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.

Claims

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 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.

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 constant conductivity value.

4. 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.

5. 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.

6. 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.

7. The method according to claim 1, characterized in that the solution with increasing conductivity is a solution with increasing sodium chloride concentration.

8. The method according to claim 1, characterized in that the solution with increasing conductivity has a conductivity that increases linearly or step-wise.

9. 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.

10. The method according to claim 1, characterized in that the erythropoietin is human erythropoietin.

11. The method according to claim 10, characterized in that the human erythropoietin has the amino acid sequence of SEQ ID NO: 01 or SEQ ID NO: 02.

12. 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.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 Elution chromatogram of a purification of a PEGylated erythropoietin preparation with a method as reported in Example 1 (wash at pH 2.5; no additional wash step).

(2) FIG. 2 Elution chromatogram of a purification of a PEGylated erythropoietin preparation with a method as reported in Example 3 (wash at pH 2.5; additional wash step at pH 2.8).

(3) FIG. 3 Magnification of the elution chromatogram (FIG. 2) of a purification of a PEGylated erythropoietin preparation with a method as reported in Example 3 (wash at pH 2.5; additional wash step at pH 2.8).

(4) FIG. 4 Elution chromatogram of a purification of a PEGylated erythropoietin preparation with a method as reported in Example 2 (wash at pH 3.0; no additional wash step).

(5) FIG. 5 Elution chromatogram of a purification of a PEGylated erythropoietin preparation with a method as reported in Example 4 (wash at pH 2.5; additional wash step at pH 3.0).

EXAMPLE 1

Chromatography of a PEGylated Erythropoietin Preparation with a Toyopearl? SP-650 M Chromatography Material without an Additional Wash Step (pH 2.5)

(6) The PEGylated Erythropoietin Chromatography was performed as shown below.

(7) PEGylated Erythropoietin Chromatography: resin: SP Toyopearl 650 M bed volume: 19.6 ml sample loading: 1.3 mg/ml resin flow rate: 1.3 ml/min solutions: A: 100 mM potassium phosphate, 100 mM sodium chloride, adjusted to pH 2.5, adjusted to LF=19 mS/cm with 5 m sodium chloride B: 100 mM potassium phosphate, 375 mM sodium chloride, adjusted to pH 2.5 application solution: 100% A wash solution: 100% A wash volume: 100 ml (5 column volumes (CV)) wash solution (wash 2): none wash volume (wash 2): none linear gradient elution solution: 100% B linear gradient: within 196 ml (10 column volumes) to 100% B wavelength: 280 nm

(8) The elution chromatogram for this method is shown in FIG. 1.

EXAMPLE 2

Chromatography of a PEGylated Erythropoietin Preparation with a Toyopearl? SP-650 M Chromatography Material without an Additional Wash Step (pH 3.0)

(9) The PEGylated Erythropoietin Chromatography was performed as shown below.

(10) PEGylated Erythropoietin Chromatography: resin: SP Toyopearl 650 M bed volume: 19.6 ml sample loading: 1.3 mg/ml resin flow rate: 1.3 ml/min solutions: A: 100 mM potassium phosphate, 100 mM sodium chloride, adjusted to pH 3.0, with a conductivity value=20.5 mS/cm B: 100 mM potassium phosphate, 375 mM sodium chloride, adjusted to pH 2.5 application solution: 100% A wash solution: 100% A wash volume: 100 ml (5 column volumes (CV)) wash solution (wash 2): none wash volume (wash 2): none linear gradient elution solution: 100% B linear gradient: within 196 ml (10 column volumes) to 100% B wavelength: 280 nm

(11) The elution chromatogram for this method is shown in FIG. 4.

