Removal of Prions by Plasma Fractionation Processes Henry Baron, M.D. Senior Director Medical and Scientific Affairs Aventis Behring

CJD/vCJD and Human Blood: Key Message Currently no scientific evidence to substantiate that persons with pre-clinical or clinical CJD, including vCJD, carry infectious prions in their blood or have transmitted them through blood or plasma products. Therefore, this risk is considered theoretical. Dozens of studies with classical CJD : Experimental (animal) Epidemiological (human) Medical observation (human) Several decades of experience Less experience with variant CJD : Oral, food-borne transmission of BSE prions

Variant CJD and Human Blood: Current State of Knowledge Whole blood, red blood cells and platelets from UK donors have been and continue to be administered to UK recipients (estimated 30 to 40 million transfusions in UK over past 10 years). Several patients with vCJD received transfusions but none could be linked to a donor who had CJD or vCJD. No cases of CJD or vCJD have been noted among identified recipients of blood or plasma products from known vCJD donors to date. To date, there is no evidence in the UK (where ~ 98% of all reported BSE and ~ 95 % of all reported vCJD worldwide have occurred) that vCJD has been transmitted through blood or plasma products.

Two Lancet publications report that prions were undetectable in blood, plasma and buffy coat of patients with vCJD, despite detection of prions in their lymphoid tissues. Bruce et al. Detection of variant Creutzfeldt-Jakob disease infectivity in extraneural tissues. Lancet, 2001; 358:208-209. Wadsworth et al. Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay. Lancet, 2001; 358:171-180.

A responsible approach to the manufacture of plasma protein therapies is to treat this theoretical risk as though it were real. RATIONAL, SCIENCE-BASED PRECAUTIONARY POLICIES TO MINIMIZE THE THEORETICAL RISK : Individual donor deferral criteria. Geographic plasma rejection criteria. Withdrawal/notification policies. EXTREMELY RAPID AND HIGHLY SENSITIVE METHODS OF PRION DETECTION : Research prion infectivity in bloods of CJD, including vCJD, cases. Assessment of prion partitioning in manufacturing processes.

Prion Partitioning in the Manufacture of Human Plasma Proteins Prions never detected in nor transmitted through human blood, plasma or plasma derivatives. Therefore, no knowledge as to the biophysicochemical nature of theoretical prion contaminant in plasma. Therefore, uncertainty as to appropriate, relevant prion spiking agent for study of prion partitioning in manufacturing processes.

Key Issues In Prion Removal Validity of Scaledown Model Nature of the Spike Detection Methodology Immunoassay Infectivity bioassay Model (rodent) versus Human Prions Independent versus Coupled Process Step Removal

Scaledown of Experimental Process Experimental Process is Equivalent to Manufacturing Process. Effluent Precipitate Separate Input Solution Sample Spike Experimental Process Step Manufacturing Process Step scaled down Prove Clearance = Prove - Effluent

Spiking Agents Used Brain Homogenates Microsomes Caveolae-Like Domains (CLDs) Purified PrPSc Prion Fibrils

Prion Detection Methods PrPSc is Highly Correlated with Infectivity Immunoassays for PrPSc Western Blot Conformation Dependent Immunoassay (CDI) Infectivity Bioassays Rodents (mice, hamsters, transgenic mice) Removal Determined by Immunoassay Correlates with Removal Determined by Bioassay

PrPSc Immunoassay vs Bioassay PrP or infectivity clearance data for various plasma protein and biotechnology processing steps is plotted. For all cases presented, log clearance of either PrPSc (Western blot) or infectivity (bioassay) is similar. Lee et al, Transfusion vol 41, April 2001 > > > > > > Kogenate > Logs Cleared CRYO 3% PEG Fr II+III Fr III Fr IV-1 11.5% PEG Fr IV-4 DE2 DE3 Fractionation Step > Designates the limits of PrPSc detection for the assay

Clearance of Rodent versus Human Prions Variant CJD and other human TSE agents partition similarly to the rodent-adapted sheep scrapie (263K strain)

Clearance of Rodent versus Human Prions

Clearance of Rodent versus Human Prions

Clearance of Rodent versus Human Prions Conclusion: Data showing removal of rodent prions can be considered predictive of removal of human CJD and vCJD prions.

Prion Clearance Study - Cohn Coupled Series Steps SPIKE Fr IV-1 Separation Cryo Fr I Fr II+III Fr II+IIIw Fr III (5.2) Cryoeffluent Effluent I Effluent II+III Effluent IV-1 Cryopaste Fr I Paste Discard Fr II+III Paste Fr IV-1 Paste Removal of PrPSC by a series of processing steps can be additive PrPSC that is not removed by an initial precipitation/centrifugation step is removed by a subsequent step No fraction of PrPSC resistant to precipitation/centrifugation was identified PrPSC removal is independent of the presence of brain homogenate Steps evaluated either independent or within a series demonstrated similar magnitudes of either infectivity or PrPSc clearance. Effluent II+IIIw Fr II+IIIw Paste (4.0) Effluent III (0) Fr III Paste Discard (4.2) Partitioning determined for independent steps is consistent with partitioning determined for coupled processes.

