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Transcript of Www.pptaglobal.org Industry TSE clearance studies for plasma-derived Factor VIII (pdFVIII) Dr....
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Industry TSE clearance studies for plasma-derived Factor VIII (pdFVIII)
Dr. Thomas R. Kreil, Chair, PPTA Pathogen Safety Steering Committee
FDA TSE Advisory CommitteeThe Holiday Inn Hotel, Gaithersburg
18 and 19 September 2006
Plasma derived therapiesRecombinant therapiesManufacturing sites inUSA, Austria, Switzerland, Italy
Plasma derived therapiesManufacturing sites inAustria, France, Sweden
Plasma derived therapiesManufacturing sites inSpain, USA
Plasma derived therapiesManufacturing site inGermany
Plasma derived therapiesRecombinant therapiesManufacturing sites inUSA, Germany, Switzerland
Plasma derived therapiesManufacturing sites inUSA
Plasma derived therapiesManufacturing site inItalyRegional Member: Europe
PPTA Fractionators: Members
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
Clearance Studies: Principles
INPUT, 1
OUTPUT, 2
prions(viruses)
Reduction factor, RF = log (V1xT1) / (V2xT2)
Manufacturing plant Pathogen Safety Lab
DOWN - 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
EQUIVALENCE, large / small scale
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
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
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
• Nanofiltration
– Prion removal• 35N: ~5 log10 OR ~2 log10 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 ?
• Nanofiltration Summary
– “experimental” conditionsAddition of high concentrations of detergents, intense sonication
research into “nature of the agent”
– “manufacturing” conditionsNo detergents present, no sonication
reduction capacity widely demonstrated
Results MAY depend on setup
Validation, Standardization: Useful ?
• 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 (SHS)
• > 10E5 LD50/ml
• Low / no PrPRES (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 ?
• 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 ?
• 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 ID50/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 ?
• Advances in Science REALLY ?
– “..high blood infectivity in transgenic mice..”
GPI-anchorless PrP• not the patho-physiologically relevant form either• Truncated PrPSC 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 ?
• 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
• 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
Company A
Step MAB column Q-Sepharose chromatography
Spike Scrapie strain 263K Scrapie strain 263K
Preparation 10% brain homogenate 10% brain homogenate
Prion detection / quantification method
- Hamster bioassay- Western blot confirmation
- Hamster bioassay- Western blot confirmation
No. of independent runs
per spike preparationone one
Log reduction(s), ID50 4.6 3.5
TOTAL REDUCTION: 8.1 log10ID50
Product is licensed in the USA
Company B
Step3.5 % PEG
PrecipitationHeparin Affinity
Chromatography*Saline Precipitation and Final Filtrations
TOTAL
SpikePrPSc
263K ScrapiePrPSc
263K ScrapiePrPSc
263K Scrapie
Preparations
1) Microsomal fraction
2) Detergent treated preparation
1) Brain homogenate2) Detergent
treated preparation
1) Microsomal fraction 2) Detergent treated
preparation
Prion detection /quantification method
WB WB WB
No. of independent runs per spike preparation
2 2 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
Company C
Steps
Sequential Precipitation
Procedure
Sequential Chromatography
Procedure
Spike 263K Scrapie 263K Scrapie
Preparation Modified Crude Brain Homogenate /
Microsomal FractionMicrosomal Fraction
Prion detection/quantification method Western Blot Western Blot
No. of independent runs/spike preparation 2 2
Log reduction(s) 1.8 / 1.7 2.4 / 2.5
Mean 1.75 2.45
Product is not licensed in the USA
Company C
Steps
Sequential Precipitation Procedure
Sequential Chromatography
Procedure
Spike 263K Scrapie 263K Scrapie
Preparation Modified Crude Brain Homogenate
Microsomal Fraction
Prion detection/quantification method
Western Blot Western Blot
No. of independent runs/spike preparation
1 1
Log reduction(s) 3.2 3.1
Product is not licensed in the USA
Company D
Steps
Subsequent Precipitation Steps
Precipitation Step Followed by Polishing
Step and Sterile Filtration
Spike 263K Scrapie 263K Scrapie
Preparation Microsomes // purified PrPSc
Microsomes // purified PrPSc
Prion detection/quantification method
CDI (conformation-dependent
immunoassay)
CDI (conformation-dependent immunoassay)
No. of independent runs/spike preparation
2 per spike preparation 2 per spike preparation
Log reduction(s), Mean 3.5 // 3.9 2.9 // 4.0
Product is licensed in the USA
Company E
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
1 per spike preparation
Log reduction(s) 3.8 for cSBH spike, 3.7 for microsomal spike
Mean 3.7 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
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
Individual 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 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 …
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
PPTA Partners
BACK-UP SLIDE
Prions & Plasma Lipoproteins
• “Human prions and plasma lipoproteins”– Brain-derived PrPSC 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– PrPSC association would not change experimental results