Purification of pegylated polypeptides
AssigneeHoffmann-La Roche Inc.
InventorsJosef Burg, Klaus Reichert, Axel Schroth, Hartmut Schurig, Axel Wessner
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Abstract
The invention is a method for the purification of mono-PEGylated erythropoietin using two cation exchange chromatography steps wherein the same type of cation exchange material is used in both cation exchange chromatography steps and a method for producing a mono-PEGylated erythropoietin in substantially homogeneous form.
Claims16
A method for producing a mono-PEGylated erythropoietin comprising (i) providing an aqueous buffered solution comprising mono-, poly-, and non-PEGylated erythropoietin and low molecular weight forms of erythropoietin, (ii) performing two consecutive cation exchange chromatography steps, and (iii) recovering purified mono-PEGylated erythropoietin in the second cation exchange chromatography step, wherein the same type of cation exchange material is used in both cation exchange chromatography steps with different elution methods, said two consecutive cation exchange chromatography steps comprising:
- a) applying said aqueous, buffered solution to a first cation exchange chromatography column under conditions suitable for binding of any mono-PEGylated erythropoietin in said aqueous buffered solution to the cation exchange material contained in said first column,
- b) recovering any bound mono-PEGylated erythropoietin from the first cation exchange chromatography column by step elution with a stepwise increase of the ionic strength of flow through buffer to obtain a purified mono-PEGylated erythropoietin solution, wherein the amount of mono-PEGylated erythropoietin in the recovered, purified solution is increased compared to the solution of step (a),
- c) applying the recovered, purified solution of step b) to a second cation exchange chromatography column under conditions suitable for binding of any mono-PEGylated erythropoietin to the cation exchange material contained in said second column, whereby the cation exchange material contained in said second column is of the same type as the cation exchange material in the first column, and
- d) recovering any purified mono-PEGylated erythropoietin bound to said second cation exchange chromatography column in a substantially homogeneous form by applying a linear gradient starting with potassium phosphate buffer at a concentration of about 100 mM with about 50 mM sodium chloride at about pH 3.0 and ending with a potassium phosphate buffer of a concentration of about 100 mM with about 500 mM sodium chloride at about pH 3.0, whereby the change in the sodium chloride concentration is linear over ten column volumes.
A method for the production of a mono-PEGylated erythropoietin comprising the following steps:
- a) PEGylating erythropoietin to obtain a mixture of mono, poly, and non-pegylated erythropoietin and low molecular weight forms of erythropoietin,
- b) recovering any bound mono-PEGylated erythropoietin from the first cation exchange chromatography column by step elution with a stepwise increase of the ionic strength of flow through buffer to obtain a purified mono-PEGylated erythropoietin solution, wherein the amount of mono-PEGylated erythropoietin in recovered, purified solution is increased compared to the mixture of step (a),
- c) applying the recovered, purified solution of step 1)) to a second cation exchange chromatography column under conditions suitable for binding of any mono-PEGylated erythropoietin to the cation exchange material contained in said second column, whereby the cation exchange material contained in said second column is of the same type as the cation exchange material in the first column, and
- d) recovering any purified mono-PEGylated erythropoietin bound to said second cation exchange chromatography column in a substantially homogeneous form by applying a linear gradient starting with potassium phosphate buffer at a concentration of about 100 mM with about 50 mM sodium chloride at about pH 3.0 and ending with a potassium phosphate buffer of a concentration of about 100 mM with about 500 mM sodium chloride at about pH 3.0, whereby the change in the sodium chloride concentration is linear over ten column volumes.
Dependencies are inferred from the claim text.