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

Human plasma: a unique & complex raw material

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

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

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

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

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

Flow-chart of plasma fractionation

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

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: 2000-4000L

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: 2000-4000L Air-classified environment

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

QUARANTINE – QUALITY CONTROL LABELLING – PACKAGING BOXING - SHIPMENT

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

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

Cryoprecipitation Batch size: 2000-4000L

Processing of cryoprecipitate

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

Ethanol precipitations and chromatography

Evolving production methods of IVIG to improve recovery Radosevich & Burnouf Vox Sang. 2010:98:12-28 Traditional method

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)

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

Ethanol fractionation 4000 Liters Stainless-steel tank

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

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

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

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

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

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

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

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.

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

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.

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

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

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

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

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

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)

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)

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;

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 VIRUS PROTEIN

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

Combination of treatments SD Pasteurisation Acid pH Nanofiltration Dry-heat F VIII or FIX X F VIII vWF AT IgG

Combination of treatments SD 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

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

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.)

“Good implementation viral reduction practices”

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

Thank you for your attention