Methods for producing aflibercept in chemically defined media having reduced aflibercept variants

Patent metadata

Field	Value
Publication number	US-11535663-B2
Application number	US-202117383322-A
Country	US
Kind code	B2
Filing date	Jul 22, 2021
Priority date	Dec 6, 2019
Publication date	Dec 27, 2022
Grant date	Dec 27, 2022

Abstract

The present disclosure pertains to compositions comprising anti-VEGF proteins and methods for producing such compositions.

First claim

What is claimed is:

1. A method of producing aflibercept harvested from a host cell cultured in a chemically defined medium (CDM), comprising:
a) culturing said host cell in said CDM under conditions suitable in which said host cell expresses said aflibercept, wherein the cumulative concentration of copper in said CDM is less than or equal to 0.8 μM or about 0.8 μM and the cumulative concentration of nickel in said CDM is less than or equal to 0.4 μM, and one or more of the following:
i) the cumulative concentration of iron in said CDM is less than or equal to 55.0 μM;
ii) the cumulative concentration of zinc in said CDM is less than or equal to 56.0 μM;
iii) the cumulative concentration of cysteine in said CDM is less than or equal to 10.0 mM; and
iv) said CDM includes anti-oxidants where the cumulative concentration of an antioxidant is about 0.001 mM to about 10.0 mM for any single anti-oxidant; and

b) harvesting aflibercept produced by said host cell.

2. The method of claim 1 , wherein said harvest has a color no more yellow-brown than European Color Standard BY2, wherein the aflibercept concentration is 5.0 g/L.

3. The method of claim 1 , wherein said anti-oxidants are taurine, hypotaurine, glycine, thioctic acid, glutathione, choline, hydrocortisone, Vitamin C, Vitamin E or combinations thereof.

4. The method of claim 1 , wherein culturing said host cell in said CDM increases titer of said aflibercept harvested compared to a CDM, wherein the cumulative concentration of copper in said CDM is more than 0.8 μM.

5. The method of claim 1 , wherein aflibercept titer, viable cell concentration, viability, ammonia or osmolality is substantially unchanged.

6. The method of claim 1 , wherein said host cell is selected from the group consisting of CHO, NS0, Sp2/0, embryonic kidney cell and BHK.

7. The method of claim 1 , wherein said harvest comprises one or more aflibercept variants, wherein said variants have at least one oxidized amino acid residue.

8. The method of claim 7 , wherein said oxidized amino acid residue is selected from the group consisting of methionine, tryptophan, histidine, phenylalanine, tyrosine and a combination thereof.

9. The method of claim 7 , wherein said aflibercept variant comprises a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 and combinations thereof.

10. The method of claim 1 , wherein the harvest has a color that is:
a) no more yellow-brown than European Color Standard BY2;
b) no more yellow-brown than European Color Standard BY3;
c) no more yellow-brown than European Color Standard BY4;
d) no more yellow-brown than European Color Standard BY5;
e) between European Color Standard BY2 and BY3; or
f) between European Color Standard BY2 and BY4, and
wherein the aflibercept concentration in the harvest is 5.0 g/L.

Assignees

• Regeneron Pharma

Inventors

• Lawrence Shawn
• Johnson Amy
• Casey Meghan
• Mastrogiacomo Jaimie
• Wang Shunhai
• Li Ning

Classifications

• A61K38/179 Primary

  for growth factors; for growth regulators · CPC title

• C07K14/71 Primary

  for growth factors; for growth regulators · CPC title

• C07K2319/33

  fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies · CPC title

• C12N15/66

  General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease · CPC title

• C07K16/28

  against receptors, cell surface antigens or cell surface determinants · CPC title