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

 Albumin
IgG
Plasma IgG
Fibrinogen
Fibrinogen Alpha 2 Macro
Alpha 1 AT
Others
Fibronectin
Alpha 1 AT
Antithrombin
Plasminogen
C1-inhibitor
Prothrombin
vWF
FXI
FVIII
FIX
PC
Albumin
FVII
Close to 55 out of 60 g have FVIII
established clinical value
 Modern Fractionation:
interconnection of production lines
Produce several products from each pool
for optimal use of plasma and selective hemotherapy
 Plasma product range
• Coagulation factors
• Factor VIII WHO model list
• Prothrombin complex of essential medicines
• Fibrinogen
• Von Willebrand Factor
• Factor VII
• Factor XI
• Factor XIII
• Albumin
• Protease inhibitors
• Alpha 1-antitrypsin
• C1-inhibitor
• Anticoagulants
• Antithrombin
 Immunoglobulins
• POLYVALENT
• Intravenous
• Intramuscular
• Sub-cutaneous
WHO model list
of essential medicines
• HYPERIMMUNE
• Anti-D (Rhesus)

• Anti-Hepatitis B
• Anti-tetanus
• Anti-Rabies
• Anti-Varicella/Zoster
• Anti-Cytomegamovirus
• Anti-hepatitis A
Flow-chart of plasma
fractionation
PREPARATION OF PLASMA
RAW MATERIAL

Storage of plasma
donations

[Freezer,  - 25 - 30°C]

Preparation of plasma
donations for pooling
 POOLING and
LARGE-SCALE PROCESSING
Batch size: 2000-4000L

Opening of bags
• Cryoprecipitation (2-4°C)

Bulk fractionation steps

(Ethanol fractionation +
chromatography)

Protein purification and viral

Inactivation

In-process filtration steps

 POOLING and
LARGE-SCALE PROCESSING
Batch size: 2000-4000L

Opening of bags
• Cryoprecipitation (2-4°C)

Bulk fractionation steps

(Ethanol fractionation + e nt
onm
chromatography)
vi r
e n
i fi ed
s s
Protein lpurification
a and viral
i r-c
A Inactivation
In-process filtration steps
 (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
 Integrated plasma protein
cryoprecipitation fractionation
process

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 • Removal of viral

several therapeutic inactivating agents
protein products (solvent/detergent)
• Removal of unwanted
proteins (e.g. IgA; FXIa)
 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
 Ethanol precipitations
Protein purification Viral safety

• Separate proteins into pre- • Contributing factor to

purified fractions: albumin, the removal/inactivation
IgG, alpha 1-antitrypsin of some viruses
• These fractions can be
stored frozen
 Viral safety keys

1. Donor screening

2. Testing of donations and

manufacturing plasma pool

3. Viral reduction treatments

4. GMP
26
Viral reduction technologies

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

• Two major methods to ensure viral safety

1. Inactivation = virus destruction/kill by

alteration of its capacity to replicate

2. Removal = partitioning of viruses and

plasma proteins in different fractions

28
Viral reduction treatments
• Inactivation = virus destruction by alteration of:
– Lipid structure
– Proteins (enzymes)
– Nucleic acids

• Examples:
– Chemicals
– Heat
– Low pH
– Irradiation (UV)
29
Viral reduction treatments
• Removal = virus partitioning/separation from
the protein of interest

1. Dedicated/ on purpose treatment

• Nanofiltration
2. Non dedicated/non specific treatments,
e.g.
• Centrifugation
• Chromatography

30
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.

31
 Viral reduction treatments
Inactivation Removal

• Solvent-detergent • Nanofiltration

• Pasteurisation

• Low pH
• Chromatography Non-
• Caprylic acid Dedicated,
Contributing
• Precipitation steps
• Dry-heat treatment
 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

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
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

PROTEIN
VIRUS
42
43
 Dry heat treatment
(historical use)

60 +/- 0.1°C for 96 hrs

HIV inactivation
68 +/- 0.1°C for 96 hrs

80 +/- 0.1°C for 72 hrs HCV inactivation

100 +/- 0.1°C for 30 min HAV /B19 inactivation

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

IgG X X X

IgG X X
 Combination of treatments
SD Pasteurisation Acid Nanofiltration Dry-heat
pH
F VIII or X X
FIX
F VIII X X
Combine treatments with different mechanisms
vWF X of viral inactivation or removal
X X
AT X X

IgG X X X

IgG X X
 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