For more details on the Cohn Alcohol Fractionation Procedure with respect to purifying immunoglobulin (IVIG)  see “alcohol precipitation” right hand panel.

Common plasma fractionation processes are the Cohn fractionation method (Cohn, 1946, J Am Chem Soc. 68: 459) and its modification (e.g., Kistler and Nitschmann, 1962, Vox Sang, 7: 414-2424). This process begins with cryoprecipitation to remove some of the coagulation factors. The resultant cyroprecipitate-depleted plasma pool is treated to precipitate IgG fraction (Fraction 1 accordingly to the Kistler and Nitschmanna method at 19% ETOH, pH 5.85 and -5C or the equivlaent Fraction II+III according to the Cohn method at 25% ETOH, pH 6.9 and -5C). The remaining impurities are removed by precipitation of Fraction IV at 40% ETOH, pH 5.85 and -5C, according to the Kistler and Nitschman method or a two step process accoding to Cohn (Fr IV-1 at 18% ETOH, pH 5.2, -5C, followed by Fr IV-4 at 40% ETOH, pH 5.8, -5C). Lowering the pH of the fraction IV supernatant to 4.8 and then dropping the temperature from -5C to -10C while maintaining the ETOH concentration at 50% causes the rpeciptiation of Cohn fraciton V. 

Fractionation Procedure for Plasma Proteins

1. collection of blood and separation of plasma: The preparation of the concentrates bigins with the combining or “pooling” of individual plasma donations. Following the collection, the blood is centrifuged to cause the blood cells to settle and separate from the plasma. Interactions between blood cells and plasma proteins are a potential source of trouble during the fractionation of blood. The plasma should thus be separated form the cell as soon as possible. Optionally, platelets may be harvested at this stage by a second centrifugation. The plasma is then frozen, either by refrigeration or by immersion in a mixture of dry ice and ethanol or a similar solvent. After this, the frozen plasma is thawed and then centrifuged once again to settle solid material in the cold thawed plasma. This solid material is known as a cryoprecipitate which contains the coagulation factors FVIII and von Willebrand factor in enriched form but may also serve as a source for the recovery of fibrinogen and other plasma proteins, such as fibronectin.(Kumpalume, US 2009/0292114). As a result of the slow, controlled de-freezing procedure, a cryoprecipitate is formed comprising Factor VIII (complexed with the von willebrand factor, vWF) and fibrinogen and a supernatant comprising the builk of the proteins remains in the plasma. The cryoprecipitate fraction may be resolubilised to form a protein solution and the proteins may be isolated by contacting it with an adsorbent, In particular, Factor VIII, vWF and fibrinogen may be isolated from the resolubilised fraction. (called cryo-poor plasma). Lihme (US2007/0299251)

Subjecting plasma to controlled thawing at 2-3C is known as cryo-precipitation. The “cryosupernatant” (or “cryo-poor palsma”) may uundergo chromatographic adsorption to isolate the proteins of the prothrombin complex, antithrombin or C1-inhibitor. (Burnouf, “Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sanguinis (2010) 98, 12-28. 

2. Fraction I precipitation (Removal of fibrionogen): The supernatant from (1) above, cryo-poor plasma may be supplemented with alchol and fraction I (a precipitate) and supernatant I are formed. Lihme (US2007/0299251)

The first precipitation step, referred to as Fraction I precipitation, is performed at high pH (7.2) and low ethanol concentration 8-10% w/v) to precipitate protein such as fibrinogen and Factor XIII away from IgG and A1PI, which remain in the supernatant.  (Bruckschwaiger US 13776448) 

After the conventional centrifugation, the plasma, which is now cryoprecipitate-poor is separated leaving behind the Factor VIII rich cryoprecipitate. The Factor VIII rich cryopricipitate is the source of an important therapeutic agent for hemophilia (i.e., AHF) and that cryoprecipitate cryo-poor plasma is also used for the preparation of the other concentrates. 

Radowitz (US4216205) dicloses removal of  fibrionogen from plasma by adding cold ethanol up to a concentration of 8%, at temperature maintained at form -2.5 to -3.0 degrees C. The precipitate (fibrinogen) is removed by centrifuging and the supernatant further processed.

3. Fraction II/III (gamma globulin fraction): Supernatant I may be supplmented with more alchol and precipitated fraction II+III and supernatant II+III are foremd. Freaction II+III may be separated and resolubilised to form a protein solution and proteins of interest may be isolated by contacting it with an adsorbent. Proteins such as immunglobuilins (subh as IgG) may be isolated form the protein solution obtained from fraction II+III. Supernatant II+III will mianly compirse albuman and alpha-1-proteinase inhibitor. Lihme (US2007/0299251)

IgG is next precipitated from the Fraction I supernatant above in a second precipitation reaction, refrrered to as a Fraction II+III precipitation, performed at moderate pH (6.8) and high ethanol concentration (20-25%). The bulk of the A1pI remains in the supernatant of the Fraction II+III precipitation reaction and is subsequently separated from albumin in a third initial precipitation reaciton, referred to as a Fraction I-IV-1 precipitation, performed at low pH (5.2) and moderate ethanol concentration (18%). (Bruckschwaiger US 13776448) 

Radowitz (US4216205) discloses further treatment of the supernatant from above with ethanol at temperature of -7 C and pH to 5.8. This fraction includes the immune globulins and other proteins. The precipitate is removed by centrifuging and the supernatant processed further.

a. Further processing of globulin fraction II/III

–PEG precipitation: For example, Radowitz (US4216205) discloses resuspending the precipitate above in a citrate -phosphate bufer at a pH of from about 7.0-7.4 at termpature held at 15C. 3% of polyethylene glycol (PEG) are added. Upon agitation and reaction time from 1/2 to 4 hours, the mixture is centrifuged and supernatant (S5) and fraction III-1 (precipitate P5) are formed. Supernatant S5 is against reated under modified conditions (pH 4.6, PEG 4k up to 5.5%, temperature 15C) and fractionated. Fraction III-2 (precipiate P6) and superantant (S6) are formed, the later containing primarily the IgG. 

4. Fraction IV: 

For example, Radowitz (US4216205) disclcoses further treating the supernatant form above with ethanol at concentration set to 40%, temerpature at -7C and pH 5.8. By centrifuging, the precipitate, Fraction IV is obtained which  contains the alpha and beta globulins. The suepernatant is further processed. 

–Fraction IV-1: The superantant II+III may be further supplemented with alcohol and forming supernatant IV-1 and precipitated fraction IV-1. Fraction IV-1 may be separated and resolubilised and form a protein solution and proteins may be isolated by contacting it with an adsorbent. In particular plasma proteins such as alpha-1-proteinase inhibitor, anti-thrombin III and Factor IX complex may be isolated form the protein solution obtained from fraction IV-1. 

–Fraction IV-4: The supernatant IV-1 may be supplemented with more alcohol to form precipitated fraction IV-4 and supernatant IV-4. Fraction Iv-4 may be separated and resolubilised to form a protein solution and the proteins of interest may be isolated by contacting it with an adsorbent. In particular Butyryleholinesterase may be isolated form the protein solution obtained from fraction IV-4. Lihme (US2007/0299251)

5. Fraction V: Supernatant VI-4 may be further supplmented with alochol to provide precipitated fraction V which may be separated and resolubilised to form a protein soluiton and proteins of interest may be isolated by contacting it with an adsorbent. In particular albumin may be isolated form the protein solution obtained from fraciton V. Lihme (US2007/0299251)