1. Plasma fractionation and viral inactivation/removal procedures
Thierry Burnouf, PhD

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. established clinical value
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
Clotting factors
Anti-Proteases
Anti-coagulants

6. 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
WHO model list
of essential medicines

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

8. Flow-chart of plasma fractionation

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

10. LARGE-SCALE PROCESSING
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: L

11. Air-classified environment
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: L
Air-classified environment

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

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

14. Fractionation methods
Step
Protein target
Cryoprecipitation
Factor VIII, vWF, Fibrinogen
Chromatography
Coagulation factors, Protease inhibitors
Anticoagulants
Ethanol fractionation
Albumin, IgG, alpha 1-AT

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

16. Cryoprecipitation
Batch size: L

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 improve recovery
Radosevich & Burnouf
Vox Sang. 2010:98:12-28
Traditional method

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

22. Chromatographic methods
1 – 500 liters column
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 24

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

26. Viral safety keys Donor screening
Testing of donations and manufacturing plasma pool
Viral reduction treatments
GMP 26 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 its capacity 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)
Nucleic acids
Examples:
Chemicals
Heat
Low pH
Irradiation (UV)

30. Viral reduction treatments
Removal = virus partitioning/separation from the protein of interest
Dedicated/ on purpose treatment
Nanofiltration
Non dedicated/non specific treatments, e.g.
Centrifugation
Chromatography

31. Target of viral reduction treatments
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
Solvent-detergent
Pasteurisation
Low pH
Caprylic acid
Dry-heat treatment
Nanofiltration
Chromatography
Precipitation
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
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. Elimination of the SD agents
Proteins + SD
Hydrophobic interaction chromatography
Chromatographic
column
SD are
adsorbed
proteins

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

37. Elimination of the SD agents
Oil extraction
Oil + SD
Proteins + SD +
OIL
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. 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.
Layer Layer Layer150
VIRUS
PROTEIN

44. Dry heat treatment (historical use)
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
HIV inactivation
HCV inactivation
HAV /B19 inactivation

45. Combination of treatmentsSD
Pasteurisation
Acid pH
Nanofiltration
Dry-heat
F VIII or FIX X
F VIII
vWF AT
IgG

46. Combination of treatmentsSD
Pasteurisation
Acid pH
Nanofiltration
Dry-heat
F VIII or FIX X
F VIII
vWF AT
IgG
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”

51. Product batch release specifications
Potency / Specific activity
Residual “contaminant” proteins
Specialized assays [e.g. thrombogenicity]
Ingredients [stabilizers]
Residual content in virus sterilizing agents
Physicochemical tests
Sterility
Pyrogen / endotoxin tests
Toxicity assays
Quality and safety of each batch is intimately dependent
upon process validation and process monitoring through GMP

52. Thank you for your attention