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Proteolytic Inactivation of prions; a biological solution to TSE decontamination. Dickinson J*, Murdoch H*, Sutton JM*, Crabb, W. D.#, Bott, R#, Penet, C .#, and Raven N.D.H*. *- Health Protection Agency, UK # -Genencor International. Decontamination of TSE agents; practical issues.

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proteolytic inactivation of prions a biological solution to tse decontamination

Proteolytic Inactivation of prions;a biological solution to TSE decontamination.

Dickinson J*, Murdoch H*, Sutton JM*, Crabb, W. D.#, Bott, R#, Penet, C.#, and Raven N.D.H*

*- Health Protection Agency, UK

# -Genencor International

decontamination of tse agents practical issues
Decontamination of TSE agents;practical issues
  • Are any of the available methods effective or applicable in the real world ?
  • Issues of practicality
      • Procedures time consuming
      • Use of large amounts of caustic and hazardous chemicals
      • Damaging to sensitive surgical instruments
      • Operator safety
      • Environmental considerations
      • Not compatible with existing practice
      • Balance of cost vs risk
  • Genuine need for cheap, clean, “user and environment friendly” alternative.
digestion of bse by thermostable proteases
Digestion of BSE by thermostable proteases
  • Conditions, protease and standard of assessment critical:
  • Proteases: Properase (~3-log reduction infectivity) and MC3 (>7-log reduction infectivity)
  • Model: mouse-passaged BSE strain 301-V (infectious mouse brain homogenate (iMBH) >109 iu / mg)
  • Assessment: Western blot (6H4 and PAb2) and bioassayed in VM mice
  • Process: 10% iMBH digested at pH12, 600C for 30 minutes
high molecular weight prp isoforms are digested by mc3 but not properase

pH12 PrP

pH12 Prt PrP

52kDa

52kDa

33kDa

33kDa

19kDa

19kDa

High molecular weight PrP isoforms are digested by MC3 but not Properase.

Properase 6H4

Properase PAb2

pH12 Prt PrP

MC3 6H4

MC3 PAb2

pH12 Prt PrP

mc3 digestion at 60 0 c ph 12 reduces infectivity by more than 7 logs
MC3 digestion at 600C pH 12 reduces infectivity by more than 7-logs

1.0E-01

1.0E-03

100

1.0E-05

1.0E-06

1.0E-07

1.0E-08

90

Properase

80

MC3 Infectivity

70

MC3:

66% survival

500 days

60

Percentage survival

50

40

30

20

10

0

100

150

200

250

300

350

400

450

Incubation time (days post inoculation)

validation of inactivation methods raising the standard
Validation of inactivation methods;raising the standard
  • In vitro assessment of PrPSc degradation not enough to validate performance
    • Both Properase and MC3 completely remove all 6H4 immunoreactive material
    • BUT significant difference between levels of observed infectivity
  • Choice of model for bioassay important
      • relevant combination of agent and animal model
      • significant reduction in infectious dose
decontamination of surgical instruments summary
Decontamination of surgical instruments; summary
  • Good progress made towards a practical solution to decontamination of instruments
    • >7-log reduction in infectious dose, in vivo, with BSE-301V
    • Further studies underway
    • Compatible with current disinfection practice without major investment
    • Safe, environmentally friendly process
    • Demonstrates capabilities of protein engineering to address issue
    • Enzyme (MC3) under development/scale up
    • Work ongoing to evaluate MC3 for rendering/bone meal applic.
acknowledgements
Neil Raven

Joanne Dickinson

Anne McLeod

Heather Murdoch

Dawn Taylor

Clive Buswell

Jean Carr

Graham Hall

Mike Dennis

Biological Investigations Group HPA-Porton Down

Mark Sutton

Funding:

EC/DEFRA

Genencor International

Acknowledgements: