TSE Task Force Prion reduction evaluation in the manufacturing of plasma protein therapies Dr. Henry Baron, Chair, PPTA TSE Task Force FDA TSE Advisory Committee The Hilton Hotel, Silver Spring 14 October 2004

FVIII / vWF Products Study Parameters Prion strain: 263K hamster adapted Scrapie –Spike preparations: 1.Microsomes 2.Purified PrP Sc 3.Detergent solubilized brain homogenate 4.10 % brain homogenate –Prion detection / quantification method: CDI; Western blot, bioassay –2 - 3 independent runs/spike preparation

FVIII / vWF Products Results Product A: Consecutive salt precipitation (Preliminary data on Prion Reduction Factor): –Microsomes: 2.5 log 10 /3.2 log 10 /2.5 log 10 [mean: 2.7 log 10 ] –Purified PrP Sc : 2.9 log 10 /3.3 log 10 /2.8 log 10 [mean: 3.0 log 10 ] Product B: 3 % PEG precipitation (Published data on Prion Reduction Factor): –2.2 log 10 infectivity –3.0 log 10 PrP RES by Western blot Product C: (Heparin-affinity purified): 3.5 % PEG precipitation (Preliminary data on Prion Reduction Factor): –Microsomes: 3.5 log 10 /3.5 log 10 –Detergent solubilised BH: 4.2 log 10 /4.2 log 10

MAb purified F VIII Prion strain: 263K hamster adapted Scrapie –Spike preparations: 1.Brain homogenate (Mab affinity step) 2.SD treated and filtered brain homogenate (DEAE step) –Prion detection / quantification method: Hamster bioassay Manufacturing stage studied: MAb affinity chromatography and DEAE Sephadex –2 independent runs/spike preparation Result (Prion Reduction Factor): –MAb affinity chromatography: 4.1 log 10 –DEAE Sephadex: 3.5 log 10

FIX Products Study Parameters Prion strain: 263K hamster adapted Scrapie –Spike preparations: 1.Microsomes 2.Purified PrP Sc 3.Detergent solubilized brain homogenate –Prion detection / quantification method: CDI; Western Blot –2 independent runs/spike preparation

FIX Products Results Product A: Manufacturing stage studied: Planova filters in series –35N –15N –Result for detergent solubilized brain homogenate (Prion Reduction Factor): ≥ 4.1 log 10 mean

FIX Products Product B: Manufacturing stage studied: Nanofiltration (YM 100) –Result (Preliminary data on Prion Reduction Factor): 1.Microsomes:  3.3 log 10 /  3.7 log 10 [mean:  3.5 log 10 ] 2.Purified PrP Sc :  3.6 log 10 /  3.6 log 10 / [mean:  3.6 log 10 ] Product C: Manufacturing stage studied: Salt precipitation –Result (Preliminary data on Prion Reduction Factor): 1.Microsomes:  3.8 log 10 /  3.6 log 10 [mean:  3.7 log 10 ] 2.Purified PrP Sc :  2.9 log 10 /  3.1 log 10 / [mean:  3.0 log 10 ]

IGIV Prion Clearance by Coupled vs Independent Steps a Bioassay n = 1, confirmed by Western blot measurement of PrP RES, n =3 b Spiked once at the beginning of coupled steps consisting Cryo, Frac. 1, and Frac. III separation c Spiked at the beginning of each independent step d Not determined by bioassay, the number is derived from the coupled study. Western blot consistently shown ~1 log clearance Conclusion: Prion infectivity reduction can be additive for these steps

Single Steps vs. Combination of Steps Immunglobulin Production LRFLRF combined steps Depth filtration Depth filtration Sum7.3 Brain homogenate spiked to intermediates Prion reduction factor determinded by infectivity Gregori et al. 2004

Risk of vCJD Donors in the UK Donor Population To date, 15 blood donors diagnosed with vCJD, of which 9 contributed to ~20 pools used to manufacture plasma protein therapies Thus from *, the incidence of vCJD donors amongst the donor population was: –15 / (1,907,000 # x 18 years) = –0.44 vCJD donors per million donors per year * Based on the time period of potential exposure to BSE until when UK plasma was no longer used for manufacture # In 1997 the yearly number of UK donors was 1,907,000 (DNV vCJD risk assessment; 1999)

BSE Exposure: EU vs. UK Number of UK BSE cases up to 2000 detected by passive surveillance* 180,845 Number of EU cases detected by passive surveillance 1,777 EU vs. UK exposure~100 fold lower i.e. ~100-fold lower EU vs UK vCJD exposure * All UK vCJD infected donors contributed prior to the introduction of active testing for BSE.

Active vs. Passive Detection of BSE (EU excluding UK) ~4-fold increase in BSE detection due to active testing From: Report on the Monitoring and Testing of Ruminants for the Presence of Transmissible Spongiform Encephalopathy (TSE) in the EU in 2003, including the results of the survey of Prion Protein Genotypes in Sheep Breeds (EU report)

Conclusions Comparability of data from different PPTA member companies using different investigative approaches gives confidence that current systems are working to ensure efficient prion removal, even though prions have never been shown to exist in human plasma. These efforts made by PPTA member companies represent enormous investments in applying the precautionary principle and providing reassurance on the safety of plasma products. Balanced approaches are necessary to ensure both safety and availability of life-saving plasma protein therapies