The present invention relates to a method for purifying fibroblast growth factor (hereinafter also briefly referred to as FGF) proteins.
FGF was first isolated as a factor exhibiting strong growth promoting action on fibroblasts such as BALB/c3T3 cells [D. Gospodarowicz, Nature 249, 123 (1974)]. It is now known that the FGF exhibits growth promoting action on almost all cells derived from mesoblast. FGF is classified into basic FGF (hereinafter briefly referred to as bFGF) and acidic FGF (hereinafter briefly referred to as aFGF), based on the isoelectric point thereof. bFGF and aFGF both have strong growth promoting action and plasminogen activator inducing action on vascular endothelial cells. Together, these actions suggest a potential for the application thereof as a drug for promoting angiogenesis, as a therapeutic drug for traumas, and as a preventive and therapeutic drug for thrombosis, arteriosclerosis, etc.
Previously, the FGFs were purified to homogeneity from organs derived from animals, such as bovine pituitary. However, supply of these FGFs was limited, and there was a fear of antigenicity due to their heterozoic origin. The FGFs were also obtained from culture supernatants of various animal cell lines, but their supply was also limited. Recently, there has been developed a method for producing FGF in large quantities. The method involves using recombinant DNA techniques to express a cloned human FGF gene in microorganisms or in animal cells. [FEBS Letters 213, 189-194 (1987); European Patent Publication (hereinafter also referred to as EP Publication) No. 237,966)].
FGFs have been purified using heparin affinity column chromatography [Science 223, 1296-1299 (1984); Journal of Biological Chemistry 261, 1924-1928 (1986)]. FGFs have been purified using heparin affinity column chromatography, from bovine and human brain; bovine pituitary; bovine retina; bovine, human and avian cartilage; rat chondrosarcoma culture supernatant; human melanoma culture supernatant; and human hepatoma culture supernatant [Journal of Biological Chemistry 261, 1924-1928 (1986)].
The heparin affinity column chromatography has also been used for the purification of the FGF prepared by the recombinant DNA technique [Biochemical and Biophysical Research Communications 146, 470-477 (1987)].
However, the carrier used in the heparin affinity column chromatography discussed above, such as a conjugate of crosslinked agarose and heparin, has several disadvantages: (1) heparin is liable to be liberated from the carrier (crosslinked agarose), and (2) the carrier undergoes serious deterioration with repeated use. Heparin affinity column chromatography is therefore unsuitable for the purification of FGF on a large scale commercial production.
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