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 co-purify using conventional chromatographic media. These species consist 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 chromatography procedure using a 5-micron C18 column, an acetonitrile-trifluoroacetic acid (TFA) isocratic elution, and elevated temperature gives baseline resolution of the four species. Using this analytical method as a development tool, a process-scale chromatography step was established. The 5-micron 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 dissociate, a combination of acetic acid and NaCl was substituted. Isocratic separations are not good process options due to problems with reproducibility and control. A shallow gradient elution using premixed mobile phase buffers at the same linear velocity was found to give an equivalent separation at low load levels and minimized solvent degassing. However, at higher loading there was a loss of resolution. A matrix of various buffers was evaluated for their effects on separation. Elevated pH resulted in a significant shift in both the elution order and relative 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 evaluated with regard to particle size, shape and pore diameter using the improved mobile phase. The new conditions were scaled up 1305-fold and resulted in superimposable chromatograms, 96% recovery and > 99% purity. Thus, by optimizing the pH, ionic strength and temperature, a high-capacity preparative separation of rhIGF-1 from its related fermentation variants was obtained.
Olson
C V
CV
Department of Recovery Process Research and Development, Genentech Inc., South San Francisco, CA 94080.
Reifsnyder
D H
DH
Canova-Davis
E
E
Ling
V T
VT
Builder
S E
SE
D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet
D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I