Industry TSE clearance studies for plasma-derived Factor VIII (pdFVIII) Thomas R. Kreil, Ph.D. Chair, PPTA Pathogen Safety Steering Committee FDA TSE Advisory Committee December 15, 2006 www.pptaglobal.org

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

Plasma-derived FVIII, pdFVIII Separation of cryoprecipitate:  centrifugation “cryosupernatant”  FIX, PCC, C1INH Cohn products immunoglobulins alpha-1-AT albumin “cryoprecipitate”  FVIII preparations: FVIII FVIII + vWF Increasing temperature:  slow thawing up to 2°C

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

 full EQUIVALENCE between production & lab scale 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  full EQUIVALENCE between production & lab scale

 many sources of VARIATION Investigational Prion Clearance Studies Choice of spiking agent Preparation of spike material Brain homogenate Partially-purified prion preparations homogenized detergent-treated sonicated etc. Choice of assay for prion quantification In vivo: animal bio-assay In vitro: Western blot, CDI  many sources of VARIATION

Prion Quantification: Controlled Quality control for critical reagents Good laboratory practices not necessarily certified by national authorities Standard Operating Procedures (SOP)s for Preparation of spiking material Assay performance Acceptance criteria for assay results Internal controls Positive / Negative / Interference  assay SUITABILITY

 even virus studies are not fully standardized … Prion Clearance Studies 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  even virus studies are not fully standardized …

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

Q-Sepharose chromatography Company A Step MAB column Q-Sepharose chromatography Spike Scrapie strain 263K Preparation 10% brain homogenate Prion detection / quantification method - Hamster bioassay - Western blot confirmation No. of independent runs per spike preparation one Log reduction(s), ID50 4.6 3.5 TOTAL REDUCTION: 8.1 log10ID50  Product is licensed in the USA

Company B  Product is licensed in the USA TOTAL REDUCTION ≥9.05 Step 3.5 % PEG Precipitation Heparin Affinity Chromatography* Saline Precipitation and Final Filtrations TOTAL REDUCTION Spike PrPSc 263K Scrapie Preparations 1) Microsomal fraction 2) Detergent treated preparation 1) Brain homogenate Prion detection / quantification method WB No. of independent runs per spike preparation 2 Log reduction(s) 3.21 – 3.43 ≥3.44 – ≥3.45 2.08 – 2.47 Mean 3.32 ≥3.45 2.28 ≥9.05 * Preliminary results  Product is licensed in the USA

Sequential Precipitation Procedure Sequential Chromatography Procedure Company C Steps Sequential Precipitation Procedure Sequential Chromatography Procedure Spike 263K Scrapie Preparation Modified Crude Brain Homogenate / Microsomal Fraction Microsomal Fraction Prion detection/quantification method Western Blot No. of independent runs/spike preparation 2 Log reduction(s) 1.8 / 1.7 2.4 / 2.5 Mean 1.75 2.45  Product is not licensed in the USA

Sequential Precipitation Procedure Sequential Chromatography Procedure Company C Steps Sequential Precipitation Procedure Sequential Chromatography Procedure Spike 263K Scrapie Preparation Modified Crude Brain Homogenate Microsomal Fraction Prion detection/quantification method Western Blot No. of independent runs/spike preparation 1 Log reduction(s) 3.2 3.1  Product is not licensed in the USA

Company D UPDATED Dec 14, 2006  Product is licensed in the USA Steps Subsequent Precipitation Steps Precipitation Step Followed by Polishing Step and Sterile Filtration Spike 263K Scrapie Preparation Microsomes // purified PrPSc Prion detection/quantification method CDI (conformation-dependent immunoassay) No. of independent runs/spike preparation 2 per spike preparation Log reduction(s), Mean 3.5 // 3.9 2.9 // 4.0 TOTAL REDUCTION: 6.4 // 7.9 log10  Product is licensed in the USA

Company E UPDATED Dec 14, 2006  Product is licensed in the USA Steps Adsorption, Precipitation, and Chromatography Spike 263K Scrapie Preparation Clarified 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 Total 4 experimental runs, 2 per spike preparation Log reduction(s) 4.0 for cSBH spike, 3.9 for microsomal spike Mean 3.9 to 4.0 Comments: Consistent results were also obtained from partially combined experiments. TOTAL REDUCTION: 3.9 - 4.0 log10 (partial process)  Product is licensed in the USA

