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An Overall View of Standardization

An Overall View of Standardization. May 26, 2004 Indira Hewlett, Ph.D. CBER/FDA. NAT and blood screening. NAT assays are used to screen blood and plasma donations for multiple viruses NAT can reduce viral burden in plasma for further manufacturing

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An Overall View of Standardization

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

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

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

  4. Standardization of NAT-con’t • 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) • Assay validation • sensitivity, specificity, precision • clinical specificity and sensitivity • Quality control testing of components, final test kit, instrumentation • Acceptance criteria, specifications • Good manufacturing and good laboratory practices

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

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

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

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

  10. Current status of CBER NAT standards • HIV-1, HBV and HCV NAT panels currently available for lot release of licensed NAT • HIV-1 subtype panel in final stages of formulation • HIV-2 panel in initial stages of testing in-house • 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 104, and 2.5 x 105 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. 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 • 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. Isolate ID Phenotype Source 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 NAa NANANANANA 10207 9697 NSI SI NSI SI NSI SI NSI SI NSI SI NKb NK NK NSI NSI NK NK NK NK NSI WRAMC NIBSC WRAMC CDC NIBSC NIBSC NIBSC WRAMC WRAMC WRAMC NIBSC NIBSC Sinnousi Spain Spain German German German US US CBER HIV-1 RNA Panel -con’t Information of virus isolates a NA= not available b NK= not known

  15. HIV-1 subtype Meana Min Max Range SD CV(%) A A2 C C2 D D2 E E2 F F2 G1 G2 N O1 O2 O3 O4 O5 6.90b 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 9.15 8.32 9.46 8.23 9.40 8.18 10.08 8.18 9.65 8.95 8.26 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 0.75 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 7.2 15.3 7.1 10.1 15.8 16.4 20.0 15.9 17.4 Data Summary HIV-1 RNA copy numbers of the subtype panel a The mean is the results from 5 independent labs b Values expressed as log10

  16. CBER HCV Panel Stock • A window-period plasma unit • Negative for anti-HCV, anti-HIV & HBsAg • No detectable HIV RNA, HBV DNA, TTV DNA and B19 DNA • HCV genotype 1b • Entire HCV sequence determined • 5 x 107 copies/mL by 5 laboratories

  17. CBER HCV RNA Panel • 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, 103, 104, and 105copies/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. 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 • Require validation as an analytical test and approve it under relevant product’s license • Proposed limit: <104 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 • <104 GE/mL in non-transmitting lots • Viral neutralization by anti-B19 in pools • Viral clearance by manufacturing procedures

  20. CBER B19 DNA Standard • Derived from a window-period plasma unit, ~1012 GE/mL • 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 • ~106 IU/mL (1 mL/vial) stored at  -70 °C

  21. WHO/NIBSC Collaborative Study International Standard for B19 DNA Candidate Log GE/mL Log IU/mL Preparation Targeted Mean AA (NIBSC, FD)* 65.92 6 BB (NIBSC, FD) 65.82 CC (CBER, Liquid) 6 5.89 6 DD (CLB, Liquid) 7 - 87.7

  22. CBER HAV RNA Standard (I) • Derived from a window-period plasma unit, ~106 copies/mL • 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. 104 copies/mL; consensus level determined by the WHO/NIBSC collaborative study

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

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

  26. Need for Viral Titer Standardization • 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 • Copy number determination necessary to define analytical sensitivity and infectivity

  27. Correlation between Copies/mL and PFU/mL

  28. Collaborative study for copy number determination

  29. Data Summary • FDA-NY99 and FDA-Hu2002 stocks have a viral titer of 1010 copies/mL • PFU titers were 2.5 logs lower than RNA copy numbers • Heat treatment results in loss of infectivity by PFU and 2 to 3 log reduction of copy number as determined by TaqMan • Final panel specifications will be established through ongoing collaborative studies of a prototype panel

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

  31. Summary • FDA has established panels for HIV, HCV, HBV, B19 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

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

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