multifaceted lecture, good for PG students as well as for guidance latest data incorporated.

1. COHN’S FRACTIONATION
DR AKSHAYA TOMAR
DEPARTMENT OF IMMUNOHEMATOLOGYAND
BLOOD TRANSFUSION
AFMC,PUNE

2. INTRODUCTION
• The procedure that continues to have a pivotal role in
plasma fractionation was developed by Cohn in 1940s.
• In terms of global demands , the volume of plasma
fractionated has been steadily increasing in the last few
years.
• The developing world uses a small portion of the
fractionated plasma products as a result of product
shortages and lack of purchasing power for these
sophisticated medicinal products

3. INTRODUCTION
• In India number of units of whole blood collected is
approx 5-10units/1000 people
• WHO estimated that about 9.3 million liters of recovered
plasma is wasted or discarded yearly worldwide.
• Currently therapeutically important proteins are:
- Human serum albumin
- Igs
- Coagulation factor VIII
- Protease inhibitors

4. INTRODUCTION
• Human serum albumin is one of the most abundant
proteins in plasma and together with Igs constitutes 80%
of all plasma proteins
• The average HSA consumption in the developed world is
200-400 kg per million population and IVIG use was at
20-40 kg IgG per million
• The volume of plasma fractionated has been steadily
increasing in the last few years, and has now reached
close to 35 million liters worldwide, compared to
approximately 25 million liters some 10 years ago

5. INTRODUCTION
• Cohn’s method for producing albumin achieved 2
outcomes:-
- It allowed the industrial scale manufacture of albumin
- It generated a series of fractions that immediately aroused
therapeutic interest

6. INTRODUCTION
• Plasma fractionation is unique in a way that it requires
integrated downstream purification and viral reduction
processes.
• Extraction processes should be mild to avoid the risks of
alterations that could lead to protein antigenicity or
activation that results in
- Hypotension
- Fever
- Thromboembolic events

7. Emerging Viral Threats
• West nile virus
• Dengue virus
• Chikungunya virus
• SARS virus
• Hepatitis E virus
It seems scientifically reasonable to conclude that potential
residual infectious risks for most plasma products would
be restricted to emerging resistant small (<15-20 nm)
non-enveloped viruses

8. Scope of Plasma Fractionation

9. Scope of Plasma Fractionation

10. History
• Cohn’s developed stable plasma protein solution for the
treatment of battlefield injuries in the 2nd World War.
• Resulting albumin product remained the mainstay of the
plasma fractionation

11. EVOLUTION OF PLASMA
FRACTIONATION

13. Safety issues
• Until the late 1970s safety was not a major concern for
plasma protein therapies
• Albumin and Ig were associated with hepatitis but it was
obviated by heating the product at 60°C for 10 hrs
• But hemophiliacs who are on continuous replacement
therapy receiving less purified fractions were carrying
major risk of transmission
• This leads to introduction of NAT for Plasma fractions

14. The Cohn Process
• It is based on the differential precipitation of plasma
proteins by manipulation of
- ethanol concentration
- pH of a Low ionic strength concentration
- maintained at subzero temperature
• Each Cohn fraction is an enriched , albeit crude , source
of various plasma protein need further purification to
generate a therapeutic product.

16. Variation to Cohn’s Process
• Kistler and Nitschmann in 1962
• To maximize Albumin yield and decrease ethanol use
• This method is adopted by CSL Behring

17. Ethanol 19%
pH 5.8
Ethanol 40%
pH 5.8
Temp -8
Ethanol 40%
pH 4.8
Temp -8
Ethanol 10%
pH 4.6
Temp -3

18. Viral removal procedures
• It is achieved by precipitation of fractions
• Regulatory requirements however require inclusion of at
least 2 dedicated orthogonal viral removal procedure
• Viral Filters: -15-20nm pore size , thereby ensuring the
removal of small non-enveloped viruses such as
Parvovirus B19
- 35nm pore size, removing large enveloped viruses
such as hepatitis B and C and HIV

19. Viral removal procedures(Other)
• Solvent Detergent Method – For enveloped viruses
• Nanofilteration – for Non enveloped viruses as well as
Prions
• Pasteurization – Heating at 60°C for 10 hours
• Caprylic acid treatment – Incubation at low pH at 30°C
for 10 hours

20. Chromatographic Procedures
• Cohn fractionation generate highly purified albumin and
immunoglobulin products
• Chromatographic techniques allow purification of
proteins because of their differences in molecular weight
, charge , hydrophobicity and specific affinity for ligands
(eg: diethylaminoethyl, quarternary amino ethyl)
• This involves the use of size exclusion, ion exchange,
hydrophobic interaction and affinity chromatography
respectively

21. Chromatographic Procedures
• Ion exchange chromatographic resins exist in either the
anion or cation forms
• Net charge of a protein depends on pH and this
determines the degree of interaction with an ion
exchange resin
• Chromatographically purified Albumin is of green color

22. Chromatographic Procedures
• Interaction can be further modulated by changes in ionic
strength
• Thus by utilizing conditions that promote differential
binding to ion exchange resins , coupled with defined
elution conditions fractionation of a mixture of proteins
can be achieved

23. Chromatographic Procedures
• Proteins exhibit differences in their hydrophobic profile
• This difference is exploited in hydrophobic interaction
chromatography (HIC)
• Solution conditions can be manipulated such that more
hydrophobic proteins are retained on a column while less
hydrophobic proteins flow through

24. Advantages of Cohn’s Fractionation
• Ethanol is cheap and easily available
• Accepted, processes and products are registered
• Process under bacteriostatic conditions
• Products may be regarded as safe in therapeutic use
• Suited to large-scale production

25. Disadvantages of Cohn’s Fractionation
• Cold areas or refrigerated tanks required
• Protein losses occur in occluded liquor
• Not suited to small scale production
• Requires high quality starting material

26. PRODUCTS WITH ESTABLISHED
CLINICAL USE
• Albumin
• Immunoglobulins
• vWF
• Factor IX , X , XIII
• Prothrombin complex concentrates(PCC)
• Fibrinogen
• α1 Proteinase inhibitor
• C1 esterase inhibitor

27. Plasma Fractionation in India

28. Plasma Fractionation in India

29. CONCLUSION
• In medium income countries like ours, RBCs need will
surpass that of FFP and Cryoprecipitate by 2025
• This will generate lot of recovered plasma but due to
underutilization it will get wasted
• One major issue is that currently collected plasma in low-
and medium-income countries typically does not meet
specifications for fractionation and is therefore discarded

30. CONCLUSION
• Contract Fractionation : A pragmatic way to go
it can overcome
- Technological challenges
- Economic considerations
• It can be utilized for interim period of 5 years by
domestic fractionation facility
• Minimum Plasma required is 10,000L / annum but most
fractionators prefers at least 30,000 to 50,000L/year (due
to logistics and licensing issues)

31. CONCLUSION
• It is important that both plasma collection and
fractionation are performed following standards
established at a global level
• It is likely that the future will be as eventful as the period
from Cohn to the present day

32. THANK YOU