EXAMPLE 3

Chromatography of a PEGylated Erythropoietin Preparation with a Toyopearl? SP-650 M Chromatography Material with an Additional Wash Step with a Solution with a pH of 2.8 and a Conductivity of about 19 mS/Cm

(12) The PEGylated Erythropoietin Chromatography was performed as shown below: resin: SP Toyopearl 650 M bed volume: 19.6 ml sample loading: 1.3 mg/ml resin flow rate: 1.3 ml/min solutions: A: 100 mM potassium phosphate, 100 mM sodium chloride, adjusted to pH 2.5, adjusted to LF=19 mS/cm with 5 m sodium chloride B: 100 mM potassium phosphate, 375 mM sodium chloride, adjusted to pH 2.5 Additional wash (wash 2): 100 mM potassium phosphate, 100 mM sodium chloride, adjusted to pH 2.8, adjusted to LF=19 mS/cm with water application solution: 100% A wash solution: 100% A wash volume: 100 ml (5 column volumes (CV)) wash solution (wash 2): 100% Additional wash (wash 2) wash volume (wash 2): 100 ml (5 column volumes (CV)) wash solution (wash 3): 100% A wash volume (wash 3): 60 ml (3 column volumes (CV)) linear gradient elution solution: 100% B linear gradient: within 196 ml (10 column volumes) to 100% B wavelength: 280 nm

(13) The elution chromatogram for this method is shown in FIG. 2 and a magnification is shown in FIG. 3.

EXAMPLE 4

Chromatography of a PEGylated Erythropoietin Preparation with a Toyopearl? SP-650 M Chromatography Material with an Additional Wash Step with a Solution with a pH of 3.0 and a Conductivity of about 19 mS/Cm

(14) The PEGylated Erythropoietin Chromatography was performed as shown below: resin: SP Toyopearl 650 M bed volume: 19.6 ml sample loading: 1.3 mg/ml resin flow rate: 1.3 ml/min solutions: A: 100 mM potassium phosphate, 100 mM sodium chloride, adjusted to pH 2.5, adjusted to LF=19 mS/cm with 5 m sodium chloride B: 100 mM potassium phosphate, 375 mM sodium chloride, adjusted to pH 2.5 Additional wash (wash 2): 100 mM potassium phosphate, 100 mM sodium chloride, adjusted to pH 3.0, adjusted to LF=19 mS/cm with water application solution: 100% A wash solution: 100% A wash volume: 100 ml (5 column volumes (CV)) wash solution (wash 2): 100% Additional wash (wash 2) wash volume (wash 2): 100 ml (5 column volumes (CV)) wash solution (wash 3): 100% A wash volume (wash 3): 60 ml (3 column volumes (CV)) linear gradient elution solution: 100% B linear gradient: within 196 ml (10 column volumes) to 100% B wavelength: 280 nm

(15) The elution chromatogram for this method is shown in FIG. 5.

EXAMPLE 5

Chromatography of a PEGylated Erythropoietin Preparation with a Toyopearl? SP-650 M Chromatography Material with an Additional Wash Step with a Solution of Different pH Values (pH 2.8, 2.9 or 3.0) and a Conductivity of about 17 mS/Cm

(16) The PEGylated Erythropoietin Chromatography was performed as shown below: resin: SP Toyopearl 650 M bed volume: 19.2 ml sample loading: 0.7 mg/ml resin flow rate: 150 cm/h solutions: A: 100 mM potassium phosphate, 100 mM sodium chloride, adjusted to pH 2.5, adjusted to LF=17 mS/cm with 5 m sodium chloride B: 100 mM potassium phosphate, 375 mM sodium chloride, adjusted to pH 2.5 Additional wash (wash 2): 100 mM potassium phosphate, 100 mM sodium chloride, adjusted to pH 2.8, 2.9 or 3.0, adjusted to LF=17 mS/cm with water application solution: 100% A wash solution: 100% A wash volume: 96.2 ml (5 column volumes (CV)) wash solution (wash 2): 100% Additional wash (wash 2) wash volume (wash 2): 96.2 ml (5 column volumes (CV)) wash solution (wash 3): 100% A wash volume (wash 3): 57.7 ml (3 column volumes (CV)) linear gradient elution solution: 100% B linear gradient: within 192 ml (10 column volumes) to 100% B wavelength: 280 nm

(17) The results are shown below:

(18) TABLE-US-00002 Wash 2 Wash 2 Wash 2 (additional (additional (additional wash) pH 2.8, wash) pH 2.9, wash) pH 3.0, 17 mS/cm 17 mS/cm 17 mS/cm Purity Pool 98.81 99.50 99.72 MonoPEG EPO [%] Yield 98.11 95.40 82.81 MonoPEG EPO [%]

(19) Yields are calculated based on the monoPEGylated EPO content in the starting material.

