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Critical Factors Influencing Prion Decontamination Using NaOH – PPTA Collaborative Study. Kang Cai, Ph.D. Virology/TSE, R & D Talecris Biotherapeutics (Formerly Bayer Biological Products). TSE Advisory Committee Meeting Washington, Sept 18, 2006. Minimizing TSE-related Risks.
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Critical Factors Influencing Prion Decontamination Using NaOH – PPTA Collaborative Study Kang Cai, Ph.D. Virology/TSE, R & D Talecris Biotherapeutics (Formerly Bayer Biological Products) TSE Advisory Committee Meeting Washington, Sept 18, 2006
Minimizing TSE-related Risks Safety Step 2Control of Source Material Plasma Donation Center Donor Safety Step 1Donor Deferral Quality Assurance & GMP Begin Manufacturing Cleaning/Sanitization Patient Safety Step 3Prion Clearance Complete Manufacturing Safety Step 4Equipment Cleaning Safety Step 5Post-marketing Surveillance
Advantages of NaOH as a Cleaning Agent • Readily available • Inexpensive • Rapid • Effectiveat moderate concentrations • Compatible with equipment
NaOH Inactivates Prions *Infectivity Assay *** Dried on stainless steel coupons ** Western blot assay **** in the presence of detergent
NaOH (M) RF SBH Temp Time (Log) (%) (°C) (min) 3.5 - 3.9 1 0.1 18 15 - 60 A Small Amount of Residual Signal Sometimes Observed After NaOH Treatment NaOH Treatment 1 2 3 4 5 6 7 8 9 10 Not measured (NM) – + NM PPTA-collaborative Study on Prion Inactivation conducted at BioReliance
Evaluate the Effect of NaOH on Prion • Mix clarified SBH and NaOH to final concentrations of 1% and 0.1M, with or without 2% sarkosyl. • Incubate at 4° or 18° for 0, 15 or 60 minutes. • Withdraw samples and neutralize pH. (Control is neutralized before addition of SBH.) • Treat with proteinase K to remove PrPC, then concentrate samples by centrifugation. • Resuspend pellets in 2X SDS sample buffer, and serially dilute (by half logs) for electrophoresis.
NaOH + Sarkosyl Eliminates Residual Signal in Western Blot Assay 4° 18° LANE: 1 2 3 4 5 6 7 8 9 10 11 LANE: 1 2 3 4 5 6 7 8 9 10 11 Control no sarkosyl LRF: 3.1 LRF: 3.3 60 minutes Control + sarkosyl LRF: 3.7 LRF: >4.5 15 minutes 60 minutes LRF: >4.6
+ + PK PrPSc + NaOH and sarkosyl + PK Sarkosyl Improves Accessibility to NaOH PrPSc + NaOH Unfolded Protected
Inactivation Unfolded or degraded prion protein Prion Inactivation Model TSEs are transmitted by misfolded prion protein. Misfolding Normal cellular prion protein PrPC Pathogenic prion protein PrPSc
0.1 M NaOH Changes Prion Structure PK Treatment + - - LRF = 0.5 LRF = 3.5 Sarkosyl Treatment + LRF > 4.5 LRF = 1.5
NaOH, t = 0 NaOH, t = 0 Buffer Buffer Input Input + + IP IP IP PrPRES NOT immunoprecipitated + NaOH, t = 60 Gdn SCN Input Unfolded PrPRES immunoprecipitated by NaOH or GdnSCN 0.1 M NaOH Unfolds Prion PrPC immunoprecipitated
Critical Factors Influencing Decon Using NaOH • Concentration of NaOH • Presence of detergent • Temperature and time NaOH works by unfolding and degrading of the structure of PrPSc
Acknowledgements Talecris PPTA Collaborators Lothar Biesert, Ph.D., Octapharma Herbert Dichtelmüller, Ph.D., Biotest AG Fabrizio Fabbrizzi, Ph.D., Kedrion SpA Albrecht Gröner, Ph.D., ZLB Behring Christoph Kempf, Ph.D., ZLB Behring Juan Jorquera, Ph.D., Grupo Grifols Rodrigo Gajardo, Ph.D., Grupo Grifols Thomas R. Kreil, Ph.D., Baxter BioScience Ilka von Hoegen, Ph.D., PPTA Pat Bauman, Ph.D. Michele Kislan, B.S. Michael Burdick, Ph.D. Michael Mink, Ph.D. Mark Nelson, B.S. Renea’ Oquendo, B.S. Dominique Pifat, Ph.D. Steve Petteway, Ph.D. Contributors Andy Bailey, Ph.D. Henry Baron, Ph.D. Chris Stenland, Ph.D. Leslie Lawrence, B.S.
