Presentation on theme: "Removal of Prions by Plasma Fractionation Processes"— Presentation transcript:
1Removal of Prions by Plasma Fractionation Processes Henry Baron, M.D.Senior Director Medical and Scientific AffairsAventis Behring
2CJD/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 experienceLess experience with variant CJD :Oral, food-borne transmission of BSE prions
3Variant 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.
4Two 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:Wadsworth et al. Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay. Lancet, 2001; 358:
5A 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.
6Prion 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.
7Key Issues In Prion Removal Validity of Scaledown ModelNature of the SpikeDetection MethodologyImmunoassayInfectivity bioassayModel (rodent) versus Human PrionsIndependent versus Coupled Process Step Removal
8Scaledown of Experimental Process Experimental Process is Equivalent to Manufacturing Process.EffluentPrecipitateSeparateInput SolutionSampleSpikeExperimental Process StepManufacturing Process Stepscaled downProveClearance = Prove - Effluent
10Prion Detection Methods PrPSc is Highly Correlated with InfectivityImmunoassays for PrPScWestern BlotConformation Dependent Immunoassay (CDI)Infectivity BioassaysRodents (mice, hamsters, transgenic mice)Removal Determined by Immunoassay Correlates with Removal Determined by Bioassay
11PrPSc 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 ClearedCRYO3% PEGFr II+IIIFr IIIFr IV-111.5% PEGFr IV-4DE2DE3Fractionation Step> Designates the limits of PrPSc detection for the assay
12Clearance of Rodent versus Human Prions Variant CJD and other human TSE agents partition similarlyto the rodent-adapted sheep scrapie (263K strain)
15Clearance of Rodent versus Human Prions Conclusion:Data showing removal of rodent prions can be considered predictive of removal of human CJD and vCJD prions.
16Prion Clearance Study - Cohn Coupled Series Steps SPIKEFr IV-1SeparationCryoFr IFr II+IIIFr II+IIIwFr III(5.2)CryoeffluentEffluent IEffluent II+IIIEffluent IV-1CryopasteFr I PasteDiscardFr II+IIIPasteFr IV-1PasteRemoval of PrPSC by a series of processing steps can be additivePrPSC that is not removed by an initial precipitation/centrifugation step is removed by a subsequent stepNo fraction of PrPSC resistant to precipitation/centrifugation was identifiedPrPSC removal is independent of the presence of brain homogenateSteps evaluated either independent or within a series demonstrated similar magnitudes of either infectivity or PrPSc clearance.Effluent II+IIIwFr II+IIIwPaste(4.0)Effluent III(0)Fr III PasteDiscard(4.2)Partitioning determined for independent stepsis consistent with partitioning determined forcoupled processes.
17Prion Removal Factors Major product categories F VIII Immunoglobulins AlbuminProtease inhibitors
18Prion 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), (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), (5)1 Foster et al., Vox. Sang.  78: Lee et al., J Virol Meth  84: 773 Lee et al., Transfusion  41: Rohwer / Baxter & ARC, internal report5 Vey et al., Biologicals  30: Brown et al., Transfusion  39: 11697 Rohwer / Baxter & ARC, preliminary results 8 Bayer, internal report.9 Biotest, internal report.
20Prion 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), (4) , (5)Precipitation of fraction IV: 3.0 (1),3.0(6), 3.9 (7), 4.6 (3), 4.1 (3), (5)Depth filtration: 4.9 (1)Albumin: 5.8 (7), 11.5 (1), 16.0 (3), (5)1 Foster et al., Vox. Sang.  78: 862 Lee et al., J Virol Meth  84: 773 Lee et al., Transfusion  41: 4494 Rohwer / Baxter & ARC, preliminary results5 Vey et al., Biologicals  30: 1876 ZLB, internal report7 Baxter, internal report
21Prion Removal Factors Proteinase inhibitor Cryoprecipitation: 1.0 (1,2), < (3)Precipitation of fraction I: 1.1 (1,2), < (3)Ppt. of fr. (I+II+)III: (2), (3)PEG precipitation: 5.4 (2)Proteinase inhibitor: 12.2 (2), (3)1 Lee et al., J Virol Meth  84: 772 Lee et al., Transfusion  41: 4493 Vey et al., Biologicals  30: 187
22Conclusions Removal of prions by plasma manufacturing processes: is very significant, and further minimizes the theoretical risk addressed by donor deferraldonor deferral - 1 log10 reduction of exposure riskis process specific and demonstrated across all prion spike materials, different prion assay systems, and manufacturing step specificsis very substantial as compared to the – still theoretical – level of risk !