Plasma fractionation and viral inactivation/removal procedures

1. Plasma fractionation and viral inactivation/removal procedures Thierry Burnouf, PhD tburnou@attglobal.net

2. Human plasma: a unique & complex raw material

3. Human plasma: a unique & complex raw material +/- 60 g proteins / liter 2 abundant proteins hundreds of other proteins (some present as traces) ~20 medicinal, plasma-derived products

4. Close to 55 out of 60 g have established clinical value

5. Modern Fractionation: interconnection of production lines Produce several products from each pool for optimal use of plasma and selective hemotherapy albumin IgG Clottingfactors Anti-Proteases Anti-coagulants

6. WHO model list of essential medicines Plasma product range Coagulation factors Factor VIII Prothrombin complex Fibrinogen Von Willebrand Factor Factor VII Factor XI Factor XIII Albumin Protease inhibitors Alpha 1-antitrypsin C1-inhibitor Anticoagulants Antithrombin

7. Immunoglobulins WHO model list of essential medicines POLYVALENT Intravenous Intramuscular Sub-cutaneous HYPERIMMUNE Anti-D (Rhesus) Anti-Hepatitis B Anti-tetanus Anti-Rabies Anti-Varicella/Zoster Anti-Cytomegamovirus Anti-hepatitis A

8. Flow-chart of plasma fractionation

9. PREPARATION OF PLASMA RAW MATERIAL Storage of plasma donations [Freezer,  - 25 - 30°C] Preparation of plasma donations for pooling

10. POOLING and • LARGE-SCALE PROCESSING • Opening of bags • Cryoprecipitation (2-4°C) • Bulk fractionation steps • (Ethanol fractionation + chromatography) • Protein purification and viral Inactivation • In-process filtration steps Batch size: 2000-4000L

11. POOLING and • LARGE-SCALE PROCESSING • Opening of bags • Cryoprecipitation (2-4°C) • Bulk fractionation steps • (Ethanol fractionation + chromatography) • Protein purification and viral Inactivation • In-process filtration steps Batch size: 2000-4000L Air-classified environment

12. (Nanofiltration) ASEPTIC DISPENSING Sterile filtration (0.2 m) Aseptic filling +/- Freeze-drying

13. QUARANTINE – QUALITY CONTROL LABELLING – PACKAGING BOXING - SHIPMENT

14. Fractionation methods

15. Integrated plasma protein fractionation process cryoprecipitation Ethanol precipitation Burnouf, T. Transfusion Medicine Reviews, 2007;21:101-117

16. Cryoprecipitation Batch size: 2000-4000L

17. Processing of cryoprecipitate

18. Capture of labile proteins from cryo-poor plasma PCC, PC, PS C1-esterase Antithrombin Ethanol

19. Ethanol precipitations and chromatography

20. Evolving production methods of IVIG to improverecovery Radosevich & Burnouf Vox Sang. 2010:98:12-28 Traditionalmethod

21. Chromatography Protein purification Viral inactivation Removal of viral inactivating agents (solvent/detergent) • Fractionation into several therapeutic protein products • Removal of unwanted proteins (e.g. IgA; FXIa)

22. 1 – 500 liters column Chromatographic methods • Anion-exchange • Cation-exchange • Affinity (e.g. heparin; copper; gelatin) • Immuno-affinity (anti-FVIII; -FIX) • Hydrophobic interaction • Size-exclusion

23. Ethanol fractionation 4000 Liters Stainless-steel tank

24. Protein separation Separation of Precipitates by depth Filtration (or centrifugation) 24

25. Ethanol precipitations Protein purification Viral safety Contributing factor to the removal/inactivation of some viruses • Separate proteins into pre-purified fractions: albumin, IgG, alpha 1-antitrypsin • These fractions can be stored frozen

26. Viral safety keys Donor screening Testing of donations and manufacturing plasma pool Viral reduction treatments GMP 26

27. Viral reduction technologies HIV HBV HCV HAV B19V WNV, DENV, CHIKV, etc. robustness

28. Viral reduction • Two major methods to ensure viral safety • Inactivation = virus destruction/kill by alteration of itscapacity to replicate • Removal = partitioning of viruses and plasma proteins in different fractions

