Anti-VEGF protein compositions and methods for producing the same

Patent metadata

Field	Value
Publication number	US-11753459-B2
Application number	US-202217858724-A
Country	US
Kind code	B2
Filing date	Jul 6, 2022
Priority date	Dec 6, 2019
Publication date	Sep 12, 2023
Grant date	Sep 12, 2023

Abstract

The present disclosure pertains to compositions comprising anti-VEGF proteins.

First claim

What is claimed is:

1. An oxo-aflibercept composition produced in a cell culture media comprising a chemically defined media (CDM), wherein one or more amino acid residues of aflibercept is oxidized and
wherein said one or more amino acid residues is histidine and/or tryptophan, and
wherein said oxo-aflibercept has a glycosylation profile based on molar percentage selected from the group consisting of about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 2% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans.

2. The oxo-aflibercept composition of claim 1 , wherein said oxo-aflibercept has about 40% to about 50% total fucosylated glycans.

3. The oxo-aflibercept composition of claim 1 , wherein said oxo-aflibercept has about 30% to about 55% total sialylated glycans.

4. The oxo-aflibercept composition of claim 1 , wherein said oxo-aflibercept has about 2% to about 15% mannose-5.

5. The oxo-aflibercept composition of claim 1 , wherein said oxo-aflibercept has about 60% to about 79% galactosylated glycans.

6. The oxo-aflibercept composition of claim 1 , wherein said oxo-aflibercept is glycosylated at one or more Asparagines as follows: G0-GlcNAc glycosylation; G1-GlcNAc glycosylation; G1S-GlcNAc glycosylation; G0 glycosylation; G1 glycosylation; G1S glycosylation; G2 glycosylation; G2S glycosylation; G2S2 glycosylation; G0F glycosylation; G2F2S glycosylation; G2F2S2 glycosylation; G1F glycosylation; G1FS glycosylation; G2F glycosylation; G2FS glycosylation; G2FS2 glycosylation; G3FS glycosylation; G3FS3 glycosylation; G0-2GlcNAc glycosylation; Man4 glycosylation; Man4_A1G1 glycosylation; Man4_A1G1S1 glycosylation; Man5 glycosylation; Man5_A1G1 glycosylation; Man5_A1G1S1 glycosylation; Man6 glycosylation; Man6_G0+Phosphate glycosylation; Man6+Phosphate glycosylation; and/or Man7 glycosylation.

7. A culture media composition comprising chemically defined media (CDM) and oxo-aflibercept, wherein one or more amino acid residues of aflibercept is oxidized and
wherein said one or more amino acid residues is histidine and/or tryptophan, and
wherein said oxo-aflibercept has a glycosylation profile based on molar percentage selected from the group consisting of about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 2% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans.

8. The oxo-aflibercept composition of claim 7 , wherein said oxo-aflibercept has about 40% to about 50% total fucosylated glycans.

9. The oxo-aflibercept composition of claim 7 , wherein said oxo-aflibercept has about 30% to about 55% total sialylated glycans.

10. The oxo-aflibercept composition of claim 7 , wherein said oxo-aflibercept has about 2% to about 15% mannose-5.

11. The oxo-aflibercept composition of claim 7 , wherein said oxo-aflibercept has about 60% to about 79% galactosylated glycans.

12. The oxo-aflibercept composition of claim 7 , wherein said oxo-aflibercept is glycosylated at one or more Asparagines as follows: G0-GlcNAc glycosylation; G1-GlcNAc glycosylation; G1S-GlcNAc glycosylation; G0 glycosylation; G1 glycosylation; G1S glycosylation; G2 glycosylation; G2S glycosylation; G2S2 glycosylation; G0F glycosylation; G2F2S glycosylation; G2F2S2 glycosylation; G1F glycosylation; G1FS glycosylation; G2F glycosylation; G2FS glycosylation; G2FS2 glycosylation; G3FS glycosylation; G3FS3 glycosylation; G0-2GlcNAc glycosylation; Man4 glycosylation; Man4_A1G1 glycosylation; Man4_A1G1S1 glycosylation; Man5 glycosylation; Man5_A1G1 glycosylation; Man5_A1G1S1 glycosylation; Man6 glycosylation; Man6_G0+Phosphate glycosylation; Man6+Phosphate glycosylation; and/or Man7 glycosylation.

13. The oxo-aflibercept composition of claim 7 , wherein the N-glycosylation of the F(ab′) 2 portion of said oxo-aflibercept at Asn36 is between 98.3% and 99.4%.

