Patent ID: 6207806
Filing Date: 2001-03-27
Classification: C07K,G01N

Abstract:
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.