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

3. 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 log 10 reduction value

4. 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

5. 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

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

8. Published TSE Clearance Studies for Plasma Fractionation 1.Brown, P et al, Transfusion 1998 38:810-6 2.Brown, P et al, Transfusion 1999 39: 1169-78 3.Lee, DC et al, J. Virol. Meth. 2000 84: 77-89 4.Foster, PR et al, Transfusion Science 2000 22:53-56 5.Foster, PR et al, Vox Sanguinis 2000 78:86-95 6.Lee, DC et al, Transfusion 2001 41: 449-55 7.Cai, K et al, Biochem Biophys. Acta 2002 1597: 28-35 8.Stenland, JS et al, Transfusion 2002 42:1497-1500 9.Vey, M et al, Biologicals 2002 30:187-96 10.Reichl, HE et al, Vox Sanguinis 2002 83:137-45

9. 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 –Prp Sc 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

10. 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 Prp Sc –Bridging in vivo to in vitro results (Transfusion 2001 41: 449-55) Mass balance – retention TSE agents by columns; loss of mass balance

11. 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

12. 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

13. Example (1) Prp Sc 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 Prp Sc in supernatant

14. Parameters Influencing Prp sc Partitioning Precipitation best at: a.Mildly acidic pH b.With EtOH c.At higher pH, with salt and EtOH Cai, K. et. al. Biochem Biophys Acta 1597(1): 28-35, 2002

15. Example (2) Cryoprecipitation: variable clearance among studies with different conditions 1.FVIII partitions with cryoprecipitate 2. Clearance of Prp Sc 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)

16. Example (3) Clearance PrP sc (microsomal spike) by Depth Filtration – Influence of Starting Materials and Filter Starting MaterialDepth FilterReduction Factor (log 10 ) Fr V (albumin)Seitz KS80> 4.9 Fr V (albumin)CUNO Delipid 12.3 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 spikeSupra P80> 2.4 Vey et al, Biologicals 30:187-96, 2002

17. 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

18. 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

19. 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

20. 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