1. 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

2. 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

3. 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

4. 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)

5.  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

6.  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

7. 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

8.  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 …

9. 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 …

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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: log10 (partial process)
 Product is licensed in the USA

16. 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

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

18. 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 …

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

20. 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.

21. 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.

22. 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.

23. 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.

24. 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

25. 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

26. 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

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

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

29. 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.

30. 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

31. PPTA Partners