1. PLASMA FRACTIONATION
Presenter:-Dr. Nippun Prinja, Senior Resident
Moderator:-Dr. Hari Krishan Dhawan, Assistant Professor
DEPARTMENT OF TRANSFUSION MEDICINE,PGIMER CHANDIGARH
3. INTRODUCTION
• Plasma is the liquid component of whole blood and makes up 50%-55% of the
total blood volume
• Fractionation is the process by which plasma is separated into individual protein
fractions, that are further purified for medicinal use.
• The scientific development of this field began in the laboratories of Edwin Cohn
at Harvard University in the 1930s
• With World war-2 on the horizon, the US Navy took a lead in organizing studies
and supporting Cohn’s work and partnerships with industry were developed.
Thus began the field of plasma fractionation, with its first phase dominated by
albumin
Reference:-Rossi
4. EVOLUTION
S.No. Phase Duration Main contributor Dominated by
1 Phase 1 1930-1970 Edwin Cohn Albumin
2 Phase 2 1970-1990 Judith Poole AHF (Cryoprecipitate)
3 Phase 3 1990-till date ------- IvIg
Edwin Cohn
(17/12/1892 TO 1/10/1953)
Edwin J. Cohn and his collaborators during World
War II developed the cold ethanol method of
plasma fractionation
6. Plasma derived product Major use
Albumin Burns, shock
Intravenous Immunoglobulin (IVIg) Immunodeficiency
Intramuscular Immunoglobulin Prevention of infections
Rh(D) immunoglobulin Rh negative pregnancy
Factor VIII Haemophilia A; von Willebrand’s disease
Factor IX Haemophilia B
Prothrombin complex concentrate Multiple factor deficiency
Clinical use
Ref:-ROSSI
9. INDIAN SCENARIO Ref: WHO plasma fractionation
• 18% of the world’s population
• 1.3 % of the world’s total plasma collection
• 65% of plasma unused
• 1988 – Hemophilia tragedy / HIV testing made mandatory
• 1989 – National Plasma Fractionation Centre (NPFC), Mumbai
• 1989 : Indian Government suspended manufacturing licenses
• 2005- recovered plasma collection permission
• 2006 – contract plasma fractionation started
• INTAS,RELIANCE,HEMARUS
10. SOURCES OF PLASMA
• Methods used to obtain plasma for fractionation:
1. Recovered plasma : Plasma recovered by centrifugal separation from the cells
and cells in whole blood
2. Apheresis plasma for source plasma/ serial plasma : Plasma is exclusively
collected for fractionation into plasma components. This plasma is obtained by
apheresis in which plasma is separated and the formed elements are returned
to the donor.
Ref: WHO plasma fractionation
11. Characteristic Recovered plasma Apheresis (Source) Plasma
Definition Plasma obtained after processing
of whole blood into red cell &
plasma components
Plasma obtained by apheresis after all red
cells and almost all WBC are returned to
donor .
This plasma is intended for further
manufacture into injectible or diagnostic
products
No. of donations/yr by single
donor
4 times (India)
6 times (USA)
24 times (INDIA)
Volume/yr by single donor 1-1.5 L 90 L (USA) ,25 L (EUROPE)
Volume / single donation 100-260 ml 450-880 ml
Factor VIII Concentration 66-84.5 U/dl 100 U /dl
IgG Concentration 9.4 g /dl 8.2 g/dl
Immediate decrease in pH Seen Not seen (due to continuous mixing & lower
concentration of anticoagulant)
Plasma to citrate ratio 1:7 1:16
Release of proteolytic enzymes More Less
Protein content, g/l (each
donation)
≥ 50
(but greater than source plasma)
≥ 50 Ref: WHO plasma fractionation
12. Donor Requirements
• Informed consent,Medical history & physical examination
• serologic testing for TTI
• At the time of first procedure and at 4-month interval thereafter
– A total plasma or serum protein determination
– Serum protein electrophoresis
– Quantitative immuno-diffusion test to determine immunoglobulin composition
– Total protein level > 6g/dl
• At least 48hrs between procedures,Not >2 procedures within 7 days
• Red cell loss <200ml Ref:AABB MANUAL
13. MEASURES TO EXCLUDE INFECTIOUS DONATIONS
The safety and quality of plasma for fractionation results from the combination of
several cumulative prevention measures
1. Appropriate selection of blood/plasma donors
2. Testing of blood/plasma donations
3. Epidemiological surveillance of the donor population
4. Strict adherence to Good Manufacturing Practices (GMP)
5. Post-donation information system
14. MANUFACTURING STEPS
I . Plasma Collection Process:
•Recovered Plasma
•Apheresis Plasma
II. Shipping Process :
•Plasma is stored between -20 C to – 30 C after completion of all testing &
verification of all information. This is standard starting material for fractionation
•Frozen plasma reduces bacterial growth
•Plasma is held for 60 days after collection, then process begins under aseptic
condition in a manufacturing plant
II. Manufacturing process :
•The plasma is expelled from container & thawed at 4 C to produce cryo.
