IGF-I purification process
AssigneeCephalon, Inc.
InventorsRussell A. Brierley, Joan N. Abrams, John M. Hanson, Francis C. Maslanka
Life of this patent
Estimating the term…
Abstract
Disclosed is an improved process for obtaining purified, monomeric, intact, correctly-folded insulin-like growth factor-I (also known as somatomedin-C). The improvements, consisting primarily of the addition of an IGF-I unfolding/refolding step and the substitution of a reverse phase chromatography step for a gel filtration chromatography step result in a three-fold increase in final yield. The process includes the following steps, in order: first cation exchange, unfolding/refolding, hydrophobic interaction chromatography, second cation exchange, and reverse phase chromatography.
Claims25
A process for purifying authentic, properly folded monomeric insulin-like growth factor-I (IGF-I) from a medium containing IGF-I peptides, comprising the steps of:
- (a) contacting the medium with a sufficient quantity of a first cation exchange matrix under conditions allowing adsorption of substantially all forms of IGF-I from the medium;
- (b) washing the IGF-I-loaded first cation exchange matrix with a first cation exchange wash buffer which has a sufficiently low ionic strength to provide for retention of substantially all authentic and nonauthentic IGF-I forms by said matrix;
- (c) eluting substantially all forms of adsorbed IGF-I from the cation exchange matrix of step (a) by contacting said cation exchange matrix with a sufficient quantity of a first cation exchange elution buffer, which has a sufficiently high pH or ionic strength to displace substantially all of said authentic and non-authentic IGF-I forms from said cation exchange matrix;
- (d) transferring the IGF-I-forms-containing eluate from step (c) into an unfolding/refolding buffer, which:
- (i) reduces the intrachain disulfide bonds of IGF-I protein and promotes unfolding without permanent denaturation;
- and
- (ii) permits refolding of the IGF-I and reoxidation to form properly-paired intrachain disulfide bonds;
- (e) contacting the refolded IGF-I from step (d), after transfer into a suitable solvent system, with a sufficient quantity of a hydrophobic interaction chromatography matrix under conditions allowing adsorption of at least about 95% of said IGF-I from said eluate;
- (f) washing the IGF-I-loaded hydrophobic interaction chromatography matrix with a hydrophobic interaction wash buffer having an ionic strength sufficiently low to remove most of the non-authentic IGF-I from the hydrophobic interaction chromatography matrix while retaining substantially all of the adsorbed authentic IGF-I on said matrix;
- (g) eluting the adsorbed IGF-I from said hydrophobic interaction chromatography matrix by contacting said matrix with a hydrophobic interaction elution buffer, which has a sufficiently elevated pH, or sufficiently low ionic strength, to cause displacement of substantially all of the adsorbed authentic IGF-I from said matrix;
- (h) contacting the eluate from step (g) with a sufficient quantity of a second cation exchange matrix under conditions allowing adsorption of IGF-I from the eluate;
- (i) washing the IGF-I-loaded second cation exchange matrix with a cation exchange wash buffer having a sufficiently high ionic strength, or sufficiently high pH, to remove non-authentic IGF-I forms from said matrix while retaining substantially all of the adsorbed authentic IGF-I on said matrix;
- (j) eluting the adsorbed IGF-I from said second cation exchange matrix by contacting said matrix with a second cation exchange elution buffer, which has a sufficiently high ionic strength, or sufficiently high pH, to displace substantially all of the adsorbed authentic IGF-I from said matrix;
- (k) contacting the eluate from step (j), in an aqueous buffer, with a suitable quantity of a reverse phase chromatography matrix under conditions allowing adsorption of at least about 95% of the authentic IGF-I from the eluate;
- (l) washing the IGF-I-loaded reverse phase chromatography matrix with an aqueous/organic reverse phase wash buffer having an organic solvent concentration sufficiently high to remove non-authentic IGF-I forms from said matrix while retaining substantially all of the adsorbed authentic IGF-I on said matrix;
- (m) eluting the adsorbed IGF-I from said reverse phase chromatography matrix with an aqueous/organic buffer having an organic solvent concentration high enough to remove substantially all of the authentic IGF-I without removing substantially all of the non-authentic forms of IGF-I from said matrix.
A method for refolding an insulin-like growth factor-I (IGF-I) polypeptide derived from a yeast cell medium to yield an authentic, properly folded IGF-I polypeptide, wherein the method comprises:
- (a) isolating an IGF-I mixture from said yeast cell medium;
- and (b) denaturing and renaturing IGF-I species present in the IGF-I mixture from said yeast cell medium by adding an unfolding/refolding buffer which comprises about 1.5 to about 3 M urea, about 1 mM to about 15 mM sodium borate, about 1 M to about 3 M sodium chloride, about 5% to about 20% ethanol and about 0.005 mM to about 10 mM dithiothreitol, under conditions which allow for the reduction and subsequent oxidation of disulfide bonds, thereby producing an authentic, properly folded IGF-I polypeptide.
Dependencies are inferred from the claim text.