1. www.pptaglobal.org Industry TSE clearance studies for plasma-derived Factor VIII (pdFVIII) Dr. Thomas R. Kreil, Chair, PPTA Pathogen Safety Steering Committee FDA TSE Advisory Committee The Holiday Inn Hotel, Gaithersburg 18 and 19 September 2006

2. Plasma derived therapies Recombinant therapies Manufacturing sites in USA, Austria, Switzerland, Italy Plasma derived therapies Manufacturing sites in Austria, France, Sweden Plasma derived therapies Manufacturing sites in Spain, USA Plasma derived therapies Manufacturing site in Germany Plasma derived therapies Recombinant therapies Manufacturing sites in USA, Germany, Switzerland Plasma derived therapies Manufacturing sites in USA Plasma derived therapies Manufacturing site in Italy Regional Member: Europe PPTA Fractionators: Members

3. Plasma-derived FVIII, pdFVIII “cryoprecipitate”  FVIII purification, intermediate: w vWF high purity: FVIII only Separation of cryoprecipitate:  centrifugation Increasing temperature:  slow thawing up to 2°C “cryosupernatant”  FIX, PCC, C1INH  Cohn products immunoglobulins alpha-1-AT albumin

4. Clearance Studies: Principles INPUT, 1 OUTPUT, 2 prions (viruses) Reduction factor, RF = log (V1xT1) / (V2xT2) Manufacturing plant Pathogen Safety Lab DOWN - SCALE

5. Down Scale: Validation Intermediate from production or pilot scale Product parameters –Protein concentration, activity –Impurity profile Process parameters –Temperature, time (stirring, incubation), ppt.-agent conc. –Pressure, flow, volume per filter area –pH, conductivity, ionic strength –Linear flow rate, resin contact time  EQUIVALENCE, large / small scale

6. Prion Clearance Studies Choice of spiking agent Preparation of spike –Brain homogenate –Partially-purified prion preparations: microsomal, detergent-treated, sonicated etc. Choice of assay for prion quantification –In vivo –In vitro: Western blot, CDI

7. Prion Quantification: Controlled Quality control for critical reagents Good laboratory practices (not necessarily certified) SOPs for preparation of spiking material, performance / acceptance criteria of assay Internal controls –Positive / Negative / Interference  Assay SUITABILITY

8. Validation, Standardization: Useful ? Conditioning –Up- vs. down-stream processes 1.Prior S/D treatment might suggest detergent-treated spikes 2.Prior filtration might suggest more dispersed spikes –Up- vs. down-stream processes: additive effect of sequential steps ?  Expert judgement and justification required, case-by-case Results  MAY depend on setup

9. Nanofiltration –Prion removal 35N: ~5 log 10 OR ~2 log 10 removal, w or w/o sarkosyl 15N, 10N: complete removal also with detergent –S. Satoh, CHI Blood Safety & Screening, Mc Lean/VA 1998 –J. Tateishi et al., Biologicals [2001] 29: 17 –Minimal prion removal 35N to 15N  minimal removal (sonicated / detergent-treated) –R.G. Rohwer (personal communication) Results  MAY depend on setup Validation, Standardization: Useful ?

10. Nanofiltration  Summary –“experimental” conditions Addition of high concentrations of detergents, intense sonication  research into “nature of the agent” –“manufacturing” conditions No detergents present, no sonication  reduction capacity widely demonstrated Results  MAY depend on setup Validation, Standardization: Useful ?

11. Advances in Science –“..soluble infectious samples from scrapie-infected brain..” 10% scrapie BH  low speed centrifugation; SN  [220.000 g / 30 min.]  high speed supernatant (S HS ) > 10E5 LD 50 /ml Low / no PrP RES (only in vivo assay possible, no WB !)  “ … suitable spiking material to use in validation …” –V.A. Berardi et al., Transfusion [2006] 46: 652 Results  MAY depend on model Validation, Standardization: Useful ?

12. Advances in Science  REALLY ? –“Further studies of blood infectivity in an experimental …” 1.Fukuoka-1 mouse plasma: 34.4 IU/ml  [17.000 g / 30 min.]  2.Pellet: 21.8 IU/ml 3.Supernatant: 6.8 IU/ml –P. Brown et al., Transfusion [1999] 39: 1169  Focus on majority of infectivity, or minor subfraction Results  MAY depend on model Validation, Standardization: Useful ?

13. Advances in Science –“..high blood infectivity in transgenic mice..”, PrP w/o GPI-anchor 1.No pathology upon i.c. scrapie inoculation 2.Prion infectivity in blood: up to > 10E7 ID 50 /ml 3.Prion accumulation in the heart, cardiac amyloidosis (?!?)  “ … sensitivity of new diagnostic kits …  … effectiveness of methods for removal” –M.J. Trifilo et al., Science [2006] 313: 94 Results  MAY depend on model Validation, Standardization: Useful ?

