IP Library › Granted Patent US 12,351,613
Granted Patent B2
US 12,351,613 · App. 17/823,883 · Granted Jul 8, 2025

Production of glycoproteins using manganese

Inventors: Christopher Kenyon Crowell (Thousand Oaks, CA); Gustavo Enrique Grampp (Thousand Oaks, CA)
Assignee: AMGEN INC.
C07K14/505C07K14/575C12N5/0018C12P21/005C12N2500/20C12N2500/32C12N2510/02
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Quick Facts
Patent No.
US 12,351,613
App. No.
17/823,883
Granted
Jul 8, 2025
Kind
B2
Abstract

Culture media comprising manganese and methods of culturing cells to improve sialylation and glycosylation of glycoproteins are provided.

Claims (20)

1. A method for producing an erythropoietic composition comprising sialylated erythropoiesis-stimulating glycoproteins, comprising the steps of:

(a) growing a manganese-responsive CHO host cell which produces recombinant erythropoiesis-stimulating glycoproteins in a culture medium containing a concentration of manganese from 0.4 to 40 μM, wherein the erythropoiesis-stimulating glycoproteins comprise a mature protein comprising amino acids 1-165 of SEQ ID NO: 2 or amino acids 1-165 of SEQ ID NO: 3; and

(b) recovering an erythropoietic composition wherein less than 5% of the erythropoiesis-stimulating glycoproteins are lower sialylated.

2. The method of claim 1 , in which the concentration of manganese is effective to increase the percentage of highly sialylated erythropoiesis-stimulating glycoproteins.

3. The method of claim 1 , wherein the concentration of manganese is effective to increase the percentage of erythropoiesis-stimulating glycoproteins which are glycosylated at potential O-linked glycosylation sites.

4. The method of claim 1 , wherein the concentration of manganese is effective to increase the percentage of galactose among the sugars attached to the erythropoiesis-stimulating glycoproteins.

5. The method of claim 1 , wherein the culture medium is essentially serum-free.

6. The method of claim 1 , wherein the concentration of manganese is from 0.4 to 10 μM.

7. The method of claim 1 , wherein the concentration of manganese is from 0.4 to 4 μM.

8. The method of claim 1 , wherein the culture medium further comprises one or more supplementary amino acids selected from the group consisting of asparagine, aspartic acid, cysteine, cystine, isoleucine, leucine, tryptophan, and valine.

9. The method of claim 1 , wherein the CHO host cells are grown in roller bottles.

10. The method of claim 1 , wherein the manganese is added after a rapid cell growth phase.

11. The method of claim 10 , wherein the rapid cell growth phase lasts for a period ranging between 2 and 20 days.

12. The method of claim 11 , wherein the manganese is added after two harvest cycles.

13. The method of claim 12 , wherein the first harvest cycle is 8 days long and the second harvest cycle is 7 days long.

14. The method of claim 1 , further comprising the step of recovering the erythropoietic composition which comprises highly sialylated recombinant erythropoiesis-stimulating glycoproteins from the host cells and culture medium.

15. The method of claim 1 , wherein the erythropoiesis-stimulating glycoproteins comprise a mature protein comprising amino acids 1-165 of SEQ ID NO: 3.

16. The method of claim 15 , wherein the erythropoiesis-stimulating glycoproteins comprise the amino acid sequence of SEQ ID NO: 3.

17. The method of claim 1 , wherein the erythropoiesis-stimulating glycoproteins comprise a mature protein comprising amino acids 1-165 of SEQ ID NO: 2.

18. The method of claim 17 , wherein the erythropoiesis-stimulating glycoproteins comprise the amino acid sequence of SEQ ID NO: 2.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 6, 2023
From: CROWELL, CHRISTOPHER K.; GRAMPP, GUSTAVO E.
To: AMGEN INC.
Reel/Frame 062603/0695 →
Continuity (8)
Continuation 16947265 · Jul 24, 2020
Continuation 15683521 · Aug 22, 2017
Continuation 15004716 · Jan 22, 2016
Continuation 14087747 · Nov 22, 2013
Continuation 13112940 · May 20, 2011
Continuation 11634506 · Dec 6, 2006
Provisional Application 60748880 · Dec 8, 2005
Related Publication 20220402986A1 · Dec 22, 2022
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