PREPARATIVE ISOLATION OF RECOMBINANT HUMAN INSULIN-LIKE GROWTH-FACTOR-1 BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

The isolation of recombinant human insulin-like growth factor 1 (rhIGF -1) is complicated by the presence of several rhIGF-1 variants which c o-purify using conventional chromatographic media. These species consi st primarily of a methionine-sulfoxide variant of the properly folded molecule and a misfolded form and its respective methionine-sulfoxide variant. An analytical reversed-phase high-performance liquid chromato graphy procedure using a 5-mu m C-18 column, an acetonitrile-trifluoro acetic acid (TFA) isocratic elution, and elevated temperature gives ba seline resolution of the four species. Using this analytical method as a development tool, a process-scale chromatography step was establish ed. The 5-mu m analytical packing material was replaced with a larger- size particle to reduce back-pressure and cost. Since the TFA counter- ion binds tightly to proteins and is difficult to subsequently dissoci ate, a combination of acetic acid and NaCl was substituted. Isocratic separations are not good process options due to problems with reproduc ibility and control. A shallow gradient elution using premixed mobile phase buffers at the same linear velocity was found to give an equival ent separation at low load levels and minimized solvent degassing. How ever, at higher loading there was a loss of resolution. A matrix of va rious buffers was evaluated for their effects on separation. Elevated pH resulted in a significant shift in both the elution order and relat ive retention times of the principal rh-IGF-1 variants, resulting in a substantial increase in effective capacity. An increase in the ionic strength further improved resolution. Several different media were eva luated with regard to particle size, shape and pore diameter using the improved mobile phase. The new conditions were scaled up 1305-fold an d resulted in superimposable chromatograms, 96% recovery and > 99% pur ity. Thus, by optimizing the pH, ionic strength and temperature, a hig h-capacity preparative separation of rhIGF-1 from its related fermenta tion variants was obtained.