Methods of purifying authentic IGF from yeast hosts
AssigneesCephalon, Inc., Chiron Corporation
InventorsCynthia Cowgill, Luis Juarbe-Osorio, Patricio Riquelme, Glenn Dorin, Christopher M. Bussineau, Robert D Kudrna, Asuman G. Ozturk, Carl Scandella, Russell A. Brierley, Joan N. Abrams, John M. Hanson, Francis C. Maslanka
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Abstract
Methods for purifying authentic, properly folded IGF polypeptides from yeast transformants are disclosed. The methods include a refolding step and provide for high yields of authentic IGF from a variety of yeast strains.
Claims16
A method for isolating an authentic, properly folded insulin-like growth factor (IGF) polypeptide from a medium into which an IGF polypeptide has been secreted by Saccharomyces cells expressing the IGF polypeptide, wherein the method comprises:
- (a) performing a first cation exchange chromatography with the Saccharomyces cell medium to yield a first IGF mixture;
- (b) denaturing and renaturing IGF species present in the first IGF mixture using a denaturation buffer comprising urea, dithiothreitol, alcohol, salt, and one or more divalent metals selected from the group consisting of Cu++, Mn++, Ni++, Zn++ and Fe++ in sufficient amounts and under conditions which allow for the reduction and subsequent oxidation of disulfide bonds, to yield a second IGF mixture;
- (c) subjecting the second IGF mixture to hydrophobic interaction chromatography to yield a third IGF mixture;
- and
- (d) performing reverse phase high performance liquid chromatography on the third IGF mixture to yield a fourth IGF mixture, wherein the fourth IGF mixture has a greater percentage of authentic, properly folded IGF than the first IGF mixture.
Dependencies are inferred from the claim text.