An Overall View of Standardization May 26 2004

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

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

Standardization of NAT u Multiple, different NAT technologies: PCR, TMA, b. DNA 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

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

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

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

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

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

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 B 19, Hepatitis A virus (HAV)

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 inhouse u WNV NAT panel in final stages of testing

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 104, and 2. 5 x 105 copies/m. L and 2 negatives – Current HIV-1 standard is 100 IU/ml for the pool test and 10, 000 IU/ml for the original donation

NIBSC/So. GAT Collaborative Study Calibration of HIV-1 Working reagents Candidate Preparation QC 105 (NRL, Australia) B 5 (CBER, USA) B 10 (CBER, USA) Pelispy (CLB, The Netherlands) PWS-1 (NIBSC, UK) PWS-3 (NIBSC, UK) IRC (Utrecht, The Netherlands) Log IU/m. L 4. 04 2. 21 3. 82 4. 43 3. 56 2. 72 4. 27 Ref: Davis et al (2003) J Virol Methods 107: 37 -44 12

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

CBER HIV-1 RNA Panel -con’t Information of virus isolates Isolate ID Phenotype Source A A 2 C C 2 D D 2 E E 2 F F 2 G 1 G 2 N O 1 O 2 O 3 O 4 O 5 B B 2 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 G 3/Nigeria HH 8793 NAa NA NA NA 10207 9697 NSI SI NSI SI NKb NK NK NSI WRAMC NIBSC WRAMC CDC NIBSC WRAMC NIBSC Sinnousi Spain German US US a NA= not available b NK= not known

Data Summary HIV-1 RNA copy numbers of the subtype panel HIV-1 subtype Meana Min Max Range SD CV(%) A A 2 C C 2 D D 2 E E 2 F F 2 G 1 G 2 N O 1 O 2 O 3 O 4 O 5 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 log 10

CBER HCV Panel Stock u. A window-period plasma unit – Negative for anti-HCV, anti-HIV & HBs. Ag – No detectable HIV RNA, HBV DNA, TTV DNA and B 19 DNA u. HCV genotype 1 b u. Entire HCV sequence determined u 5 x 107 copies/m. L by 5 laboratories

CBER HCV RNA Panel u. A 10 -member HCV panel derived from the HCV stock diluted with anti-HCV negative, defibrinated pooled plasma, genotype 1 b – 8 positives with target levels of 5, 10, 50, 100, 500, 103, 104, and 105 copies/m. L, 2 negatives Current HCV standard: 100 IU/ml and 5, 000 IU/ml for the original donation (Ref: Yu et al, Hepatology 1998; 28: 566 A) 17

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

Parvovirus B 19 NAT as an In-Process Control u Require validation as an analytical test and approve it under relevant product’s license u Proposed limit: <104 IU of B 19 DNA per m. L in all manufacturing pools – B 19 transmissions associated with S/D Treated Pooled Plasma in a phase 4 study in healthy donors » <104 GE/m. L in non-transmitting lots – Viral neutralization by anti-B 19 in pools – Viral clearance by manufacturing procedures

CBER B 19 DNA Standard u Derived from a window-period plasma 12 unit, ~10 GE/m. L u Diluted with pooled, cryo-poor plasma negative for HBs. Ag, anti-HIV, anti-HCV, anti-B 19, HIV RNA, HCV RNA, HBV DNA, B 19 DNA, and HAV RNA – ~106 IU/m. L (1 m. L/vial) stored at -70 °C

WHO/NIBSC Collaborative Study International Standard for B 19 DNA Candidate Preparation Log GE/m. L Targeted Mean AA (NIBSC, FD)* 6 5. 92 BB (NIBSC, FD) 6 5. 82 CC (CBER, Liquid) 6 5. 89 7 -8 Log IU/m. L 7. 7 DD (CLB, Liquid) 6 6 21

CBER HAV RNA Standard (I) u Derived from a window-period plasma 6 unit, ~10 copies/m. L u Diluted with a pooled, cryo-poor plasma negative for anti-HAV, HBs. Ag, anti-HIV, anti-HCV, HIV RNA, HCV RNA, HBV DNA, B 19 DNA and HAV RNA 4 – ca. 10 copies/m. L; consensus level determined by the WHO/NIBSC collaborative study

WHO/NIBSC Collaborative Study International Standard for HAV RNA Candidate Preparation Log GE/m. L AA (NIBSC, FD)* 5. 29 BB (NIBSC, FD) 5. 07 CC (CLB, Liquid) 4. 99 DD (ISS, Liquid) 5. 40 EE (CBER, Liquid) Log IU/m. L 5 4. 08 Ref: Saldanha et al, WHO ECBS Report, Feb 2003. 23

HBV NAT panel u CBER HBV DNA panel derived from a window period specimen genotype A, serotype adw 2 u Panel members are 0, 10 and 100 copies/ml u Panel tested by 3 NAT manufacturers

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

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

Correlation between Copies/m. L and PFU/m. L Sample Average (FDA & NYSDH) Copy # PFU FDA-Hu 2002 1010 5 x 107/m. L FDA-Hu 2002 60 o. C/2 hr 107 0 FDA-NY 99 1010 1 x 107/m. L FDA-NY 99 60 o. C/2 hr 106. 5 0

Collaborative study for copy number determination Sample Lab 1 Lab 2 Lab 3 Lab 4 Average FDA-Hu 2002 10 -1 109 109 109 FDA-Hu 2002 10 -4 106 ND 106 106 FDA-Hu 2002 10 -7 103 ND 103 103 FDA-Hu 2002 10 -1 60 o. C/2 hr 107 107 107 NY 99 -FDA 10 -1 109 109 109 NY 99 -FDA 10 -4 106 ND 106 106 NY 99 -FDA 10 -7 103 ND 103 103 NY 99 -FDA 10 -1 60 o. C/2 hr 107 108 106 104 106. 5

Data Summary u FDA-NY 99 and FDA-Hu 2002 stocks have a viral titer of 1010 copies/m. L 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 Taq. Man u Final panel specifications will be established through ongoing collaborative studies of a prototype panel

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

Summary u u u FDA has established panels for HIV, HCV, HBV, B 19 and HAV and standards for licensing tests Panel for WNV under development 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

Acknowledgements CBER/FDA S. Lee M. Yu O. Wood M. Rios S. Kerby R. Taffs J. Hu R. Biswas WRAIR N. Michael ARC S. Stramer CDC R. Lanciotti NYDOH L. Kramer