•Typical batches are 2000 to 4000 L Ref:-ROSSI
17. Overview of fractionation process
Reception of plasma shipped by blood establishment
Check for control of shipping parameters
Quarantine of plasma in cold room <-200
C or colder
Control of documentation
Preparation of plasma batch
Conditioning of plasma packs
Pooling and large scale processing
Opening of plasma packs
Cryoprecipitation
Bulk fractionation steps
Preparation of intermediate fractions
Downstream processing and viral reduction steps
18. Aseptic dispensing
Filling in vials/bottles
+/- freeze drying
Quarantine – quality control
Check of production batch records and documentation
Labeling – packaging
Boxing – shipment
Reference:-Rossi
19. CATEGORIES OF PLASMA FOR FRACTIONATION
• Plasma frozen within 24 hours of collection
• Plasma frozen after 24 hours of collection
• Plasma not meeting the requirement for fractionation
• Hyper-immune (antibody-specific) plasma
Ref: WHO plasma fractionation
20. CHARACTERISCTICS OF PLASMA FOR FRACTIONATION
Plasma frozen within 24 hours of collection (or apheresis) at -20C or -30C
•Optimal recovery of
labile factors (factor VIII & other coagulation factors and inhibitors)
stable plasma proteins (Albumin & Immunoglobulin)
is possible .
Plasma frozen after 24 hours of collection
Suitable for the manufacture of
stable plasma proteins
but not coagulation factors
Ref: WHO plasma fractionation
21. CHARACTERISCTICS OF PLASMA FOR FRACTIONATION
Plasma not meeting the requirement for fractionation
1.Plasma obtained by TPE (as there is enhanced risk of transmitting blood borne
disease & high risk of irregular antibody )
2.Plasma from autologous blood donation (It may have higher prevalence of viral
marker)
3.Plasma not frozen within 72 hours of collection or separation .
Ref: WHO plasma fractionation
22. CHARACTERISCTICS OF PLASMA FOR FRACTIONATION
Hyper-immune (antibody-specific) plasma :
The approaches for preparation of plasma for manufacture of specific Igs
(antibody-specific immunoglobulins) are:
1.Individuals selected from the normal population by screening of plasma units for
antibody titres. (Screening may be random or by knowledge of history of recovery
from an infectious disease like varicella)
2.Individuals with a high titre of a specific antibody resulting from prophylactic
immunization.
3.Volunteers recruited to a panel for a targeted immunization programme.
Clinically relevant antibody specific immunoglobulins include anti-D, anti-HAV, anti-
HBs, anti-tetanus, anti-varicella/herpes zoster and anti-rabies Igs
Ref: WHO plasma fractionation
24. PRINCIPLES OF PLASMA FRACTIONATION
Methods of plasma fractionation are based on:
1.Differential solubility of proteins
2.Differential interaction of protein with solid media
3.Differential Interection with physical field
Solubility of protein is determined by :
1.Charge
2.Isoelectric point
3.Size
4.Composition
5.Conformation
6.Other physical and Chemical properties Ref:-ROSSI
29. COLD ETHANOL/ COHN METHOD
• Edwin Cohn developed cold ethanol precipitation method (later modified by
Onclay & Kistler & Nitschman) ---induces changes in protein solubility to isolate
various fractions.
• Successive processing steps are carried out at specific ethanol concentration &
pH, temperature & osmolality.
• This results in selective precipitation of proteins which are then separated by
centrifugation &/ or filtration.
• Different protein fraction are obtained as ethanol concentration or pH is
changed.
• Ethanol is removed by freeze drying or ultrafiltration.