Prion Removal Factors Major product categories F VIII Immunoglobulins Albumin Protease inhibitors

Prion Removal Factors F VIII Cryoprec.: 1.0 (1), <1.0 (2,3), 1.5 (7), 1.0 (6) Aluminium-hydroxide adsorption: 1.7 (1), 1.3 (5) PEG or glycine precipitation: 2.2 (3), 3.0 (3), 1.7-3.3 (5) Ion exchange or size exclusion chromatography: 3.1 (1), 1.0 (8), 3.5 (4) Monoclonal antibody purification: 4.1 (4) 0.45 µm / 0.2 µm filtration: 1.0 (1), 1.0 (5) F VIII: 6.8 (1), 3.2 (2,3), 3.2(9), 8.0 (4), 4.8-5.5 (5) 1 Foster et al., Vox. Sang. [2000] 78: 86 2 Lee et al., J Virol Meth [2000] 84: 77 3 Lee et al., Transfusion [2001] 41: 449 4 Rohwer / Baxter & ARC, internal report 5 Vey et al., Biologicals [2002] 30: 187 6 Brown et al., Transfusion [1999] 39: 1169 7 Rohwer / Baxter & ARC, preliminary results 8 Bayer, internal report. 9 Biotest, internal report.

Prion Removal Factors Immunoglobulins Immunoglobulins Cryoprecipitation: <1 (1), 1.0 (2), <1 - 2.4 (5) Precipitation of fraction I: 1.1 (2), <1 – 3.1 (5) Precipitation of fraction (I+)III: >3.3-3.8(9), 3.5(6), >3.7 (1), >4.0 (2), >4.3 (3), 5.3 (3) PEG precipitation: >3.0(9) Depth filtration: >2.8 (1), 2.8(6), 4.4(6), 6.4 (2,3), 6.0 (4) Nanofiltration: 4.4(6) Ig:  3.0(7),  5.0-9.4(8), 6.5 (1),  6.3-6.8(9), 6.4 (2&3), 7.9 (4) Immunoglobulins Cryoprecipitation: <1 (1), 1.0 (2), <1 - 2.4 (5) Precipitation of fraction I: 1.1 (2), <1 – 3.1 (5) Precipitation of fraction (I+)III: >3.7 (1), >4.0 (2), >4.3 (3), 5.3 (3) Depth filtration: >2.8 (1), 6.4 (2,3), 6.0 (4) Ig safety margin: 6.5 (1), 6.4 (2&3), 7.7 (4) 1 Foster et al., Vox. Sang. [2000] 78: 86 2 Lee et al., J Virol Meth [2000] 84: 77 3 Lee et al., Transfusion [2001] 41: 449 4 Rohwer / Baxter & ARC, preliminary results 5 Aventis, submitted 1 Foster et al., Vox. Sang. [2000] 78: 86 2 Lee et al., J Virol Meth [2000] 84: 77 3 Lee et al., Transfusion [2001] 41: 449 4 Rohwer / Baxter & ARC, preliminary results 5 Vey et al., Biologicals [2002] 30: 187 6 ZLB, internal report. 7 Biotest, internal report. 8 Aventis Behring, internal report. 9 Baxter, internal report.

Prion Removal Factors Albumin Cryoprecipitation: <1 (1), 1.0 (2), <1 – 2.4 (5) Precipitation of fraction I: 1.1 (2) , <1 – 3.1 (5) Ppt. of fr. (I+II+)III: 1.3 (1), 1.9 (7), 2.2(6), 4.0 (2), 6.0 (3), 4.7 (3), 2.4 (4) , 3.1-4.0 (5) Precipitation of fraction IV: 3.0 (1),3.0(6), 3.9 (7), 4.6 (3), 4.1 (3), 4.5-4.6 (5) Depth filtration: 4.9 (1) Albumin: 5.8 (7), 11.5 (1), 16.0 (3), 7.7-14.1 (5) 1 Foster et al., Vox. Sang. [2000] 78: 86 2 Lee et al., J Virol Meth [2000] 84: 77 3 Lee et al., Transfusion [2001] 41: 449 4 Rohwer / Baxter & ARC, preliminary results 5 Vey et al., Biologicals [2002] 30: 187 6 ZLB, internal report 7 Baxter, internal report

Prion Removal Factors Proteinase inhibitor Cryoprecipitation: 1.0 (1,2), <1 - 2.4 (3) Precipitation of fraction I: 1.1 (1,2), <1 - 3.1 (3) Ppt. of fr. (I+II+)III: 4.7-6.0 (2), 3.1 - 4.0 (3) PEG precipitation: 5.4 (2) Proteinase inhibitor: 12.2 (2), 3.1-9.5 (3) 1 Lee et al., J Virol Meth [2000] 84: 77 2 Lee et al., Transfusion [2001] 41: 449 3 Vey et al., Biologicals [2002] 30: 187

Conclusions Removal of prions by plasma manufacturing processes: is very significant, and further minimizes the theoretical risk addressed by donor deferral donor deferral - 1 log10 reduction of exposure risk is process specific and demonstrated across all prion spike materials, different prion assay systems, and manufacturing step specifics is very substantial as compared to the – still theoretical – level of risk !