1. Robert Somerville, First slide

2. TSE infectivity reduction during gelatine extraction processes Ad Grobben Philip J. Steele David M. Taylor Robert A. Somerville Neuropathogenesis Unit Institute for Animal Health Edinburgh Funded by GME with further support from EU

3. Denaturation Heat (hydrated). Chemical exposure (detergents, chaotropes). Destruction Combustion (incineration). Oxidation (e.g. hypochlorite). Hydrolysis (extreme pH, proteases). Radiation. Mechanisms of TSE Inactivation Treatment Variables: Biological: Agent strain, Host genotype, Tissue, Tissue state. Physicochemical: Heat, pH, Detergents, Chaotropes, Proteases, Radiation. Dynamics and kinetics: Time, Concentration, Temperature.

4. Little effect of time after initial exposure. Biphasic with respect to time. Biphasic with respect to temperature. Inactivation dependent on: Temperature. TSE strain. Hydration status. High pH acts synergistically. Inactivation is not dependent on PrP genotype. Heat Denaturation Original data: Kimberlin, R. H., Walker, C. A., Millson, G. C., Taylor, D. M., Robertson, P. A., Tomlinson, A. H. & Dickinson, A.G.(1983). Journal of Neurological Sciences 59, 355-369. Re-analysis: Somerville, R. (2002). Trends in Biochemical Sciences In press.

5. Little effect of time after initial exposure. Biphasic with respect to time. Biphasic with respect to temperature. Inactivation dependent on: Temperature. TSE strain. Hydration status. High pH acts synergistically. Inactivation is not dependent on PrP genotype. Heat Denaturation Somerville, R. A., Oberthür, R. C., Havekost, U., MacDonald, F., Taylor, D. M. & Dickinson, A. G. (2002). J Biol Chem 277, 11084-11089.

6. Little effect of time after initial exposure. Biphasic with respect to time. Biphasic with respect to temperature. Inactivation dependent on: Temperature. TSE strain. Hydration status. High pH acts synergistically. Inactivation is not dependent on PrP genotype. Heat Denaturation Taylor, D., Fernie, K., Steele, P. J., McConnell, I. & Somerville, R. A. (2002). Thermostability of mouse-passaged Journal of General Virology, 83 3199-3244.

7. Heat Denaturation Little effect of time after initial exposure. Biphasic with respect to time. Biphasic with respect to temperature. Inactivation dependent on: Temperature. TSE strain. Hydration status. High pH acts synergistically. Inactivation is not dependent on PrP genotype. Somerville et al. unpublished data.

8. Known properties Little effect of time after initial exposure Biphasic with respect to time. Biphasic with respect to temperature. Inactivation is dependent on: Temperature. TSE strain. Hydration status. High pH acts synergistically. Inactivation is not dependent on: PrP genotype. Results and Conclusions Thermostability is an intrinsic property of the agent. Kinetic model of TSE agent heat denaturation. Heat may inactivate via dissociation of two components, …...and may also promote a protective reaction. Heat Denaturation Somerville, R. A., Trends in Biochemical Sciences, 27 606-612.

9. Risk reduction steps Sourcing of bones (EU requires that source material is from animals passed fit for human consumption). Butchering practices Pre-cleaning of raw materials (including removal of SRM). Standard gelatine extraction methods Sterilisation: Specific steps which remove TSE infectivity (and other contaminants) Reduction of Risk of TSE infectivity in Gelatine Challenges to removal/destruction of TSE infectivity Very high resistance to inactivation. Resistance increases on drying (etc). Available approaches Removal (filtration) Denaturation (heat, pH). Hydrolysis (very high pH).

10. Risk reduction steps (from EU........)

11. Heat Denaturation Dependent on: Temperature TSE Strain pH Prior treatment of sample (Hydration status) Little effect of time (after initial exposure) pH Hydrolysis Dependent on: pH Temperature (Time) Condition of the sample Removal Degreasing Filtration Ion exchange Removal/Inactivation of TSE infectivity

12. ME7 homogenate (>10 7 ID 50 /g) 4.5% HCl Ca(OH) 2 (sat. soln.) (20, 40 or 60 days) Combined treatment (Inveresk Research International (1998) Validation of the clearance of scrapie from the manufacturing process of gelatin. IRI Report no 14682 and IRI Report no 14683. (unpublished results obtained by courtesy of GME) Inveresk Research data Observations from Previous studies