EXAMPLE 6

Chromatography of a PEGylated Erythropoietin Preparation with a Toyopearl? SP-650 M Chromatography Material with an Additional Wash Step with a Solution of Different pH Values (pH 2.8, 2.9 or 3.0) and a Conductivity of about 18 mS/cm

(20) The PEGylated Erythropoietin Chromatography was performed as shown below: resin: SP Toyopearl 650 M bed volume: 19.2 ml sample loading: 0.7 mg/ml resin flow rate: 150 cm/h solutions: A: 100 mM potassium phosphate, 100 mM sodium chloride, adjusted to pH 2.5, adjusted to LF=18 mS/cm with 5 m sodium chloride B: 100 mM potassium phosphate, 375 mM sodium chloride, adjusted to pH 2.5 Additional wash (wash 2): 100 mM potassium phosphate, 100 mM sodium chloride, adjusted to pH 2.8, 2.9 or 3.0, adjusted to LF=18 mS/cm with water application solution: 100% A wash solution: 100% A wash volume: 96.2 ml (5 column volumes (CV)) wash solution (wash 2): 100% Additional wash (wash 2) wash volume (wash 2): 96.2 ml (5 column volumes (CV)) wash solution (wash 3): 100% A wash volume (wash 3): 57.7 ml (3 column volumes (CV)) linear gradient elution solution: 100% B linear gradient: within 192 ml (10 column volumes) to 100% wavelength: 280 nm

(21) The results are shown below:

(22) TABLE-US-00003 Wash 2 Wash 2 Wash 2 (additional (additional (additional wash) pH 2.8, wash) pH 2.9, wash) pH 3.0, 18 mS/cm 18 mS/cm 18 mS/cm Purity Pool 99.54 100 99.24 MonoPEG EPO [%] Yield 78.60 81.82 64.11 MonoPEG EPO [%]

(23) Yields are calculated based on the monoPEGylated EPO content in the starting material.

EXAMPLE 7

Chromatography of a PEGylated Erythropoietin Preparation with a Toyopearl? SP-650 M Chromatography Material with an Additional Wash Step with a Solution of Different pH Values (pH 2.8, 2.9 or 3.0) and a Conductivity of About 19 mS/cm

(24) The PEGylated Erythropoietin Chromatography was performed as shown below: resin: SP Toyopearl 650 M bed volume: 19.2 ml sample loading: 0.7 mg/ml resin flow rate: 150 cm/h solutions: A: 100 mM potassium phosphate, 100 mM sodium chloride, adjusted to pH 2.5, adjusted to LF=19 mS/cm with 5 m sodium chloride B: 100 mM potassium phosphate, 375 mM sodium chloride, adjusted to pH 2.5 Additional wash (wash 2): 100 mM potassium phosphate, 100 mM sodium chloride, adjusted to pH 2.8, 2.9 or 3.0, adjusted to LF=19 mS/cm with water application solution: 100% A wash solution: 100% A wash volume: 96.2 ml (5 column volumes (CV)) wash solution (wash 2): 100% Additional wash (wash 2) wash volume (wash 2): 96.2 ml (5 column volumes (CV)) wash solution (wash 3): 100% A wash volume (wash 3): 57.7 ml (3 column volumes (CV)) linear gradient elution solution: 100% B linear gradient: within 196 ml (10 column volumes) to 100% B wavelength: 280 nm

(25) The results are shown below:

(26) TABLE-US-00004 Wash 2 Wash 2 Wash 2 (additional (additional (additional wash) pH 2.8, wash) pH 2.9, wash) pH 3.0, 19 mS/cm 19 mS/cm 19 mS/cm Purity Pool 100 100 100 MonoPEG EPO [%] Yield 65.57 59.06 45.02 MonoPEG EPO [%]

(27) Yields are calculated based on the monoPEGylated EPO content in the starting material.