plaque forming units (PFU) –Broad range of viral particles per PFU (1 – 1000 virions) –Need for correlation of RNA copies with PFU –Non-infectious particles (defective) may be detected by PCR but not by infectivity assays u Copy number determination necessary to define analytical sensitivity and infectivity

27. Correlation between Copies/mL and PFU/mL Sample Average (FDA & NYSDH) Copy # PFU FDA-Hu200210 5x10 7 /mL FDA-Hu2002 60 o C/2 hr 10 7 0 FDA-NY9910 1x10 7 /mL FDA-NY99 60 o C/2 hr 10 6.5 0

28. Collaborative study for copy number determination SampleLab 1Lab 2Lab 3Lab 4Average FDA-Hu2002 10 -1 10 9 FDA-Hu2002 10 -4 10 6 ND10 6 FDA-Hu2002 10 -7 10 3 ND10 3 FDA-Hu2002 10 -1 60 o C/2hr 10 7 NY99-FDA 10 -1 10 9 NY99-FDA 10 -4 10 6 ND10 6 NY99-FDA 10 -7 10 3 ND10 3 NY99-FDA 10 -1 60 o C/2hr 10 7 10 8 10 6 10 4 10 6.5

29. Data Summary u FDA-NY99 and FDA-Hu2002 stocks have a viral titer of 10 10 copies/mL u PFU titers were 2.5 logs lower than RNA copy numbers u Heat treatment results in loss of infectivity by PFU and 2 to 3 log reduction of copy number as determined by TaqMan u Final panel specifications will be established through ongoing collaborative studies of a prototype panel

30. Analytical sensitivity u FDA’s current standard for WNV NAT assays is 100 copies/ml for the individual donation u Standard may be revised as assay sensitivity improves and additional data on viremia and infectivity become available in future studies

31. Summary u FDA has established panels for HIV, HCV, HBV, B19 and HAV and standards for licensing tests u Panel for WNV under development u Standards are useful for: -quality control and quality assurance of NAT - licensing tests and post-market surveillance -Standards useful for global harmonization of NAT assays

32. Acknowledgements CBER/FDAWRAIR S. LeeN. Michael M. YuARC O. WoodS. Stramer M. RiosCDC S. KerbyR. Lanciotti R. TaffsNYDOH J. HuL. Kramer R. Biswas