14. Advances in Science  REALLY ? –“..high blood infectivity in transgenic mice..”  GPI-anchorless PrP not the patho-physiologically relevant form either Truncated PrP SC to predict behaviour of full length, physicochemical similar or different behaviour ? –M.J. Trifilo et al., Science [2006] 313: 94 Results  MAY depend on model Validation, Standardization: Useful ?

15. Validated downscale Controlled prion spike materials Controlled prion assays Further Standardization would –Inhibit process-specific investigations (depends on expert input) –Prevent novel approaches –Discourage application of improved understanding Prion Clearance Studies

16. Different manufacturing processes Not necessarily all steps investigated Detailed data for US-licensed products have been shared with the FDA Research still ongoing … Company-specific Data

17. Company A Step MAB columnQ-Sepharose chromatography Spike Scrapie strain 263K Preparation 10% brain homogenate Prion detection / quantification method - Hamster bioassay - Western blot confirmation - Hamster bioassay - Western blot confirmation No. of independent runs per spike preparation one Log reduction(s), ID 50 4.63.5 TOTAL REDUCTION: 8.1 log 10 ID 50  Product is licensed in the USA

18. Company B Step 3.5 % PEG Precipitation Heparin Affinity Chromatography* Saline Precipitation and Final Filtrations TOTAL Spike PrP Sc 263K Scrapie PrP Sc 263K Scrapie PrP Sc 263K Scrapie Preparations 1) Microsomal fraction 2) Detergent treated preparation 1) Brain homogenate 2) Detergent treated preparation 1) Microsomal fraction 2) Detergent treated preparation Prion detection / quantification method WB No. of independent runs per spike preparation 222 Log reduction(s) 3.21 – 3.43≥3.44 – ≥3.452.08 – 2.47 Mean 3.32≥3.452.28≥9.05 * Preliminary results  Product is licensed in the USA

19. Company C Steps Sequential Precipitation Procedure Sequential Chromatography Procedure Spike263K Scrapie PreparationModified Crude Brain Homogenate / Microsomal Fraction Microsomal Fraction Prion detection/quantification methodWestern Blot No. of independent runs/spike preparation22 Log reduction(s)1.8 / 1.72.4 / 2.5 Mean1.752.45  Product is not licensed in the USA

20. Company C Steps Sequential Precipitation Procedure Sequential Chromatography Procedure Spike263K Scrapie PreparationModified Crude Brain Homogenate Microsomal Fraction Prion detection/quantification method Western Blot No. of independent runs/spike preparation 11 Log reduction(s)3.23.1  Product is not licensed in the USA

21. Company D Steps Subsequent Precipitation Steps Precipitation Step Followed by Polishing Step and Sterile Filtration Spike263K Scrapie PreparationMicrosomes // purified PrP Sc Prion detection/quantification method CDI (conformation- dependent immunoassay) No. of independent runs/spike preparation 2 per spike preparation Log reduction(s), Mean3.5 // 3.92.9 // 4.0  Product is licensed in the USA

22. Company E StepsAdsorption, Precipitation, and Chromatography Spike263K Scrapie PreparationClarified Scrapie Brain Homogenate (cSBH) and Microsomal Fraction Prion detection/quantification method PK treatment, 0.5 log titration, and one-step Western blot No. of independent runs/spike preparation 1 per spike preparation Log reduction(s)3.8 for cSBH spike, 3.7 for microsomal spike Mean3.7 to 3.8 Comments: Consistent results were also obtained from partially combined experiments. An additional step is under evaluation.  Product is licensed in the USA

23. Company F StepsSeparation of Cryo Ppt Plus Al(OH)3 Adsorption Spike263K Scrapie PreparationSupernatant of Centrifuged 10% Brain Homogenate Prion detection / quantification method WB No. of independent runs per spike preparation 1 Log reduction(s)3.5  Product is not licensed in the USA

24. Summary / 1 Plasma-derived FVIII products  Manufacturing processes remove prions  Individual reduction factors depend on Specific manufacturing process Number of steps investigated Experimental design

25. Summary / 2 Safety margin  Level of risk: unknown, but likely low  No evidence for transmission by pdFVIII products  High level of pharmacovigilance  Exposure: low, and getting lower  Reduction by all pdFVIII manufacturing processes  Quantification of reduction vs. unknown / low level of risk: an open equation at this point …

26. Conclusion  Unsubstantiated level of risk for pdFVIII: not a rational basis for additional measures Additional steps may adversely impact product characteristics, clinical safety, and availability  Industry continues to be committed to research

27. PPTA Partners

28. BACK-UP SLIDE

29. Prions & Plasma Lipoproteins “Human prions and plasma lipoproteins” –Brain-derived PrP SC bind to (V)LDL / apoB  J. Safar et al., PNAS [2006] 103: 11312 “The Plasma Proteins” –Fractionation of ß-lipoproteins into Cohn III ppt. Mostly, a waste fraction (F. Putnam (Editor), 1960; AP) Spiking studies –Plasma-derived matrices also contain (V)LDL / apoB –PrP SC association would not change experimental results