Use of size-exclusion and ion-exchange high-performance liquid chromatography for the isolation of biologically active growth factors

Abstract

Size-exclusion and ion-exchange high-performance liquid chromatography were used to purify biologically active growth factors as measured by the ability of the factors to stimulate DNA synthesis in 3T3 cells. Chromatography was performed in aqueous buffer and at neutral pH to avoid possible inactivation of biological activity. The growth factors analyzed were chondrosarcoma growth factor (CHSA-GF), human milk growth factor (HMGF), retinal-derived growth factor (RDGF) and mouse epidermal growth factor (EGF). CHSA-GF, HMGF, and RDGF were eluted from TSK 2000 columns as well-defined peaks of activity with molecular weights of 12,000–15,000, 5000–6000, and 16,000–18,000, respectively. EGF was found to have an abnormally low molecular weight after chromatography on TSK 2000. However, incorporation of guanidine · HCl into the TSK column resulted in the elution of EGF at its known molecular weight of ca. 6000. Anion-exchange high-performance liquid chromatography on AX 300 was used for the purification of HMGF and RDGF, and cation-exchange high-performance liquid chromatography on CM 300 was used for the purification of CHSA-GF. The results show that size-exclusion and ion-exchange chromatography can be used without organic solvents or extremes in pH to purify a number of different growth factors successfully with retention of biological activity.