Method for the production of a glycosylated immunoglobulin

Patent metadata

Field	Value
Publication number	US-11021728-B2
Application number	US-202016790177-A
Country	US
Kind code	B2
Filing date	Feb 13, 2020
Priority date	Oct 26, 2009
Publication date	Jun 1, 2021
Grant date	Jun 1, 2021

Abstract

Herein is reported a method for the production of an immunoglobulin comprising the following steps: a) providing a eukaryotic cell comprising a nucleic acid encoding the immunoglobulin, b) cultivating the eukaryotic cell in a cultivation medium wherein the amount of glucose available in the cultivation medium per time unit is kept constant and limited to less than 80% of the amount that could maximally be utilized by the cells in the cultivation medium per time unit, and c) recovering the immunoglobulin from the culture.

First claim

What is claimed:

1. A composition comprising Tocilizumab protein with mannose-5 glycostructure (M5) attached to Asn 297 of the Tocilizumab protein, wherein the fraction of M5 is in a range from 2.8% to 6% of the sum comprising M5, G(0), G(1), and G(2) oligosaccharide attached to Asn 297 of the Tocilizumab protein, wherein the fraction equals area % fraction determined in a liquid chromatography method.

2. The composition according to claim 1 , wherein the Tocilizumab protein with M5 attached thereto has been produced by a recombinant Chinese Hamster Ovary (CHO) cell.

3. The composition according to claim 2 , wherein the CHO cell is cultured in cell culture at cell density of 10 5 cells/ml or more.

4. The composition according to claim 3 , wherein the cell density is 8-12×10 5 cells/ml or more.

5. The composition according to claim 2 , wherein the relative antibody concentration produced by the CHO cell is greater than 100% of the antibody concentration resulting from constant feeding of CHO cells.

6. The composition according to claim 2 , wherein the Tocilizumab protein is produced by the CHO cell cultured in cell culture at cell density of 10 5 cells/ml or more.

7. The composition according to claim 2 , wherein the Tocilizumab protein is produced by a cell culture of CHO cells at a cultivation volume from 10,000 L-50,000 L.

8. The composition according to claim 1 , wherein the M5 fraction is in a range from 2.8 to 4%.

9. A composition comprising Tocilizumab protein with mannose-5 glycostructure (M5) attached to Asn297 thereof, wherein the fraction of M5 is in a range from 2.8 to less than 5% of the sum comprising M5, G(0), G(1), and G(2) oligosaccharide attached to Asn297 of the Tocilizumab protein, wherein the fraction equals area % fraction determined in a liquid chromatography method.

10. The composition according to claim 9 , wherein the Tocilizumab protein with M5 attached thereto has been produced by a recombinant Chinese Hamster Ovary (CHO) cell.

11. The composition according to claim 10 , wherein the CHO cell is cultured in cell culture at cell density of 10 5 cells/ml or more.

12. The composition according to claim 11 , wherein the cell density is 8-12×10 5 cells/ml or more.

13. The composition according to claim 10 , wherein the relative antibody concentration produced by the CHO cell is greater than 100% of the antibody concentration resulting from constant feeding of CHO cells.

14. The composition according to claim 10 , wherein the Tocilizumab protein is produced by the CHO cell cultured in cell culture at cell density of 10 5 cells/ml or more.

15. The composition according to claim 10 , wherein the Tocilizumab protein is produced by a cell culture of CHO cells at a cultivation volume from 10,000-50,000 L.

16. The composition according to claim 9 , wherein the M5 fraction is in a range from 2.8 to 4%.

Assignees

• Hoffmann La Roche
• Chugai Pharmaceutical Co Ltd

Inventors

• Franze Reinhard
• Hirashima Chikashi
• Link Thomas
• Takagi Yoshinori
• Takuma Shinya
• Tsuda Yuriko

Classifications

• C07K16/2866 Primary

  against receptors for cytokines, lymphokines, interferons · CPC title

• C07K2317/41

  Glycosylation, sialylation, or fucosylation · CPC title

• C07K2317/14

  Specific host cells or culture conditions, e.g. components, pH or temperature · CPC title

• A61K39/395

  Antibodies (agglutinins A61K38/36 {; as drug carriers A61K47/50}); Immunoglobulins; Immune serum, e.g. antilymphocytic serum · CPC title

• C07K2317/24

  containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered · CPC title