Presentation on theme: "Removal of Prions by Plasma Fractionation Processes Henry Baron, M.D. Senior Director Medical and Scientific Affairs Aventis Behring."— Presentation transcript:
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.
4 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. EffluentPrecipitate Separate Input Solution Sample Spike Experimental Process Step Manufacturing Process Step scaled down Sample Prove Clearance = Prove - Effluent
Prion Detection Methods PrP Sc is Highly Correlated with Infectivity Immunoassays for PrP Sc –Western Blot –Conformation Dependent Immunoassay (CDI) Infectivity Bioassays –Rodents (mice, hamsters, transgenic mice) Removal Determined by Immunoassay Correlates with Removal Determined by Bioassay
PrP Sc Immunoassay vs Bioassay > Designates the limits of PrP Sc detection for the assay PrP or infectivity clearance data for various plasma protein and biotechnology processing steps is plotted. For all cases presented, log clearance of either PrP Sc (Western blot) or infectivity (bioassay) is similar. Lee et al, Transfusion vol 41, April 2001 Fractionation Step Logs Cleared 3% PEGCRYOFr II+IIIFr IIIFr IV-1Fr IV-411.5% PEGDE3DE2 > > > > > > > Kogenate
Clearance of Rodent versus Human Prions Variant CJD and other human TSE agents partition similarly to the rodent-adapted sheep scrapie (263K strain)
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 Separation Fr I Separation Fr II+III Separation Fr II+IIIw Separation Fr III Separation Effluent IV-1CryoeffluentEffluent IEffluent II+III Effluent II+IIIw Fr I Paste Discard Fr II+III Paste Fr II+IIIw Paste Fr III Paste Discard Effluent III Fr IV-1 Paste Cryopaste (5.2) (4.0) (4.2) (0) Removal of PrP SC by a series of processing steps can be additive PrP SC that is not removed by an initial precipitation/centrifugation step is removed by a subsequent step No fraction of PrP SC resistant to precipitation/centrifugation was identified PrP SC removal is independent of the presence of brain homogenate Steps evaluated either independent or within a series demonstrated similar magnitudes of either infectivity or PrP Sc clearance. 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.  78: 86 2 Lee et al., J Virol Meth  84: 77 3 Lee et al., Transfusion  41: 449 4 Rohwer / Baxter & ARC, internal report 5 Vey et al., Biologicals  30: 187 6 Brown et al., Transfusion  39: 1169 7 Rohwer / Baxter & ARC, preliminary results 8 Bayer, internal report. 9 Biotest, internal report.
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) Prion Removal Factors 1 Lee et al., J Virol Meth  84: 77 2 Lee et al., Transfusion  41: 449 3 Vey et al., Biologicals  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 log 10 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 !