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 Toyopearl® SP-650 chromatography material and employing a second wash step with an increased pH value compared to the first wash step a fusion protein of erythropoietin and a single poly (ethylene glycol) residue can be obtained in a single step with high purity and yield and suitability for large scale applications.

1 . A method for purifying 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 a chromatography material that has a matrix of methacrylate with a sulfopropyl as functional group, to which a first solution with a pH of about 2.4 to about 2.7 has been applied,   b) applying a second solution with an increased H value with respect to the first solution,   c) applying a solution with increased or increasing conductivity to the column and thereby recovering the protein, which comprises erythropoietin and a single poly (ethylene glycol) residue.   
     
     
         2 . The method according to  claim 1 , characterized in that the method further comprises the step of re-applying the first solution with a pH of about 2.4 to about 2.7 after step b) and before step c). 
     
     
         3 . The method according to  claim 1 , characterized in that the second solution with an increased pH value is a solution with a pH of about 2.7 to about 3.0. 
     
     
         4 . The method according to  claim 1 , characterized in that the second solution with an increased pH value is a solution with a constant conductivity value. 
     
     
         5 . The method according to  claim 1 , characterized in that the second solution with an increased pH value and the first solution with a pH of about 2.4 to about 2.7 have about the same constant conductivity value. 
     
     
         6 . The method according to  claim 1 , characterized in that the second solution with an increased pH value and/or the first solution with a pH of about 2.4 to about 2.7 have a constant conductivity value of about 17 mS/cm to about 19 mS/cm. 
     
     
         7 . The method according to  claim 1 , characterized in that the second solution with an increased pH value has a pH of about 2.7 to about 3.0 and has a conductivity value of about 17 mS/cm to about 19 mS/cm. 
     
     
         8 . The method according to  claim 1 , characterized in that 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 value of about 19 mS/cm. 
     
     
         9 . The method according to  claim 1 , characterized in that the solution with increasing conductivity is a solution with increasing sodium chloride concentration. 
     
     
         10 . The method according to  claim 1 , characterized in that the solution with increasing conductivity has a conductivity that increases linearly or step-wise. 
     
     
         11 . The method according to  claim 1 , characterized in that the method is used in large scale protein preparations wherein the chromatography column of step a) has a diameter of at least 30 cm. 
     
     
         12 . The method according to  claim 1 , characterized in that the erythropoietin is human erythropoietin. 
     
     
         13 . The method according to  claim 12 , characterized in that the human erythropoietin has the amino acid sequence of SEQ ID NO: 01 or SEQ ID NO: 02. 
     
     
         14 . The method according to  claim 1 , characterized in that the single poly (ethylene glycol) residue has a molecular weight of from 20 kDa to 40 kDa.