Prion Removal Capacity of Plasma Protein Manufacturing Processes

Transcription

1 Prion Removal Capacity of Plasma Protein Manufacturing Processes 1 A data collection from PPTA member companies Presented by Nathan Roth, Ph.D. Director, Global Pathogen Safety CSL-Behring on behalf of the PPTA Pathogen Safety Working Group Kang Cai, Douglas Lee: Grifols, RTP NC, USA Rodrigo Gajardo, Juan I. Jorquera: Grifols, Parets del Vallès, Spain Nathan Roth, CSL Behring, King of Prussia PA, USA Albrecht Gröner: CSL Behring, Marburg, Germany Christoph Kempf: CSL Behring, Berne, Switzerland Herbert Dichtelmüller, Eckhard Flechsig: Biotest AG, Dreieich, Germany Thomas Kreil, Gerhard Pölsler: Baxter BioScience, Vienna, Austria Fabrizio Fabbrizzi, Mila Moscardini: Kedrion, Castelvecchio Pascoli, Italy Ilka von Hoegen: PPTA, Brussels, Belgium May 21, 2014 IPFA/PEI 21 st International Workshop, Rome

2 Member Companies

3 The Drivers for Studying Prion Removal Capacity # of BSE Cattle Entering Food Chain Heightened Risk? BSE vcjd HIV >200 prion removal studies by PPTA member companies Modified from Ludlam and Turner, 2006

4 Biochemical Differences Between Prions and Plasma Proteins Prion Misfolded Hydrophobic Polymerized Associated with lipids Insoluble Plasma Protein Naturally folded Hydrophilic Monomeric Devoid of lipids Soluble

5 Scale Down Characterization Critical Process Parameters Biochemical Characterization Starting Material Scale Down Process Step(s) Output ph temperature ionic strength precipitation rates contact time filter load ratio activity/content total protein sp. activity impurities SEC yield Waste Demonstrate that Scale Down Process is a Valid Representation of Full Scale Manufacturing Process

6 Prion Clearance Studies Biochemical BioAssay Half log dilutions Log dilutions Prion Spike X X Starting Material Input PrP RES X X Scale Down Process Step(s) Output PrP RES Waste Reduction (LRV)= Input - Output Western blot of pathogenic prion protein, Lee et al PrP RES X X X X

7 Spikes for Studying Prion Reduction Blood Spike Direct model Not readily available Low titer Spike may alter composition of intermediate: invalid scaledown model Biochemical assays for prion are not sensitive enough Brain Spike Indirect model Readily available High titer Nonetheless it shares feature of: misfolding hydrophobicity polymerization lipid association insolubility infectivity Very limited studies Large amount of studies that may reveal trends

8 Prion Brain Spike Preparations Preparation Abbrev Characteristics Crude brain homogenate Clarified brain homogenate Microsomes Caveolae-like domains (CLDs) Detergent treated Purified cr cl mi ca de pu Mechanically generated brain homogenate, typically 10% w/v Centrifuged at low speed to remove large debris Centrifuged multiple times to selectively concentrate microsomal membrane structures Centrifuged using gradient to enrich CLD structures Treated with detergent and/or nuclease to generate membranepoor prion Purified with multiple steps including detergent and nuclease treatments, purity > 90% All prion spike preparations were derived from brains of syrian hamsters infected with 263K strain of TSE, unless otherwise noted

9 Purification Steps Studied for Prion Reduction Pooled Plasma Cryopoor Plasma Effluent II+III Effluent IV Cryo Precipitate Adsorption Precipitation Depth Filtration Chromatography Nanofiltration FVIII/vWF, Fibrinogen Adsorption Precipitation Depth Filtration Chromatography Nanofiltration FVII, FIX, PCC, Fibrinogen, Protein C, Thrombin FXIII, ATIII C1-INH Fraction (I)+II+III Adsorption Precipitation Depth Filtration Chromatography Nanofiltration IVIG, SCIg, IGIM, Hyperimmunes Fraction IV Precipitation Depth Filtration Chromatography Nanofiltration A 1 PI, AT III, C1-INH Fraction V Albumin

10 PEG Precipitation Reduction (log10) PEG 3.5% 4.0% 8.3% 11.5% ph Assay BC=4, BA=1 BC=5, BA=1 BC=2, BA=0 BC=3, BA=1 Spike mi, de mi, cr, de de cl Solid symbols = measured using biochemical assays (BC) Open symbols = measured using bioassay (BA) Triangles = prion is removed to the limit of detection Dashes = the lowest and highest reduction of a group Spikes used: mi = microsomes; pu = purified; cr = crude brain homogenate; de = detergent treated; cl = clarified brain homogenate PEG precipitation consistently removes prion complete removal obtained at higher PEG concentrations

