1. TSE Clearance Studies for pdFVIII: Study Methods and Clearance Levels TSE Advisory Committee September 18, 2006 Dorothy Scott, M.D. Office of Blood Research and Review/CBER

2. TSE Safety Concerns Theoretically, plasma derivatives might transmit vCJD or other TSE agents Any such risk is thought to be very low based on the fact that no cases of vCJD have been reported worldwide in any recipients of plasma derivatives, including in the UK, where vCJD risk is greatest FDA seeks to assure the safety of plasma derivatives, especially pdFVIII, against risk for transmission of TSE

3. Importance of TSE Clearance Clearance of TSE agents in manufacturing of pdFVIII and other plasma derivatives has a major impact on estimated risk: –FDA risk assessment for pdFXI, 2005 ( http://www.fda.gov/ohrms/dockets/ac/05/briefing/200 5-4088b1.htm ) However, standardized methods and assessment criteria for TSE clearance have not been defined.

4. Issue for the TSEAC FDA seeks the advice of the Committee whether standardized methods and assessment criteria are feasible and appropriate for determining TSE clearance in the manufacturing processes for plasma-derived FVIII (pdFVIII) products.

5. Items for Discussion Feasibility and scientific value of adopting standardized methods to assess TSE clearance in manufacturing of pdFVIII products Whether a minimum TSE agent reduction factor might reasonably serve as an appropriate standard for demonstrating vCJD safety of pdFVIII products; and if so Actions FDA should consider if only lower levels of clearance can be demonstrated for a given pdFVIII product

6. FDA’s Evaluation of Sponsors’ Voluntary Studies of TSE Clearance FDA discussed TSE clearance with the TSEAC in 2/03. FDA has engaged in case by case review of the following types of information on TSE clearance: Rationale for animal model selected Rationale for selection of spiking preparation Characterization of the spiking agent Demonstration of accurately scaled-down processes Robust and reproducible experiments Well-characterized assay for TSE infectivity

7. FDA’s Evaluation of Sponsors’ Voluntary Studies of TSE Clearance (continued) Estimated logs TSE clearance by processing steps Demonstration of “mass balance” (accounting for all input infectivity) Demonstration that mechanistically similar clearance steps are or are not additive Account for “conditioning” of infectivity where a prior step may affect physical state of TSE agent and in turn affect clearance step downstream

8. TSE Clearance Labeling Approvals for Plasma-derived Products ApprovedStepsRF CarimuneNF precipitations 7.2 nanofiltration4.4 Panglobulin NFprecipitations 7.2 nanofiltration4.4 Gamunexcloth filtration + depth filtration 6.6 Thrombate IIIprecipitation6.0

9. pdFVIII manufacturing Cryoprecipitation is the first step in manufacturing pdFVIII Other steps can include: precipitations, column purifications, some of which may result in further TSE clearance

10. Starting material for pdFVIII (cryoprecipitate) is precipitated early in plasma fractionation schemes

11. Experimental Clearance of PrP TSE and Infectivity by Cryoprecipitation Reference Log 10 Reduction Assay Brown et al., Transfusion 38: 810-16, 1998 < 1.0Bioassay Foster et al., Vox Sang 78:86- 95, 2000 1.0PrP TSE Lee et al., Transfusion 41: 449-55, 2001 < 1.0Bioassay Vey et al., Biologicals 30: 187-96, 2002 < 1.0PrP TSE

12. TSE Clearance Issues Exogenous (“spiking”) experiments –Nature of spiking material and its relevance to blood-borne infection Endogenous experiments –Relevance and feasibility TSE strain and animal model Output measure of infectivity reduction –Bioassay –In vitro assays

13. TSE Spike Plasma CryoprecipitationCryoprecipitate (FVIII) Cryopoor Plasma Supernatant FIX, IGIV, A1PI, Albumin, etc. TSE Clearance Evaluation: Exogenous (“Spiking Experiment”) Model

14. Exogenous TSE clearance studies – form of spiking material Form infectious agent –Brain homogenate, centrifuged –Ultracentrifuged (microsomal) –Caveolae-like domains –Detergent-solubilized homogenate –Membrane-free infectious material (e.g. fibrils) Very insoluble Probably NOT representative of blood infectivity Membrane-associated

15. Spike form impacts clearance by precipitation Vey et al. Biologicals 30: 187-96, 2002 Manufacturing Process Log 10 PrP TSE Reduction (with respect to supernatant) Microsomal Spike Purified PrP- Scrapie Spike Cryoppt.0.22.4 8% EtOH ppt.0.93.1 25% EtOH ppt.3.14.0 Glycine ppt.1.73.3

16. “Conditioning”: Detergent-treatment diminishes clearance of scrapie agent by nanofiltration DetergentLog 10 RF* Feed solution- (8.13)---- + (7.32)---- Filtrate 35 nM-4.93 +1.61 15 nM-> 5.87 +> 4.21 10 nM+> 3.80 Tateishi et al, Biologicals 29: 17-35, 2001 * Determined by bioassay [Scrapie ME7]

