METHOD FOR PURIFYING PEGylated ERYTHROPOIETIN

Abstract

Herein is reported a method for the purification of a protein comprising erythropoietin and a single poly (ethylene glycol) residue from reaction by-products or not reacted starting material by a cation exchange chromatography method. It has been found that by employing a cation exchange SP Sephacryi™ S 500 HR chromatography material conditioned to a conductivity of 21 mS/cm and a linear gradient elution a fusion protein of erythropoietin and a single poly (ethylene glycol) residue can be obtained in a single step with high purity and yield.

1 . A method for obtaining a protein, which comprises erythropoietin and a single poly (ethylene glycol) residue, comprising the following steps:
 a) applying a solution comprising a mixture of erythropoietin and conjugates of erythropoietin and poly (ethylene glycol) with one or more poly (ethylene glycol) residues per erythropoietin molecule to a column, comprising SP Sephacryl™ S 500 HR chromatography material, to which a solution with a conductivity of about 21 mS/cm has been applied,   b) applying a solution with a conductivity of about 21 mS/cm to the column and thereby recovering free poly (ethylene glycol), and proteins comprising two or more poly (ethylene glycol) residues,   c) applying a solution with linearly increasing conductivity up to a final value of about 62.5 mS/cm to the column and thereby recovering separately the protein, which comprises erythropoietin and a single poly (ethylene glycol) residue, and erythropoietin, whereby the protein comprising erythropoietin and a single poly (ethylene glycol) residue is recovered first.   
     
     
         2 . The method according to  claim 1 , characterized in that the solution with a conductivity of about 21 mS/cm is a solution with a pH value of from pH 2.5 to pH 3.5. 
     
     
         3 . The method according to  claim 1 , wherein the solution with a conductivity of about 21 mS/cm is a phosphate buffered solution with a pH value of from pH 2.5 to pH 3.5. 
     
     
         4 . The method according to  claim 1 , wherein the solution comprising a mixture of erythropoietin and conjugates of erythropoietin and poly (ethylene glycol) with one or more poly (ethylene glycol) residues per erythropoietin molecule is not adjusted to a conductivity of about 21 mS/cm. 
     
     
         5 . The method according to  claim 1 , wherein the solution with linearly increasing conductivity is a solution with linearly increasing sodium chloride concentration. 
     
     
         6 . The method according to  claim 1 , wherein the solution with linearly increasing conductivity has a pH value of from pH 2.3 to pH 3.5. 
     
     
         7 . The method according to  claim 1 , wherein the erythropoietin is human erythropoietin. 
     
     
         8 . The method according to  claim 7 , characterized in that the human erythropoietin has the amino acid sequence of SEQ ID NO: 01 or SEQ ID NO: 02. 
     
     
         9 . The method according to  claim 1 , wherein the single poly (ethylene glycol) residue has a molecular weight of from 20 kDa to 40 kDa. 
     
     
         10 . A composition comprising a mixture of oligo-PEGylated erythropoietin, mono-PEGlyated erythropoietin, and non-PEGylated erythropoietin, wherein the composition comprises 12.1% oligo-PEGylated erythropoietin, 87.7% mono-PEGylated erythropoietin, and 0.2% non-PEGylated erythropoietin.