Ref:-ROSSI
30. COHN METHOD USES FIVE VARIABLES
S.No. Variables Stength
1 Ethanol concentration 8-40 %
2 pH level 4.5-7.4
3 Temperature -5 0
to -70
C
4 Ionic strength differential 0.14 to 0.01
6. Protein concentration 5.1 to 0.8%
Ref:-ROSSI
33. CHROMATOGRAPHIC (C) TECHNIQUE
• It was introduced in 1960.
USE :
1. To isolate Factor VIII from cryoprecipitate
2. To increase IgG recovery
3. To remove virus
4. To produce anti-D
It is capable of improving purity, extracting trace labile proteins, optimizing
proteins recovery & removing virus inactivation agents
• Anion exchange C, Affinity C, Immunoaffinity C are types of C.
34. Parameter Cold ethanol / Cohn method Chromatographic technique
Advantage - It facilitates large scale
production in batches
-Ethanol is readily removed
-Bacterial growth is inhibited
throughout the process
- Materials are inexpensive
Sensitivity & high yield
Disadvantag
e
Unsatisfactory for recovery &
maintenance of function of
some proteins
-Requirement for larger processing
volume
-Potential for bacterial growth
during processing
-Inability to handle high lipid
content (degrading of columns)
- Lost ligand from the columns
(leaching)
37. Available viral reduction treatments
Non specific methods Specific methods Newer methods
•Physical methods
Pasteurization
Dry heat
Other heat treatment
•Chemical inactivation
Solvent detergent
Acid ph incubation
•Neutralization by Ab
•Physiochemical methods
β propiolactone with UV
(Cold Sterilization)
β propiolactone with no UV
•Fractionation by ethanol
•Other precipitation
•Chromatography
•Nanofiltration
41. PLASMA FRACTIONATION – INDIA – Historical perspective
• BEFORE 1988
1. Small plants – Albumin and IVIG – Poor quality of factors
2. Mainly imported
• 1988 -2000
1. Professional donors banned
2. Component therapy started
3. NPFC, Mumbai
• POST 2000
1. Transfusion Medicine department – modernized – More plasma generated
2. Automation - Better IDM testing
3. Private sector initiative
42. PLASMA FRACTIONATION - INDIA
• Plasma Fractionation Centre, Chennai – First project in public sector - not
functional
• National Plasma Fractionation Centre (NPFC) has proposed to set up a
fractionation centre at Shatabdi Hospital, Govandi, Mumbai. The project is
underway and is expected to be completed in a few years. The opening of this
centre is expected to bring down the costs of plasma derivates.
• India’s first plasma fractionation facility opened up in Ahmedabad as a
collaboration between Celestial Biologicals (CBL-INTAS biopharmaceuticals) and
GE Healthcare.
• Dhirubhai Ambani Life Sciences Centre, Navi Mumbai
43. International Plasma Fractionation Association(IPFA)
• MISSION : Bridge the interest of Donors ,Collection centers,
fractionation centers and patients.
• It promotes the interests and activities of its member
organizations and countries who made a commitment to
create a safe and secure supply of blood and plasma products
based on voluntary non remunerated blood donations and not
for profit business model.
Ref:IPFA official website
47. Plasma to be collected from a licensed blood bank and manufactured in a separate premises
other than blood bank
Form 28-E & 27-E for obtaining license mentioned in part XII C of DCA
A. General requirements
Air supply through HEPA filters
Positive air pressure area
B. Collection and storage
C. Personnel
Manufacture
One year practical experience of blood component/fractionation with
MD in (microbiology/pathology/biochemistry/immunology/bacteriology) or
PG in science / pharmacology
Testing
Eighteen months practical experience in the testing of drugs, especially the blood
MD in (microbiology/pathology/biochemistry) or
PG in (science / pharmacology )
D. Production control
The production area & viral inactivation room grade C-HEPA Filters
The filling and sealing area grade-A, HEPA Filters
48.
MAXIMUM NUMBER OF PARTICLES
PERMITTED PER m3
MAXIMUM NUMBER OF
VIABLE MICROORGANISM
PERMITTED PER m3
GRADE 0.5 -5 micron Less than 5 micron
A (Class 100)
(Laminar- Airflow workstation)
3500 None Less than 1
B (Class 100) 3500 None Less than 5
C (Class 10000) 350000 2000 Less Than 100
Air classification system for manufacturing of sterile products
