Cohn fractionation

Factor VIII, immunoglobulin, and albumin are all held as protein precipitates, the first as cryoprecipitate and the others as the Cohn fractions FI + II + III (or FII + III) and FIV + V (or FV), respectively (Table 7, Fig. 2). Similarly, Fractions FIVj + FIV can provide an intermediate product for the preparation of antithrombin III and a-1-proteinase inhibitor. This abiUty to reduce plasma to a number of compact, stable, intermediate products, together with the bacteriacidal properties of cold-ethanol, are the principal reasons these methods are stiU used industrially. 

Alpha-1-proteinase inhibitor and antithrombin III are used to treat people with hereditary deficiencies of these proteins. Both can be recovered from Cohn Fraction IV (Table 7) using ion-exchange chromatography (52) and affinity chromatography (197), respectively. Some manufacturers recover antithrombin III directiy from the plasma stream by affinity adsorption (56,198,199). 

Antibodies and derivatives Immune globulin (human) against Rho (D), purified IgG Cohn fraction II BayRho-D 820 (300 pg) (full) or 137 (mini) lU vial or syringe IM NA... 

Antibodies and derivatives Tetnus immune globulin (human), isolated from solubilized Cohn Fraction II BayTet 250 U vial or syringe IM (deep) NA... 

Immune globulin (human) against Rho (D), purified IgG cohn fraction II... 

The aims of protein purification, up until the 1940s, were simply academic. To then, even the basic facts of protein structure were not fully appreciated, and pure proteins were needed just to study structure and test the rival theories of the pre-DNA days. During the Second World War, an acute need for blood proteins led to development of the Cohn fractionation procedure for purification of albumin and other proteins from serum (Cohn et al., 1946). This was the inception of large-scale protein purifications for commercial purposes Cohn fractionation continues to be used to this day. 

The iron-binding protein of serum transferrin was found in fraction IV-3,4 of human plasma when the plasma was fractionated by low temperature ethanol fractionation procedures (31, 116). By further subfractionations, serum transferrin could be concentrated in Cohn fraction IV-7 (30, 125, 126). Cohn (30) first reported the properties of the isolated protein, which he called the 3i metal-binding protein since the protein had been found to bind copper, and possibly zinc, as well as iron. Holm-berg and Laurell (66) proposed that the protein be called transferrin on the basis that the principal function of the protein was associated with the transport of iron in serum and that it was not the major copperbinding protein in human serum. 

Human serum transferrin has been prepared in the laboratories of the University of California, Davis, from Cohn fraction IV—7 obtained from a commercial company (Cutter Laboratories, Berkeley, California). Such fractions are, of course, from plasma of a large number of individuals and may have been exposed to different treatments. For example, some of these fractions have been exposed to a heating step to inactivate viruses. This type of human material, which is the usual type used for large scale preparations, certainly contains different molecular forms, because of genetic differences, as well as some artifactual materials. The procedures employed molecular filtration on Sephadex columns and sequential ion exchange chromatography on anion and cation cellulose exchangers (22, 137). 

Today, two industrial GMP processes exist for mass production of human BChE. The first one is purification of the natural enzyme from human plasma (Cohn Fraction IV). This process has been developed by Baxter Healthcare Corporation (www.baxter.com). Human plasma derived... 

Fig. 2. SDS-PAGE of affinity-purified MAbs specific for human IL-3. Track 5 shows standard mol-wt markers (from top to bottom myosin heavy chain, 200 kD p-galactosidase, 116 kD phosphorylase b, Mf 97.4 kD bovine albumin, 66 kD egg albumin, Mr 45 kD and carbonic anhydrase, M, 29 kD). Track 1 shows Cohn fractionated human IgG. The remaining three tracks show three different IgG antibodies purified using a column of human recombinant DNA-derived IL-3 coupled to Sepharose 4B. The m jor bands are kappa and gamma chains the faint higher Mr bands are caused by incomplete dissociation of heavy and li t chains.

FIGURE 14.8 Cohn fractionation process (simplified illustration). 

D4. Deutsch, H. F., Kasper, C. B., and Walsh, D. A., Rapid method for preparation of crystalline human ceruloplasmin from Cohn fraction IV-1. Arch. Biochem. Biophys. 99, 132-135 (1962). 

Bl. Bala, R. M., and Bhaumick, B., Purification of a basic somatomedin, from human plasma Cohn fraction IV-1, with physicochemical and radioimmunoassay similarity to somato-medin-C and insulin-like growth factor. Can. J. Biochem. 57, 1289-1298 (1979). 

Some of the problems surrounding the removal of glycerol from the high glycerol preparation have been overcome through the use of cell washing devices. One of these, developed by the Haemonetics Company (Braintree, MA) and based on the Cohn Fractionator, performs a continuous flow wash in a disposable bowl. The other apparatus developed by the IBM Corporation, conducts an automated batch wash in a disposable bag. Although effective. 

Bovine serum albumin (BSA) (Sigma Chemicals — Cohn fraction V) was selected as a solute material to be studied in batch cell ultrafiltration. The justification of this choice was based on the fact that BSA has been used by previous investigators their work would offer a source of comparison to our results [Blatt, et al. (1970), Kozinski and Llghtfoot (1972), Shen and Probstein (1977, 1979), Probstein (1978, 1979), Goldsmith (1971),... 

IgG, DNAse. Human, freeze dried IgG was purchased from Sigma Chemical (cat. no. HG-11) as Cohn fraction IT. The IgG was resuspended in physiological saline or cell concentration filtrate and the IgG was added to the lysed cells to a final concentration of around 0.1%-0.2%. DNAse was also purchased from Sigma Chemical. 

The pharmaceutical industry would prefer to make the gamma globulin/al-bumin separation with a UF membrane rather than Cohn fractionation (sequential precipitation with ethanol). But to do so, they would have to dilute the mixture way down to accomplish the separation. The processing of the large diluted volumes followed by concentration of the two fractions would make the membrane process more cumbersome and expensive than Cohn fractionation. 

Anti-infective, Immunomodulatory. Mixture of IgGl and other Abs derived from human plasma via Cohn fractionation. 70.3% IgGl, 24.7% IgG2, 3.1% IgG3, and 1.9% IgG4. 

Solutions of bovine serum albumin (Cohn fraction V, Miles Lab., Elkhart, IN) were made with PBS. 

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