11 Cold Ethanol Precipitation biochemical assays (BC) Reduction (log10) bioassays (BA) 0.0 EtOH 10-12% 10/0.5%FA 14% 17% 19-20% 25% 38-40% ph Assay BC=10, BA=0 BC=4, BA=0 BC=7, BA=0 BC=3, BA=1 BC=10, BA=3 BC=6, BA=0 BC = 13, BA = 0 Spike cl, mi, cr, pu, de mi, pu cl mi, cr cl, mi, cr, pu, de ca, mi, cr, pu, de ca, cl, mi, cr, pu Spikes used: mi = microsomes; pu = purified; cr = crude brain homogenate; de = detergent treated; cl = clarified brain homogenate; ca = caveolae-like domains Increasing [EtOH] and/or low ph drives precipitation and increases prion removal

12 Salt/Other Precipitation biochemical assays (BC) bioassays (BA) Reduction (log10) Precipitation Gly/NaCl Gly supe Gly supe AMS Caprylate Low ph Assay BC=6, BA=1 BC=3, BA=0 BC=3, BA=0 BC=7, BA=1 BC=5, BA=1 BC=4, BA=0 Spike mi, pu, de microsomal CJD purified CJD mi, cr, pu cl mi, cr Same process conditions but purified spike led to higher removal in this case Spikes used: mi = microsomes; pu = purified; cr = crude brain homogenate; de = detergent treated; cl = clarified brain homogenate

13 Nanofiltration Reduction (log10) Step Nanofiltration Assay BC=21, BA=2 Spike cl, mi, cr, pu, de mi, cr biochemical assays (BC) bioassays (BA) 15 to 20 nm nanofiltration steps were evaluated Complete clearance attained Spikes used: mi = microsomes; pu = purified; cr = crude brain homogenate; de = detergent treated; cl = clarified brain homogenate

14 Prion Reduction of Coupled Steps Only Example #1 Pooled plasma Suspended cryo Al(OH)3 Al(OH)3 adsorbed Gly ppt Gly ppt Al(OH)3 Fibrinogen intermediate Pasteurization Gly/NaCl ppt Depth filtration UF, Sterile filtration Fibrinogen Coupled (BC) Total (BC) 0.2 log 1.0 log 0.2 log 2.8 log 1.4 log 0.1 log 1.5 log 0.3 log 0.5 log 2.4 log Example #2 Pooled plasma Cryo ppt Cryopoor plasma Frac I ppt Super I Frac II+III ppt Frac II+III Frac II+III wash Frac III ppt Washed Frac II+III Supe III Additional purification IgG Coupled (BA) 0.7 log 0.8 log 0 log log 5.6 log Total (BA) 6.7 log Substantial overall TSE removal can be obtained by coupling multiple manufacturing steps

15 Prion Reduction of Single vs Coupled Steps Example #1 Pooled plasma Suspended cryo PEG ppt Clarified supernatant SD Heparin affinity Heparin column eluate Additional purification FVIII/vWF Single (BA) Total (BA) Coupled (BC) Total Coupled (BC) 3.2 log 3.5 log 6.7 log 2.9 log 3.0 log 5.9 log Additive Example #2 Pooled plasma Suspended cryo Al(OH)3 Heparin ppt Al(OH)3 adsorbed Gly/NaCl ppt depth Filtrate SD treatment Chromatography Eluate 0.1 μm filtration FVIII/vWF Single (BC) 0.8 log 2.9 log 1.1 log 3.0 log Total Single (BC) >7.8 log Coupled (BC) Total Coupled (BC) 1.1 log 1.2 log 4.0 log 0.3 log 1.4 log Non-Additive

16 Prion Reduction of Single vs Coupled Steps Example 3 Pooled plasma Cryo ppt Cryopoor plasma Frac I ppt Supe I Frac II+III ppt Supe II+III Additional purification Albumin Single (BA) Total Single (BA) Coupled (BA) Total Coupled (BA) 0.7 log 0.8 log 6.0 log 7.5 log Additive 0.7 log 0.8 log 5.0 log 6.6 log Assessment of overall removal capacity (coupled vs additive single step) should consider the potential influence of prion conditioning effects from upstream steps Evaluation of TSE clearance across coupled process steps is a powerful method for demonstrating TSE clearance capacity of manufacturing processes

17 Conclusions Intrinsic Prion reduction capacity of manufacturing purification processes should be considered as a major risk reduction factor with respect to the safety of plasma derived therapeutic proteins. >200 studies

18 vcjd Risk Over Time Based on Kuru ~ 30 years?? BSE Codon 129 MM, 42% of the UK population Codon 129 VM & VV 58% of the UK population 1st wave vcjd?? 2nd wave vcjd?? Human transmission confirmed blood transfusion pd FVIII transmission As of 2014, there is still no sign of the 2 nd wave Prion removal capacity manufacturing processes adds additional safety margins to donor exclusion measures Modified from Ludlam and Turner, Br J Haematol Jan;132(1):13-24

19 Acknowledgements Kang Cai for compiling, analyzing, and providing meaningful interpretation of the data. The authors pay special thanks to all of the research staff members who diligently performed prion removal studies over the past 15 years. Biochemical Differences b/w Prions and Plasma Proteins Prion Misfolded Hydrophobic Polymerized Associated with lipids Insoluble Plasma Protein Naturally folded Hydrophilic Monomeric Devoid of lipids Soluble

20 Thank you for your attention!