14. The oxo-aflibercept composition of claim 7 , wherein the N-glycosylation of the F(ab′) 2 portion of said oxo-aflibercept at Asn68 is between 51.9% and 64.9%.

15. The oxo-aflibercept composition of claim 7 , wherein the N-glycosylation of the F(ab′) 2 portion of said oxo-aflibercept at Asn123 is between 98.4% and 99.9%.

16. The oxo-aflibercept composition of claim 7 , wherein the N-glycosylation of the F(ab′) 2 portion of said oxo-aflibercept at Asn196 is between 55.1% and 98.6%.

17. A composition comprising oxo-aflibercept, wherein one or more amino acid residues of aflibercept is oxidized and
wherein said one or more amino acid residues is histidine and/or tryptophan, and
wherein said oxo-aflibercept has a glycosylation profile based on molar percentage selected from the group consisting of about 40% to about 52.4% total fucosylated glycans, about 30% to about 59.2% total sialylated glycans, about 5.2% to about 15% mannose-5, and about 60% to about 80.6% galactosylated glycans.

18. The oxo-aflibercept composition of claim 17 , wherein said oxo-aflibercept has about 40% to about 50% total fucosylated glycans.

19. The oxo-aflibercept composition of claim 17 , wherein said oxo-aflibercept has about 30% to about 55% total sialylated glycans.

20. The oxo-aflibercept composition of claim 17 , wherein said oxo-aflibercept has about 2% to about 15% mannose-5.

21. The oxo-aflibercept composition of claim 17 , wherein said oxo-aflibercept has about 60% to about 79% galactosylated glycans.

22. The oxo-aflibercept composition of claim 17 , wherein said oxo-aflibercept has about 51.4% to about 52.4% total fucosylated glycans.

23. The oxo-aflibercept composition of claim 17 , wherein said oxo-aflibercept has about 58.2% to about 59.2% total sialylated glycans.

24. The oxo-aflibercept composition of claim 17 , wherein said oxo-aflibercept has about 5.19% to about 5.24% mannose-5.

25. The oxo-aflibercept composition of claim 17 , wherein said oxo-aflibercept has about 79.6% to about 80.6% galactosylated glycans.

26. The oxo-aflibercept composition of claim 17 , wherein said oxo-aflibercept is glycosylated at one or more Asparagines as follows: G0-GlcNAc glycosylation; G1-GlcNAc glycosylation; G1S-GlcNAc glycosylation; G0 glycosylation; G1 glycosylation; G1S glycosylation; G2 glycosylation; G2S glycosylation; G2S2 glycosylation; G0F glycosylation; G2F2S glycosylation; G2F2S2 glycosylation; G1F glycosylation; G1FS glycosylation; G2F glycosylation; G2FS glycosylation; G2FS2 glycosylation; G3FS glycosylation; G3FS3 glycosylation; G0-2GlcNAc glycosylation; Man4 glycosylation; Man4_A1G1 glycosylation; Man4_A1G1S1 glycosylation; Man5 glycosylation; Man5_A1G1 glycosylation; Man5_A1G1S1 glycosylation; Man6 glycosylation; Man6_G0+Phosphate glycosylation; Man6+Phosphate glycosylation; and/or Man7 glycosylation.

27. The oxo-aflibercept composition of claim 13 , wherein the N-glycosylation of F(ab′) 2 portion of said oxo-aflibercept at SEQ ID NO. 100 is between 98.3% and 99.4%.

28. The oxo-aflibercept composition of claim 14 , wherein the N-glycosylation of F(ab′) 2 portion of said oxo-aflibercept at SEQ ID NO. 101 is between 51.9% and 64.9%.

29. The oxo-aflibercept composition of claim 15 , wherein the N-glycosylation of F(ab′) 2 portion of said oxo-aflibercept at SEQ ID NO. 102 is between 98.4% and 99.9%.

30. The oxo-aflibercept composition of claim 16 , wherein the N-glycosylation of F(ab′) 2 portion

Assignees

• Regeneron Pharma

Inventors

• Wang Shunhai
• Li Ning
• Chen Hunter
• Bhupender Bhalla Amardeep Singh
• Lawrence Shawn M
• Johnson Amy S
• Casey Meghan E
• Grapel Jaimie

Classifications

• C12N2529/10

  Stimulation by light · CPC title

• C12N15/85

  for animal cells · CPC title

• C07K1/06

  using protecting groups or activating agents {(C07K1/003, C07K1/006 take precedence)} · CPC title

• C12N2510/02

  Cells for production · CPC title

• C07K1/165

  mixed-mode chromatography · CPC title