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 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