Anti-VEGF protein compositions and methods for producing the same

1. An oxo-aflibercept composition 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 51.4% to about 52.4% total fucosylated glycans.

3. The oxo-aflibercept composition of claim 2 , wherein said oxo-aflibercept has about 58.2% to about 59.2% total sialylated glycans.

4. The oxo-aflibercept composition of claim 1 , wherein said oxo-aflibercept has about 5.19% to about 5.24% mannose-5.

5. The oxo-aflibercept composition of claim 1 , wherein said oxo-aflibercept has about 79.6% to about 80.6% 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; GIFS 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. An oxo-aflibercept composition wherein one or more amino acid residues of histidine and/or tryptophan is oxidized on the F(ab′) 2 portion of said oxo-aflibercept.

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

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

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

11. The oxo-aflibercept composition of claim 10 , wherein N-glycosylation of the F(ab′) 2 portion of said oxo-aflibercept at Asn196 is between 55.1% and 98.6%.

12. A prefilled syringe comprising aflibercept and oxidized variants of aflibercept that have at least one oxidized amino acid residue selected from the group consisting of His86, His110, His145, His209, His95, His19, His203, Trp58, Trp138 and a combination thereof.

13. The prefilled syringe of claim 12 , wherein said aflibercept and oxidized variants of aflibercept in said prefilled syringe has about 40% to about 50% total fucosylated glycans.

14. The prefilled syringe of claim 12 , wherein said aflibercept and oxidized variants of aflibercept in said prefilled syringe has about 30% to about 55% total sialylated glycans.

15. The prefilled syringe of claim 12 , wherein said aflibercept and oxidized variants of aflibercept in said prefilled syringe has about 2% to about 15% mannose-5.

16. The prefilled syringe of claim 12 , wherein said aflibercept and oxidized variants of aflibercept in said prefilled syringe has about 60% to about 79% galactosylated glycans.

17. The prefilled syringe of claim 16 , wherein said aflibercept and oxidized variants of aflibercept in said prefilled syringe has a b* value between 0.5 and 5, when the concentration of said aflibercept is normalized to 5.0 g/L.

18. The prefilled syringe of claim 17 , wherein said aflibercept and oxidized variants of aflibercept in said prefilled syringe has a BY value of at least 6 when the concentration of aflibercept is normalized to 5 g/L protein concentration.

19. The prefilled syringe of claim 16 , wherein said aflibercept and oxidized variants of aflibercept in said prefilled syringe has between 0.1% and 1% oxidized amino acid residue selected from the group consisting of tryptophan, histidine, and a combination thereof.

20. The prefilled syringe of claim 12 , wherein said aflibercept and oxidized variants of aflibercept in said prefilled syringe has between 0.2% and 1% oxidized amino acid residue selected from the group consisting of tryptophan, histidine, and a combination thereof.

21. The prefilled syringe of claim 17 , wherein said aflibercept and oxidized variants of aflibercept loaded into said prefilled syringe is formulated with (a) 40 to 250 mg/ml of aflibercept (b) a pH between 5.5 and 7.5, and (c) one or more pharmaceutically acceptable excipients selected from the group of buffering agents, preservatives, detergents and combinations thereof.

22. The prefilled syringe of claim 17 , wherein said one or more pharmaceutically acceptable excipients includes 5-40 mM of a buffer.

23. The prefilled syringe of claim 21 , further comprising 0.01% to 5.0% of an organic co-solvent.

24. The prefilled syringe of claim 21 , further comprising 30 mM to 150 mM of a tonicity agent.

25. The prefilled syringe of claim 20 , wherein 90% of said aflibercept or more by weight is not present as an aggregate.

26. The prefilled syringe of claim 22 , wherein said prefilled syringe is glass.

27. The prefilled syringe of claim 22 , wherein said prefilled syringe is an auto injector.

28. The prefilled syringe of claim 12 , wherein said 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; GIF glycosylation; GIFS 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.

29. The prefilled syringe of claim 12 , wherein said 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.

30. The prefilled syringe of claim 20 , wherein said aflibercept and oxidized variants of aflibercept loaded into said prefilled syringe is formulated with (a) 40 to 250 mg/ml of aflibercept (b) a pH between 5.5 and 7.5, and (c) one or more pharmaceutically acceptable excipients selected from the group of buffering agents, preservatives, detergents and combinations thereof.