Kang Cai, Ph.D.

1

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
2
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
3
Advantages of NaOH as a Cleaning Agent

• Readily available
• Inexpensive
• Rapid
• Effective at moderate concentrations
• Compatible with equipment

4
NaOH Inactivates Prions
Infectivity Assay Dried on stainless steel
coupons
Western blot assay
in the presence of detergent
5
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
6
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.

7
NaOH Sarkosyl Eliminates Residual Signal in
Western Blot Assay
18
4
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 gt4.5
15 minutes
60 minutes
LRF gt4.6
8
Sarkosyl Improves Accessibility to NaOH
PrPSc NaOH
Unfolded
Protected
9
Prion Inactivation Model
TSEs are transmitted by misfolded prion protein.
Misfolding
Normal cellular prion protein
PrPC
Pathogenic prion protein
PrPSc
10
0.1 M NaOH Changes Prion Structure
PK Treatment

-
-
LRF 0.5
LRF 3.5
Sarkosyl Treatment

LRF gt 4.5
LRF 1.5
11
0.1 M NaOH Unfolds Prion
PrPC
immunoprecipitated
12
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
13
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.