Separation method

Patent metadata

Field	Value
Publication number	US-11667671-B2
Application number	US-202016911511-A
Country	US
Kind code	B2
Filing date	Jun 25, 2020
Priority date	May 11, 2016
Publication date	Jun 6, 2023
Grant date	Jun 6, 2023

Abstract

The invention relates to a method of isolating an immunoglobulin, comprising the steps of:a) providing a separation matrix comprising at least 15 mg/ml multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, wherein the porous support comprises cross-linked polymer particles having a volume-weighted median diameter (d50,v) of 56-70 micrometers and a dry solids weight of 55-80 mg/ml;b) contacting a liquid sample comprising an immunoglobulin with the separation matrix;c) washing the separation matrix with a washing liquid;d) eluting the immunoglobulin from the separation matrix with an elution liquid; ande) cleaning the separation matrix with a cleaning liquid comprising at least 0.5 M NaOH.

First claim

What is claimed is:

1. A method of isolating an immunoglobulin, comprising the steps of:
a) providing a separation matrix comprising at least 15 mg/ml multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, wherein said porous support comprises cross-linked polymer particles having a pore size corresponding to an inverse gel filtration chromatography Kd value of 0.69-0.85 for dextran of Mw 110 kDa;
b) contacting a liquid sample comprising an immunoglobulin with said separation matrix;
c) washing said separation matrix with a washing liquid;
d) eluting the immunoglobulin from the separation matrix with an elution liquid; and
e) cleaning the separation matrix with a cleaning liquid comprising more than 2M NaOH,
wherein in step e) the contact time between the separation matrix and the cleaning liquid is 3 minutes or less.

2. The method of claim 1 , wherein said separation matrix has a maximum pressure of at least 0.58 MPa when packed at 300+/−10 mm bed height in a chromatography column having a diameter of 35 mm.

3. The method of claim 1 , wherein said immunoglobulin comprises an Fc fusion protein.

4. The method of claim 1 , wherein said immunoglobulin has a hydrodynamic radius of at least 6.0 nm.

5. The method of claim 1 , wherein said immunoglobulin comprises a bispecific, trispecific or polyspecific antibody.

6. The method of claim 5 , wherein said method separates half antibodies or homodimeric antibodies from said bispecific, trispecific or polyspecific antibody.

7. The method of claim 1 , wherein said immunoglobulin comprises a conjugated antibody.

8. The method of claim 1 , wherein said immunoglobulin comprises an antibody fragment.

9. The method of claim 1 , wherein the separation matrix is a Protein A chromatography column.

10. The method of claim 1 , wherein steps b)-e) are repeated at least 10 times.

11. The method of claim 1 , wherein in step b) the pH is 6-8.

12. The method of claim 1 , wherein in step b) the residence time of said liquid sample on said separation matrix is 2-20 min.

13. The method of claim 1 , which is performed in a continuous or semicontinuous multicolumn chromatography process.

14. The method of claim 1 , which is performed in a periodic countercurrent chromatography (PCC) process.

15. The method of claim 1 , wherein the separation matrix is an antibody affinity chromatography cross-linked agarose matrix having alkaline-stabilized protein A-derived ligands.

Assignees

• Cytiva Bioprocess R & D Ab

Inventors

• Forss Annika
• Ander Mats
• Bjorkman Tomas
• Blom Hans
• Hansson Jesper
• Rodrigo Gustav

Classifications

• C07K1/22

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• C07K16/00 Primary

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• B01D15/1864

  using two or more columns · CPC title

• B01D15/34

  Size-selective separation, e.g. size-exclusion chromatography; Gel filtration; Permeation · CPC title

• B01J20/285

  based on polymers · CPC title