Interferons are proteins which are produced by a number of different kinds of organisms and which are presently grouped into three major classes designated leukocyte interferon (IFN-.alpha.), fibroblast interferon (IFN-.beta.) and immune interferon (IFN-.gamma.). These interferons have antiviral and antiproliferative activites, a potent ability to confer a virus-resistant state in targeted cells and immunomodulatory activities. These biological properties have led to the clinical use of interferons as therapeutic agents for the treatment of viral infections and malignancies, which in turn has prompted a demand for a greater supply.
Interferons have been produced from natural sources such as buffy coat leukocytes and fibroblast cells, optionally using known inducing agents to increase the production of interferon. Interferons have also been produced by recombinant DNA techniques, i.e. by expression from a microorganism which has been transformed with an expression vector containing an interferon gene under the control of a promoter-operator sequence. (Leukocyte, fibroblast and immune interferons produced by recombinant techniques are designated rIFN-.alpha., rIFN-.beta. and rIFN-.gamma. respectively.)
Regardless of the method of production employed, the resulting interferon must be purified, preferably to homogeneity, before it may be employed as a therapeutic agent. Interferons may be purified to homogeneity using methods described in U.S. Pat. Nos. 4,289,689 and 4,289,690 or by monoclonal antibody affinity chromatography. Presently available purification methods are carried out under conditions which can favor the formation of dimers, trimers and higher oligomers of the interferon. These oligomeric forms of interferon result from two or more interferon molecules becoming irreversibly associated with one another through intermolecular covalent bonding, such as by disulphide linkages. This problem has been observed particularly will respect to leukocyte and fibroblast interferons.
The oligomeric form in many cases has either no biological activity or lower activity than the monomeric form, or it has the potential for causing deleterious side effects if used therapeutically. It is important, therefore, to have available a method for obtaining interferons in monomeric form.
A method for obtaining fibroblast interferon in monomeric form is described in U.S. Pat. No. 4,278,661. Serum-free human fibroblast interferon is purified by adsorption on immobilized Cibacron Blue and elution of the adsorbed interferon with an aqueous buffer solution containing ethylene glycol, to provide a mixture of interferon in monomeric and dimeric form. The dimeric form is converted to monomeric form by heating the mixture in the presence of an organic thiol compound such as thioglycolic acid, 2-mecaptoethanol or dithiothreitol. The use of elevated temperatures, however, is generally not desirable in protein preparation because of a resulting diminution or complete loss of biological activity for the heated protein.