Immunoglobulin purification

Patent metadata

Field	Value
Publication number	US-11261238-B2
Application number	US-202016780382-A
Country	US
Kind code	B2
Filing date	Feb 3, 2020
Priority date	Jun 1, 2007
Publication date	Mar 1, 2022
Grant date	Mar 1, 2022

Abstract

The current invention reports a method for purifying an immunoglobulin, wherein the method comprises applying an aqueous, buffered solution comprising an immunoglobulin in monomeric and in aggregated form to a cation exchange material under conditions whereby the immunoglobulin in monomeric form does not bind to the cation exchange material, and recovering the immunoglobulin in monomeric form from the solution after the contact with the cation exchange material.

First claim

The invention claimed is:

1. A method for obtaining an immunoglobulin in monomeric form from a solution comprising the immunoglobulin in monomeric and in aggregated form, comprising the steps of:
1) applying an aqueous, buffered solution comprising said immunoglobulin in monomeric and in aggregated form to an affinity column under conditions whereby said immunoglobulin binds to said affinity column, and recovering said immunoglobulin in monomeric and in aggregated form from said affinity column;
2) applying an aqueous, buffered solution comprising said immunoglobulin in monomeric and in aggregated form to an anion exchange material under conditions whereby said immunoglobulin does not bind to said anion exchange material, and recovering said immunoglobulin in monomeric and in aggregated form from said solution after the contact with said anion exchange material; and
3) applying an aqueous, buffered solution comprising said immunoglobulin in monomeric and in aggregated form to a cation exchange material under conditions whereby said immunoglobulin in monomeric form does not bind to said cation exchange material, and recovering said immunoglobulin in monomeric form from said solution after the contact with said cation exchange material,
wherein:
said aggregated form is an immunoglobulin molecule associated either covalently or non-covalently with a least one additional immunoglobulin molecule,
said anion exchange material is a chromatography material comprising only anionic charged groups as chromatographically active substituents, and
said cation exchange material is a chromatography material comprising only cationic charged groups as chromatographically active substituents.

2. The method according to claim 1 , characterized in that said step 3) is a chromatographic step operated in flow-through mode comprising applying an aqueous, buffered solution comprising said immunoglobulin in monomeric and in aggregated form to a cation exchange material under conditions whereby said immunoglobulin in monomeric form does not bind to said cation exchange material, and recovering said immunoglobulin in monomeric form from said solution after the contact with said cation exchange material.

3. The method according to claim 2 , characterized in that said step 2) is a chromatographic step operated in flow through-mode comprising applying an aqueous, buffered solution comprising said immunoglobulin in monomeric and in aggregated form to an anion exchange material under conditions whereby said immunoglobulin does not bind to said anion exchange material, and recovering said immunoglobulin in monomeric and in aggregated form from said solution after the contact with said anion exchange material.

4. The method according to claim 3 , characterized in that said step 1) is a chromatographic step operated in bind-and-elute mode comprising applying an aqueous, buffered solution comprising said immunoglobulin in monomeric and in aggregated form to an affinity column under conditions whereby said immunoglobulin binds to said affinity column, and recovering said immunoglobulin in monomeric and in aggregated form from said affinity column.

5. The method according to claim 1 , characterized in that in said step 3) the cation exchange material is a membrane cation exchange material.

6. The method according to claim 5 , characterized in that said membrane cation exchange material is a polyethersulfone based membrane or a regenerated cellulose based membrane modified with sulfonic acid groups or carboxymethyl groups.

7. The method according to claim 1 , characterized in that said aqueous, buffered solution of step 3 ) has a pH value of from pH 5 to pH 8.

8. The method according to claim 1 , characterized in that said aqueous, buffered solution of step 3 ) has a conductivity of from 1.0 to 15.0 mS/cm.

9. The method according to claim 8 , characterized in that said aqueous, buffered solution of step 3 ) has a conductivity of from 4.0 to 10.0 mS/cm.

10. The method according to claim 1 , characterized in that said recovering said immunoglobulin in monomeric form from the flow-through is by a method selected from precipitation, salting out, ultrafiltration, diafiltration, lyophilization, affinity chromatography, or solvent volume reduction to obtain a concentrated solution.

11. The method according to claim 10 , characterized in that said recovering is by ultrafiltration, lyophilization, or solvent volume reduction.

12. The method according to claim 1 , characterized in that of said immunoglobulin obtained from the flow-through of the membrane cation exchange material at least 95% of the immunoglobulin is in monomeric from.

13. The method according to claim 1 , characterized in that at least 90% of the immunoglobulin in monomeric form does not bind to the cation exchange material.

14. The method according to claim 1 , characterized in that said aqueous, buffered solution is a solution comprising phosphoric acid or salts thereof, citric acid or salts thereof, or histidine or salts thereof.

15. The method according to claim 1 , characterized in that said aqueous, buffered solution comprises sodium chloride or potassium chloride.

16. The method according to claim 1 , characterized in that the sum of pH value and conductivity in mS/cm of the aqueous, buffered solution in step 3 ) is in the range of from 9 to 18.

17. The method according to claim 16 , characterized in that said sum is in the range of from 10 to 15.

Assignees

• Hoffmann La Roche

Inventors

• Falkenstein Roberto
• Giessel Claudia

Classifications

• C07K16/32

  against translation products of oncogenes · CPC title

• C07K16/065 Primary

  Purification, fragmentation · CPC title

• C07K1/18 Primary

  Ion-exchange chromatography · CPC title

• C07K16/244

  Interleukins [IL] · CPC title

• C07K16/00

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