Human blood plasma is conveniently fractionated by the cold ethanol precipitation method. For this method the plasma is treated with ethanol at below 5C and by increasing the concentration of ethanol and reducing the pH, a succession of fractions are precipitated. The supernatant liquor after reach precipitation is subjected to the next. It is based on the methods described by E.J. Cohn et al., J. Amer. Chem. Soc. 68, 459-475 (1946), but the original methods have been modified over the years. (EP0440509A2). The Cohn process is essnetially a batch process where the various proteins are precipitated from the solution sequentially. 

The major components (US4,540,573)

In the Cohn fractionation method, the frist fractionation step results in fraction I which comprises mainly fibrinogen and fibronectin. The sueprnatant from this step is futher processed to precipitate out fraction II+III and then fractions III and II. Typically, fraction II+III contains about 60% IgG, together with impurities such as fibrinogen, IgM and IgA. Most of these impurities are then removed in fraction III, which is considered a waste fraction. The supernatant is then treated to preciptiate out the main IgG containing fraction, fraction II, which can contain greater than 90% IgG. 

In the Kistler & Nitschmann method, fraciton I is equivalent to fraction I of the Cohn method. The next preciptiate/fraction is referred to as precipitate A (fraction A). This preciptiate is broadly equivalent, alhtough not identical to Cohn fraciton II+III. The preciptiate is then redissolved and conditions adjusted to preciptiate out preciptiate B (fraction B) which is equivalent to Cohn fraciton III. Again, this is considered to be a waste fraciton. The preciptiate B supernatant is then processed further to produced preciptiate II, which coreesponds to Cohn fraciton II. 

Plasma is cooled to about 1C and then centrifuged to seaprate a cold insoluble precipitate from a supernatant. The supernatant is further fractionated to yeild Precipitate I and Supernatant I. Preciptiate I which consists principally of fibrinogen is discarded. Supernatant I is fruther fracitoanted to yeild Supernatant II+IIII and Precipitate II+III. Superantant II+III, which is discarrded, contains alpha and beta globulin and lipids. Precipitate II+III consists principally of beta and gamma globulins and isoagglutins, but also contains prothrombin, plasminogen, cholesterol and other lipdis.  Precipitate II+III upon further fractionation yields Supernatant II+III W and Precipitate II+IIIW. The beta globulin, cholesterol and other lipids are largely removed in Supernatant II+IIIW which is discard. Precipitate II+IIIW consists principally of gamma goblulins, isoagglutins, plasminogen and prothrombind and some beta goblulin, chloesterol and other lipids. (Van Holten, EP 1247818). Upon further fractionation, Precipitate II+IIIW yeilds Supernatant III and Precipitate III. Precipitate III, which is discarded, contains isoagglutinins, plasminogen and prothrombin. Supernatant III consists principally of gamma globulins and minor amounts of fibrinogen and lipids. The final step of teh fractionation yields Precipitate II which is essentially pure gamma G globulin almost completely free of 19S globulin, plasminogen and lipides. (Van Holten, EP 1247818)

 Cohn Fraction I (AKA Fraction I Precipitation or Kistler-Nitschman Fraciton I)

First fraction precipitate (Fraction I): contains fibrinogen and fibronectin (EP0440509A2),

Fraction I is the starting material for the preparation of fibrinogen which represents 50-60% of its total protein. The isolation of purified fribrinogen can be undertaken either by using ethanol at various pH values, or with the aid of ammonium sulfate, after a preliminary treatment with barium sulfate.

To form a Fraction I precipitate, the cryopoor plasma solution is cooled to below about 6C (typically to about 1C) and the pH adjusted to about 7-75, Pre-cooled alcohol (typicaly ethanol) is then added to a concentraiton of fromb aobut 6-10% typically while stirring the solution. After completion of the precipitation reaction, the supernatant (e.g., Supernatnat I) is then separated from the precipitate (Fraction I precipitate) by centrifugation, filtration or other suitable means. (Hofbauer et al. (US 2014/0271669). 

Cohn Fractions II+III (AKA Fraction II+III Precipitation)

To form a Fraction II+III precipitate (e.g., a Cohn Fraction II_III precipitate or Kistler-Nitschmann Precipitate A), the fraction I supernatant from above is cooled to below about 0C and the pH adjsuted to about 5.5-7.0. Precooled alcohol is added to a target concentraiton of from about 20-25. At the same time the temperature is further lowered typically between -9–5. Typically, the precipitation reaction includes an incubation time of at least about 1 hour. After completion of the precipitaiton reaction, the supernatant (e.g., Supernatant II+III) is then separated form the precipitation (e.g., Fraction II+II” precipitate) by centrifugation, filtration or other suitable means. (Hofbauer et al. (US 2014/0271669). 

Cohn Fraction II+III contains premodminantly IgG, IgA and IgM and is commonly prepared accoridng to Cohn’s method 6. 

The second (Fraction II) contains gamma-globulins (EP0440509A2),. Gamma globulins are blood protteins produced by lymphocytes and plasma cells of the immune system. Almost all gamma globulins are antibodies. The three main types are IgM, IgG and IgA. 

third (Fraction III) contains beta-globulins (EP0440509A2). Beta globulins are blood proteins. There are beta-1 and beta-2 globulins. They are simliar to alpha globulins which are produced by the liver. Beta globulins have similar functions and carry lipids, hormones and cholesterol through the bloodstream. Beta globulins also assist immune cells in mounting an immune response. 

Cohn Fraction IV

fourth (fraction IV) alpha-globulins (can be recovered under different conditions to give different products as for example IV(1) and IV(4), the latter containing more transferrin and albumins) (EP0440509A2). 

globulins were described (Cohn, J. Am. Chem. Soc., 1946, 68(3):459-475); US 2,390,074, US 6893639).

Several years later, Oncley (J. Am. Chem Soc., 1949, 71(2): 541-550) expanded upon the Cohn methods by publishing a method (method 9) that resulted in further division of these major fractions into subfractions. Fraction IV can be collected as two cuts, knwon as IV(1) and IV(4), the lattter containing more transferring and albumins.

Cohn Fraction V

Fifth (Fraction V) contains albumins (this is a large volume product collected at a 40% ethanol concentration and pH5.2 and represents a commercially valuable product). Each fraction is heavily contamined by compoents of other factions and other materials (EP0440509A2). 

The first fraction is a large volume collected at a 40% ethanol concentraiton and pH 5.2 and represents a commercially valuable product.