Patent ID: 8795533

Claim:
A method for obtaining a mono-PEGylated erythropoietin in substantially homogenous form comprising the following steps: a) PEGylating erythropoietin using an activated PEGylating reagent of a molecular weight of from 20 kDa to 40 kDa to produce PEGylated erythropoietin, b) purifying the PEGylated erythropoietin obtained in step a) with two consecutive cation exchange chromatography steps, wherein the first and second cation exchange chromatography steps employ the same type of cation exchange material, c) recovering the mono-PEGylated erythropoietin from the second cation exchange chromatography column in a substantially homogeneous form, d) regenerating the cation exchange chromatography column by: i) removing any bound polypeptide from the cation exchange column with an aqueous buffered solution comprising sodium chloride, followed by ii) flushing the column with purified water, followed by iii) applying a sodium hydroxide solution to the column, followed by iv) flushing the column with purified water, followed by v) applying a solution comprising sodium dihydrogen phosphate and phosphoric acid to the column, followed by vi) flushing the column with purified water, followed by vii) applying a 0.5 M sodium hydroxide solution to the column for at least 4 hours, and followed by viii) regenerating the cation exchange chromatography column by flushing the column with purified water.