17. “Conditioning” PrP TSE clearance by membrane filtration and depth filtration increases in presence of alcohol (evidence of aggregation) Van Holten et al, Vox Sang. 85:20-24, 2003

18. Plasma from TSE-infected animal CryoprecipitationCryoprecipitate (FVIII) Cryopoor Plasma Supernatant FIX, IGIV, A1PI, Albumin, etc. TSE Clearance Evaluation: Endogenous Infection model

19. Endogenous TSE studies: Relevance to Blood Infectivity Comparison of results from endogenous and exogenous infectivity studies suggest similar reductions for some precipitations –Limited number of endogenous studies Endogenous infectivity characteristics in plasma –Small size –Difficult to sediment (in its native form) –Poorly aggregated –May be lipid/plasma-protein associated

20. Endogenous TSE Clearance Studies Relevance to human blood highly likely Limited clearance can be demonstrated because starting infectivity is low (est. 2-30 ID/ml) – Large numbers of donor and assay animals may compensate for low titers Recipients – volume injectible i.c. for titration: 0.02 ml mice; 0.05 ml hamsters For 100 ml plasma: 5000 mice or 2000 hamsters Large animal models (sheep: Scrapie, BSE) Experimental logistics - herd management, limited locations, incubation time, availability Scale-down logistics – dedicated pilot laboratories

21. TSE Model Selection TSE’s differ in resistance to inactivation To date, clearance in plasma products demonstrated by partitioning studies only Few direct strain comparisons – EtOH precipitations – clearance similar BSE, CJD, vCJD (Stenland et al) – EtOH precipitations –Nanofiltration could be influenced if strain-related differences exist in aggregation properties (theoretical) (Vey et al) Strain differences for partitioning clearance experiments not demonstrated

22. Assays for TSE Agents Bioassay – limiting dilution titration into susceptible rodents PrP TSE proposed as surrogate marker for infectivity PrP TSE measured by Western Blot or Conformation-dependent immunoassay (based on binding of antibody to Prp TSE )

23. Rationale for Bioassay Use Binding assays detect PrP TSE –Examples of infectivity without detectable PrP TSE –Examples of PrP TSE without infectivity –Conditioning might differentially affect binding vs. infectivity Binding assays (currently) not as sensitive as bioassays (limit of detection typically 2-3 logs infectivity)

24. Challenges in TSE Clearance interpretation – how much clearance is “significant?” Viral validation (clearance) studies – typically demonstrate at least 2-3 logs greater clearance than maximum potential absolute amount of virus present Added clearance provides a margin of safety

25. TSE clearance level and safety TSE infectivity – if present, how much might be in a single plasma unit? 800 ml (plasma unit) x 2-30 ID/ml TSE infectivity = 1600 – 24,000 ID’s (total) = 3.2 - 4.4 log 10 total infectious units estimated possible infectivity in one unit of infected plasma Actual infectivity might be less due to blood- brain barrier (IC/IV ID50 1 to 1 to 1 to 10 estimated [TSEAC 10/2005]), and host susceptibility

26. Question 1A 1.A. Please comment on the feasibility and scientific value of adopting standardized exogenous (spiking) study methods to assess TSE clearance in manufacturing of pdFVIII including the following I)Optimal spiking material and its preparation from the standpoint of relevance to blood infectivity II)Selection of TSE strain and animal model III)TSE immunoassays for PrP TSE and bioassays for infectivity IV)Identification of manufacturing processes that might alter TSE agent properties

27. Question 1B 1. B. Please comment on the feasibility and scientific value of adopting standardized endogenous study methods to assess TSE clearance in pdFVIII.

28. Question 2 2. Based on the available scientific knowledge, please discuss whether a minimum TSE agent reduction factor, demonstrated using an exogenous spiking model in scaled-down manufacturing experiments, might reasonably serve as an appropriate standard for demonstrating TSE safety of the products.

29. Question 3 3.Considering the outcome of discussion on Question 2, in cases where only lower levels of clearance can be demonstrated for a pdFVIII, should FDA consider the following: A.Labeling that would differentiate the lower clearance products from other products with sufficient TSE clearance; B.Recommending addition of TSE clearance steps to the manufacturing method; C.Performance of TSE clearance experiments using endogenous infectivity models; D.Any other actions?

30. TSE Clearance Labeling Under DESCRIPTION: “Additionally, the manufacturing process was investigated for its capacity to decrease the infectivity of an experimental agent of transmissible spongiform encephalopathy (TSE), considered as a model for the vCJD and CJD agents.”

31. TSE Clearance Labeling Under DESCRIPTION: “Several of the individual production steps in the [product name] manufacturing process have been shown to decrease TSE infectivity of an experimental model agent. TSE reduction steps include [process] [logs], [process] [logs], etc. These studies provide reasonable assurance that low levels of CJD/vCJD agent infectivity, if present in the starting material, would be removed.