Method for separation of monomeric polypeptides from aggregated polypeptides

Patent metadata

Field	Value
Publication number	US-10654886-B2
Application number	US-201715707888-A
Country	US
Kind code	B2
Filing date	Sep 18, 2017
Priority date	Jul 1, 2011
Publication date	May 19, 2020
Grant date	May 19, 2020

Abstract

Herein is reported a method for obtaining a polypeptide in monomeric form comprising the steps of a) providing a solution comprising the polypeptide in monomeric form and in aggregated form, wherein the ratio of monomeric to aggregated form is 4:1 or less as determined by size exclusion chromatography, b) performing a mixed-mode chromatography in bind-and-elute mode, or a hydrophobic interaction chromatography in flow-through mode, or a size-exclusion chromatography, and c) performing a weak cation exchange chromatography in bind-and-elute mode or flow-through mode, and thereby obtaining the polypeptide in monomeric form.

First claim

The invention claimed is:

1. A method for removing aggregates from a solution with high aggregate content, comprising the steps of:
a) applying a solution comprising the polypeptide in monomeric form and in aggregated form, wherein the aggregated form is 20% or more as determined by size exclusion chromatography of the total. polypeptide in the solution, to a mixed-mode Chromatography material. and recovering the polypeptide from the mixed-mode chromatography material, wherein the polypeptide recovered from the mixed-mode chromatography material has a reduced aggregate content; and
b) applying the solution obtained in the previous step to a weak cation exchange chromatography material and recovering the polypeptide therefrom and thereby producing the monomeric polypeptide,
wherein the solution applied to the mixed-mode chromatography material and/or the weak cation exchange chromatography material has a pH value at least 0.2 pH units and at most 2.5 pH units above the isoelectric point of the polypeptide.

2. The method according to claim 1 , characterized in that the solution applied to the mixed-mode chromatography material and/or the weak cation exchange chromatography material is a buffered solution with a buffer concentration of from about 1 mmol/l to about 150 mmol/l.

3. The method according to claim 2 , characterized in that the buffer is selected from citrate buffer, acetate buffer and MES buffer.

4. The method according to claim 1 , characterized in that the recovering from the weak cation exchange chromatography material is from the eluate of the weak cation exchange chromatography material or from the flow-through of the weak cation exchange chromatography material.

5. The method according to claim 1 , characterized in that the applying to and recovering from the mixed-mode chromatography material comprises the following steps
applying to the mixed-mode chromatography material a solution comprising a buffer with a pH value of the IEP of the polypeptide plus about 0.7 pH units to about 1.3 pH units,
adjusting the solution comprising the polypeptide prior to the applying to the chromatography material to a pH value of the IEP of the polypeptide plus about 0.7 pH units to about 1.3 pH units,
applying the solution comprising the polypeptide to the mixed-mode chromatography material,
recovering the polypeptide from the mixed-mode chromatography material by applying a solution comprising a phosphate buffer with a pH value of the IEP of the polypeptide plus about 1.7 pH units to about 2.3 pH units.

6. The method according to claim 5 , characterized in that the mixed-mode chromatography buffer has a conductivity of about 1.9 mS/cm.

7. The method according to claim 1 , characterized in that the applying to the weak cation exchange chromatography material and the recovering of the polypeptide comprises the following steps
applying to the weak cation exchange chromatography material a solution comprising a buffer with a pH value of the IEP of the polypeptide plus about 0.2 pH units to about 1.0 pH units,
adjusting the solution comprising the polypeptide prior to the applying to the chromatography material to a pH value of the IEP of the polypeptide plus about 0.2 pH units to about 1.0 pH units,
applying the solution comprising the polypeptide to the weak cation exchange chromatography material,
recovering the polypeptide from the weak cation exchange chromatography material by applying a solution comprising sodium chloride or potassium chloride at a concentration of from about 210 mmol/l to about 240 mmol/l, or
recovering the polypeptide from the weak cation exchange chromatography material with a solution of a pH value of from about pH 6.0 to about pH 8.0.

8. The method according to claim 7 , characterized in that in the recovering the pH value is about pH 5.0 and the sodium chloride concentration is about 230 mmol/l, or the pH value is about pH 5.2 and the sodium chloride concentration is about 210 mmol/l.

9. The method according to claim 7 , characterized in that the recovering is by an isocratic elution.

10. The method according to claim 7 , characterized in that the recovering is by adding 10% (v/v) of an acetate buffer of a pH value of from about pH 6.0 to about pH 6.5 or the recovering is by adding 60% (v/v) of a MES buffer of a pH value of about pH 8.0.

11. The method according to claim 1 , characterized in that the applying to the weak cation exchange chromatography material and the recovering of the polypeptide comprises the following steps
applying to the weak cation exchange chromatography material a solution with a pH value of the IEP of the polypeptide plus about 0.2 pH units to about 1.2 pH units,
adjusting the solution comprising the polypeptide prior to the applying to the chromatography material to a pH value of the IEP of the polypeptide plus about 0.2 pH units to about 1.2 pH units,
applying the solution to a weak cation exchange chromatography material,
recovering the polypeptide from the flow-through.

12. The method according to claim 11 , characterized in that the adjusting is to an acetate buffer of about pH 5.1 and of from about 100 mol/1 to about 120 mmol/l, or to an acetate buffer of about pH 5.5 and of about 40 mmol/l to about 55 mmol/l, or to an acetate buffer of about pH 6.0 and of about 10 mmol/l.

13. The method according to claim 11 , characterized in that the buffer in step a) is an acetate buffer of about pH 5.3 and of about 50 mmol/l to about 75 mmol/l, or an acetate buffer of about pH 5.5 and of about 7.5 mmol/l to about 15 mmol/l.

Assignees

• Hoffmann La Roche

Inventors

• Baehner Monika
• Grossmann Adelbert
• Hepbildikler Stefan

Classifications

• C07K1/20

  Partition-, reverse-phase or hydrophobic interaction chromatography · CPC title

• C07K1/165 Primary

  mixed-mode chromatography · CPC title

• C07K1/36

  by a combination of two or more processes of different types · CPC title

• C07K2319/30

  Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto · CPC title

• C07K14/5443

  IL-15 · CPC title