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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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slide1

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

slide2

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

slide3

Advantages of NaOH as a Cleaning Agent

  • Readily available
  • Inexpensive
  • Rapid
  • Effectiveat moderate concentrations
  • Compatible with equipment
slide4

NaOH Inactivates Prions

*Infectivity Assay

*** Dried on stainless steel coupons

** Western blot assay

**** in the presence of detergent

slide5

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

slide6

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.
slide7

NaOH + Sarkosyl Eliminates Residual Signal in Western Blot Assay

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

slide8

+

+ PK

PrPSc + NaOH

and sarkosyl

+ PK

Sarkosyl Improves Accessibility to NaOH

PrPSc

+ NaOH

Unfolded

Protected

slide9

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

slide10

0.1 M NaOH Changes Prion Structure

PK Treatment

+

-

-

LRF = 0.5

LRF = 3.5

Sarkosyl Treatment

+

LRF > 4.5

LRF = 1.5

slide11

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

slide12

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

slide13

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.