Separation of Cryo Ppt Plus Al(OH)3 Adsorption Company F Steps Separation of Cryo Ppt Plus Al(OH)3 Adsorption Spike 263K Scrapie Preparation Supernatant 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

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

Summary / 2 Safety margin Level of risk: unknown, but likely low No evidence for transmission by pdFVIII products , or any other plasma product 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 …

Low, and getting lower … vCJD epidemic in the UK  Andrews, UK HPA ( July 18, 2006)

FDA questions  TSE AC / 1 FDA questions Based on available scientific knowledge, pls discuss whether a minimum TSE agent reduction factor, demonstrated using an exogenous (spiking) model in scaled-down manufacturing experiments, would enhance safety of the products. PPTA response The plasma-protein associated vCJD risk is considered very low, although not exactly known yet, and thus any level of reduction is re-assuring.

FDA questions  TSE AC / 2a FDA questions … what actions should FDA consider in cases when a licensed pdFVIII has a lower reduction factor: a) Labeling that would differentiate the lower TSE clearance products from the higher TSE clearance products. PPTA response Prion reduction factors are derived by different investigational approaches. Labeling, in our opinion, would thus provide information that cannot be meaningfully assessed out of context. Also, it might suggest a safety differential, which in light of the remaining uncertainties we feel cannot be substantiated.

FDA questions  TSE AC / 2b FDA questions … what actions should FDA consider in cases when a licensed pdFVIII has a lower reduction factor: b) Recommending addition of clearance steps to the manufacturing method. PPTA response The introduction of additional clearance steps would likely require clinical testing to confirm safety & efficacy product characteristics, with unsubstantiated benefit to the patients involved. Also, production yields would be negatively affected.

FDA questions  TSE AC / 2c FDA questions … what actions should FDA consider in cases when a licensed pdFVIII has a lower reduction factor: c) Performance of TSE clearance experiments using endogenous infectivity models. PPTA response Using (low-titered) endogenous infectivity limits demonstrable prion reduction to levels lower than those already shown for pd FVIII. Animal and human plasma are different (significantly).  We thus believe that such experiments would result in immense animal consumption and effort, without changing product safety.

Results  MAY depend on model Standardization: Useful ? Advances in Science “..high blood infectivity in transgenic mice..”, PrP w/o GPI-anchor No pathology upon i.c. scrapie inoculation Prion infectivity in blood: up to > 10E7 ID50/ml 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

Results  MAY depend on model Standardization: Useful ? Advances in Science  REALLY ? “..high blood infectivity in transgenic mice..” GPI-anchorless PrP not the patho-physiologically relevant form truncated PrPSC physicochemically dissimilar, thus behaviour is likely different, also natural PrPsc is hydrophobic, i.e. poorly soluble, whereas this GPI-deficient molecule is relatively soluble M.J. Trifilo et al., Science [2006] 313: 94 Results  MAY depend on model

Of mice and men … Mouse vs human plasma has: 420% Factor X 270% Factor VII 250% Factor VIII 250% fibrinogen 150% Factor IX 120% Factor XI 80% prothrombin 75% Factor XII Behringwerke, unpublished

Different animals and men … Animal vs human plasma: Karges et al., Drug Research [1994] 44: 793

Different animals and men … Animal vs human plasma: Karges et al., Drug Research [1994] 44: 793

FDA questions  TSE AC / 2d FDA questions … what actions should FDA consider in cases when a licensed pdFVIII has a lower reduction factor: d) Any other actions ? PPTA response Member companies remain committed to further investigate prions and their reduction by the plasma product manufacturing processes. We believe that open exchange of data benefit all stakeholders, and wish for continued dialogue with the agency & advisors. Given remaining uncertainties and increasingly reassuring epidemiological information, we feel that for now further actions are not justified.

Conclusion The unsubstantiated level of prion risk for pdFVIII is not a rational basis for any additional measures. Minimum TSE reduction factors versus an unquantified, but considered very low, level of risk is not necessary. Quantitative prion reduction labeling including a threshold would not provide meaningful safety information. Introduction of additional manufacturing steps may adversely impact clinical product safety, and lower yield, and would require patient exposure with unclear benefits. Endogenous prion reduction studies would not change the prion safety profile of a product. Industry committed to research & dialogue

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