13. Manske et al, Drugs made in Germany 40, 32-36 (1997) Demonstrated the reduction of nerve-specific proteins under industrial degreasing conditions. Reduction in infectivity titre measured by acid, alkali and combined treatment of ME7 homogenate. Combined treatment is more effective than either single treatment but they are not additive. Time of exposure to Ca(OH) 2 does not affect infectivity titre. Unrepresentative of conditions in actual process. Observations from Previous studies Inveresk data - summary Data which support the contribution to inactivation of individual steps, include; when testing the industrial degreasing process, Manske and others (1997) who found a 98-99% reduction in the amount of brain specific proteins present in the raw materials. Laboratory scale testing of this process showed 300 to 800 fold reductions of these proteins (Grobben unpublished data).

14. GME studies NPU, Edinburgh, Scotland Four Gelatin extraction processes 301V alkaline(NPU 1) 263K alkaline(NPU 2) 301V acid (NPU 3) 301V acid + NaOH(NPU 3b) ID-DLO, Lelystadt, Netherlands: Dutch heat and pressure methods only Rohwer, Baltimore, USA UHT sterilisation experiments

15. Rationale of experimental design TSE source: –used a high titre BSE derived model, thermostable, readily assayed. Total titre of spike exceeds likely BSE contamination event during industrial processing of gelatine. –Short incubation periods, but occasional extended incubation periods after heat treatments. –Limits of detection depend on concentration and toxicity of sample. –Near optimum demonstrable clearance levels from this model. Scaledown to simulate typical Gelatine manufacturing conditions. (Protocol reviewed by International panel prior to initiation). Quality of gelatin checked.

16. Gelatine manufacturing process - lab scale  Raw materials: 1,500g fresh crushed bone. 500g intact calf backbone. Spiked with approx. 10g of TSE-infected brain homogenate. Half spike injected into spinal column, remainder smeared on bones and dried onto surface. Backbone sawn into pieces.  Degreasing: Bone chips washed in water at ~85°C to remove soft tissue and fat. Dried in hot air @ 120°C.  Demineralisation: Bones treated for 96 hours with HCl: 24 hrs with 0.5%, 24 hrs with 2.5% and 48 hours with 4%. Hydrochloric acid (  4%) pumped through vessel containing bone chips. Ossein remains.  Liming: Ossein exposed to saturated Ca(OH) 2 (~pH 12.5), minimum 3 weeks. (alkaline process)Neutralised.  Acid treatment:Ossein left overnight at pH 3, washed with water. (acid process)  NaOH treatment:Ossein exposed to 0.3M NaOH (pH 13) for 2 hours. (acid process)  Extraction:Ossein stirred gently with water at 60, 70, 80, 90°C. to final gelatine concentration of 2-8%.  Purification:Depth filtration, ion-exchange, heat-sterilisation* and drying.  All steps accurately represent the conditions of the industrial process. * Indirect heating in lab, direct steam injection in industrial scale.

17. Gelatine validation studies: spike material Pool Titre ID 50 /g Pool 1, first titration7.7 Pool 1, second titration7.6 Pool 27.8 Titrations of Pool macerates TSE strain: 301V, derived by serial passage of BSE in VM mice. 301V is the most thermostable TSE strain tested so far. All clinically negative animals observed for > 600 days,......then examined for pathological lesions of TSE infection.

18. Fresh crushed bone Degreasing Demineralisation Liming Extraction Filtration Ion-exchange Concentration Drying Gelatine 301V alkaline process (NPU1) 301V spike pool 1 10 7.7 ID 50 /g (Total infective load =10 8.7 ) DCP effluent 658 dpi 10-1 0/9  10 2.3 ID 50 /g 10-2 2/8 (217) 10-3 0/10 Extract sample 666 dpi neat 10/18 (283)  10 1.8 ID 50 /ml 10-1 1/14 (378) 10-2 0/17 Total recovery  10 5.0 Sterilised sample 604 dpi 10-1 0/18  10 1.3 ID 50 /ml 10-2 0/17 10-3 0/18 Total recovery  10 3.8 Liming Neutralisation Sterilisation