29. Viral reduction treatments • Inactivation = virus destruction by alteration of: • Lipid structure • Proteins (enzymes) • Nucleicacids • Examples: • Chemicals • Heat • Low pH • Irradiation (UV)

30. Viral reduction treatments • Removal= virus partitioning/separationfrom the protein of interest • Dedicated/ on purposetreatment • Nanofiltration • Non dedicated/non specifictreatments, e.g. • Centrifugation • Chromatography

31. Target of viral reductiontreatments • Balanced compromise between the extent of microbial kill and the unwanted side effects on active ingredients of the product: • Coagulation factors • Immunoglobulins • Albumin • Etc.

32. Viral reduction treatments Inactivation Removal Nanofiltration Chromatography Precipitation • Solvent-detergent • Pasteurisation • Low pH • Caprylic acid • Dry-heat treatment Non- Dedicated, Contributing steps

33. Solvent Detergent • Incubation of plasma protein solution in the presence of Tri n-butyl phosphate (TnBP) and detergent(s) [e.g.Tween-80 and/or Triton X-100] • 25 – 35 ˚C (validated) • 1 - 6 hr (validated) • Target: HIV, HBV, HCV, WNV, DENV, CHIKV etc. • Coagulation factors, IVIG, alpha 1-AT, etc.

34. SOLVENT-DETERGENT TREATMENT AT LARGE SCALE Solvent + detergent Up to 1% TnBP Up to 1% detergent (Tween 80, Triton X-100, Triton X-45) 20-35°C 1 to 6 hrs Depending upon validation data Protein solution Mixing device

35. Proteins + SD Elimination of the SD agents Chromatographic column • Hydrophobic interaction chromatography SD are adsorbed proteins

36. Proteins + SD Elimination of the SD agents Chromatographic column • Ion-exchange chromatography Proteins are adsorbed S/D

37. Elimination of the SD agents Oil + SD Proteins + SD + OIL • Oil extraction Mixing and decantation proteins

38. Pasteurisation • Heat-treatment of a protein solution • 60˚C • 10 hr • Protein stabilizers • Target: HIV, HBV, HCV, WNV, • DENV, HAV, B19V • Albumin, FVIII, IVIG, alpha 1-AT • Risk of protein denaturations to be • controlled

39. Low-pH treatment • Treatment in the liquid state: • pH 4.0 • 30-37°C • > 24 hrs • HIV, HBV, HCV, [HAV, B19V] • Only IgG products (historically performed to allow IV infusion)

40. Caprylic acid treatment • Treatment in the liquid state: • < pH 6.0 • 1 hr • 20-25°C • HIV, HBV, HCV, WNV, CHIKV etc. • Only IgG products (also a purification method)

41. Nanofiltration • Filtration on dedicated small pore-size filters (15, 20, or 35 nm, or equivalent) • HIV, HBV, HCV, WNV, DENV, CHIKV, HAV, B19V • Coagulation factors, IgG, AT, alpha 1-AT, etc;

42. PROTEIN VIRUS Removal mechanism Multi-step filtration with multi-layered structure • Product solution passes through capillary-void structure repeatedly. • Product, smaller than the size of capillary, passes through effectively, whereas, viruses relatively larger than the size of capillary, are trapped effectively. Layer1 Layer2 Layer150

44. Dry heattreatment(historical use) HIV inactivation • 60 +/- 0.1°C for 96 hrs • 68 +/- 0.1°C for 96 hrs • 80 +/- 0.1°C for 72 hrs • 100 +/- 0.1°C for 30 min HCV inactivation HAV /B19 inactivation

45. Combination of treatments

46. Combination of treatments Combine treatments with different mechanisms of viral inactivation or removal

47. Each treatment has limits:Viral validations are needed (relevant viruses and model viruses)

48. In vitro validation of viral reduction treatments • Scientific understanding of the capacity of a process to inactivate / remove viruses in a robust and consistent manner • Determination of process robustness • > 4 logs of reduction of infectivity under conditions demonstrated to be not significantly affected by potential process variations (pH, temperature, content of inactivating agents, etc.)

50. “Good implementation viral reduction practices”