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TSE Agent Clearance Issues

TSE Agent Clearance Issues

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TSE Agent Clearance Issues

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  1. TSE Agent Clearance Issues TSE Advisory Committee February 20, 2003 Dorothy Scott, M.D. DH/OBRR/CBER/FDA

  2. Paradigm: Validation of Virus Removal/inactivation Includes: • Scaling down process steps • Spiking appropriate steps with high titer of infectious agent (actual or model) • Determination reduction factors for each step • Summing reduction factors [from orthogonal processes] to give a total log10 reduction value

  3. Scale-Down of Purification Steps • Usually 1/10 to 1/100 scale; no set guidelines • Must keep buffers, pH, protein concentration, and product the same as full scale manufacturing • Must keep operation parameters as close to full scale as possible (e.g., bed height, flow rate) • Must show product is identical to production scale

  4. Criteria for Acceptable Pathogen Detection Assays • Accuracy • Assay repeatability and reproducibility • Linearity • The limit of detection (LOD) • The limit of quantitation (LOQ) • Assay robustness and reproducibility

  5. TSE Clearance Evaluation: Example TSE Spike Plasma Cryoprecipitation Cryoprecipitate (FVIII) Cryopoor Plasma Supernatant Albumin, IGIV, 1PI, ATIII, etc.

  6. Published TSE Clearance Studies for Plasma Fractionation • Brown, P et al, Transfusion 1998 38:810-6 • Brown, P et al, Transfusion 1999 39: 1169-78 • Lee, DC et al, J. Virol. Meth. 2000 84: 77-89 • Foster, PR et al, Transfusion Science 2000 22:53-56 • Foster, PR et al, Vox Sanguinis 2000 78:86-95 • Lee, DC et al, Transfusion 2001 41: 449-55 • Cai, K et al, Biochem Biophys. Acta 2002 1597: 28-35 • Stenland, JS et al, Transfusion 2002 42:1497-1500 • Vey, M et al, Biologicals 2002 30:187-96 • Reichl, HE et al, Vox Sanguinis 2002 83:137-45

  7. Challenges in Studies of Clearance of TSE Agents • What source of infectivity to use • Brains preparations from experimentally infected animals most easily available • Hamsters (scrapie) • Mice (GSS, BSE) • BSL-3 facility needed to study vCJD, BSE • PrpSc partitioning similar when source is human (CJD, vCJD), or animal TSE’s (Stenland et al, Transfusion 42: 1497-1500, 2002; single study) • What “form” of infectious agent most relevant to blood? • Brain homogenate • Subcellular membrane fractions • Membrane-free infectious material

  8. Challenges in Studies of Clearance of TSE Agents • The lower limits of assay sensitivity (2-3 logs), and upper limits of titers available for spiking • Range of infectivity removal detectable 4-5 logs • “Throughput” experiments to assess additiveness of clearance steps therefore have limitations • What assays are best to measure outcomes • In vivo infectivity (time, expense) • In vitro surrogates – measurements of PrpSc • Bridging in vivo to in vitro results (Transfusion 2001 41: 449-55) • Mass balance – retention TSE agents by columns; loss of mass balance

  9. Challenges in Evaluating Clearance of TSE Agents • How much reduction is “enough? (risk assessment) • How many disparate clearance steps should there be? • What steps can be summed, which cannot? • Summed reduction factors for similar steps, e.g. EtOH precipitation

  10. TSE Clearance depends upon specific characteristics of starting material and process conditions: Examples • Partitioning of infectivity depends upon pH, ionic strength, and alcohol concentration • Cryoprecipitation methods may influence degree of clearance • Depth filtration effectiveness depends upon filter used and/or properties of starting material

  11. Example (1) PrpSc Partitioning is condition-dependent Cai, A. et. al. Biochem. Biophys. Acta 597: 28-35, 2002 • Scrapie brain homogenate spiked into buffers with varied: • EtOH concentrations • Salt concentrations • pH • Incubation • Centrifugation • Measurement PrpSc in supernatant

  12. Parameters Influencing Prpsc Partitioning • Precipitation best at: • Mildly acidic pH • With EtOH • At higher pH, with salt and EtOH Cai, K. et. al. Biochem Biophys Acta 1597(1): 28-35, 2002

  13. Example (2) Cryoprecipitation: variable clearance among studies with different conditions • FVIII partitions with cryoprecipitate • 2. Clearance of PrpSc in cryoprecipitation • - 1 log clearance in effluent(Lee et al., • Transfusion 41: 449-55, 2001) • 1 log clearance in effluent(Brown et al., • Transfusion 38: 810-16, 1998) • <1 – 1.7 logs clearance in precipitate • (Foster et al., Vox Sang 78:86- 95, 2000)

  14. Example (3) Clearance PrPsc (microsomal spike) by Depth Filtration – Influence of Starting Materials and Filter Starting MaterialDepth FilterReduction Factor (log10) Fr V (albumin) Seitz KS80 > 4.9 Fr V (albumin) CUNO Delipid 1 2.3 S I + III (IGIV) Millipore AP20 < 1 Fr II (IGIV) Seitz K200 > 2.8 Foster et. al., Vox Sang 78: 86-95, 2000 Fr I supernatant (IGIV, albumin) Supra P80 < 1 Fr V supernatant (albumin) Supra P80 > 1.1 Fr V supernatant (albumin) – Prp-sc spike Supra P80 > 2.4 Vey et al, Biologicals 30:187-96, 2002

  15. TSE Clearance and the Manufacturing Process • Manufacturing processes are highly individual • Cohn-Oncley process variations • Other fractionation methods • Variations in downstream processing/purification of products (e.g. column chromatography) • Rigorous demonstrations of TSE clearance therefore need to be based upon the specific manufacturing process • Published studies may prove useful to identify steps with potential for TSE clearance

  16. Evaluation of TSE clearance studies from industry, to support labeling claims of lowering possible TSE risk • Characterization of spiking agent • Accurately scaled-down processes • Robust and reproducible experiments • Well-characterized assay for TSE infectivity • Bridging binding assays to bioassays • Estimated logs clearance of TSE by processing steps (reduction factor and clearance factor) • Demonstration of mass balance • Demonstration, where relevant, that non- orthogonal (similar) clearance steps are/are not additive

  17. Evaluation of Submissions to Support Labeling Claims • Clearance “beltline” to support labeling • At least 2 orthogonal steps with > 4 logs clearance (total 8 logs) • At least 2 steps demonstrated to be additive with > 4 logs clearance/step (total 8 logs) • ? At least 2 steps (orthogonal or demonstrated to be additive) with > 3 logs/step (total 6 logs) • Is a single clearance step of > 4 logs sufficient if robust and reproducible? • Are clearance steps of > 2 logs reliable if they are robust and reproducible? • Cumulative clearance/risk analysis

  18. Labeling for TSE Risk • Current proposal: “Because this product is made from human plasma, it carries a risk of transmitting infectious agents, e.g. viruses, and, theoretically the vCJD agent. It has been demonstrated that [the manufacturer’s] manufacturing process provides substantial clearance of agents similar to those causing CJD and vCJD. Thus the theoretical risk of transmission of CJD or vCJD is extremely remote.” • Future improvements in risk assessment, understanding of plasma infectivity, and study methods could provide a basis for additional labeling content