19. 263K alkaline process (NPU 2) Fresh crushed bone Degreasing Demineralisation Extraction Gelatine solution 263K spike pool 519dpi 10 8.0 ID 50 /g (Total infective load =10 9.0 ) DCP effluent 712dpi 10-1 2/3(268)  10 2.7 ID 50 /g 10-2 2/9 (347) 10-3 0/11 Extract sample 644 dpi neat 2/9 (221)  10 1.1 ID 50 /g 10-1 1/9 (199) 10-2 0/9 Total recovery  10 4.3 Liming Neutralisation

20. 301V acid process (NPU 3) Fresh crushed bone Degreasing Demineralisation Extraction Filtration Ion-exchange Concentration Drying Gelatine 301V spike pool 2 10 7.8 ID 50 /g (Total infective load =10 8.8 ) Extract sample 658 dpi neat 15/15 (193) 10 2.8 ID 50 /g 10-1 10/17 (213) 10-2 0/18 Total recovery 10 6.2 Sterilised sample 604dpi 10-1 0/16  10 1.3 ID 50 /g 10-2 0/17 10-3 0/17 Total recovery  10 4.0 Acid treatment Sterilisation

21. Fresh crushed bone Degreasing Demineralisation Extraction Gelatine solution 301V spike pool 1 10 7.7 ID 50 /g (Total infective load =10 8.7 ) Extract sample 666dpi neat 0/16  10 0.3 ID 50 /g 10-1 0/17 10-2 0/15 Total recovery  10 3.3 301V acid process + NaOH treatment (NPU 3b) Acid treatment NaOH treatment

22. Fresh crushed bones Degreasing Demineralisation Liming Neutralisation Extraction Gelatine Solution Filtration Concentration Sterilisation Drying Gelatine Ion Exchange Spike DCP Crude Gelatine extract Purified Gelatine 8.7  5.0  3.8 (no +ves) NPU 1: 301V Alkaline process NPU 2: 263K Alkaline process Fresh crushed bones Degreasing Demineralisation Liming Neutralisation Extraction Gelatine Solution Spike DCP Crude Gelatine extract 9.0  4.3 Fresh crushed bones Degreasing Demineralisation Acid treatment Extraction Gelatine Solution Filtration Concentration Sterilisation Drying Gelatine Ion Exchange Spike DCP Crude Gelatine extract Purified Gelatine 8.8 6.2  4.0 (no +ves) NPU 3: 301V Acid process Fresh crushed bones Degreasing Demineralisation Acid treatment NaOH treatment Extraction Gelatine Solution Spike DCP Crude Gelatine extract 8.7  3.3 (no +ves) NPU 3b: 301V Acid process

23. Results - summary

24. Summary of results Acid bone process: Substantial removal of infectivity measured before purification (NPU3) but complete clearance after purification including sterilization (NPU3). Complete clearance (no infectivity detected) before purification if additional NaOH-treatment is included (NPU3b). Limed bone (alkaline) process: Greater removal of infectivity than after equivalent acid hydrolysis procedure (NPU1 & 2). Complete clearance (no infectivity detected) after purification including sterilization (NPU1).

25. Conclusions The gelatin manufacturing procedure was successfully scaled down: normal bone gelatin was produced. Both, degreasing and the standard acid and alkaline treatments alone removed most, but not all of the applied infectivity before final purification of gelatine. Lime (alkaline) procedure was more effective. Additional NaOH step in acid procedure inactivated residual detectable infectivity before purification steps. After purification all samples did not show any detectable infectivity. Removal of infectivity is cumulative but not necessarily additive.

26. Fresh crushed bones Degreasing Pre-heating 3 bar/20min/133°C Extraction Gelatine Solution Filtration Concentration Sterilisation Drying Gelatine Ion Exchange Spike Crude Gelatine extract Purified Gelatine 9.2  0.2 No +ves Not tested ID-DLO 1: 301V Heat-pressure process Clearance factor > 6.8

27. Risk reduction and Gelatin Sourcing - use only animals passed fit for human consumption. Omission of head bones and vertebrae from source material in BSE countries. Removal/inactivation of TSE infectivity during gelatin extraction and purification. Species barrier reduces effective titre. Intracerebral route is most efficient route of infection, oral routes are less efficient.

28. Risk reduction steps (from EU - SSC)

29. Baltimore Study Data and Comments to be agreed with Bob Rohwer

30. The End