Method for purifying PEGylated erythropoietin
AssigneeHoffmann-La Roche Inc.
InventorsRoberto Falkenstein, Bernhard Spensberger
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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.
Claims12
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 pH 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,
- wherein 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.
Dependencies are inferred from the claim text.