Anti-VEGF protein compositions and methods for producing the same
The present disclosure pertains compositions comprising anti-VEGF proteins and methods for producing such compositions.
1. A method of producing aflibercept MiniTrap, comprising:
a) producing aflibercept using cells cultured in a chemically defined medium (CDM);
b) binding said aflibercept to a first capture chromatography;
c) eluting said aflibercept of step b) and subjecting said aflibercept to enzymatic cleavage to remove its Fc domain thereby forming MiniTrap;
d) subjecting said formed MiniTrap of step c) to a second capture chromatography, wherein said second capture chromatography step is subjected to one or more washes to obtain a first flowthrough fraction;
e) subjecting said first flowthrough fraction of step d) to an anion exchange chromatography (AEX) using an AEX column; and
f) washing said AEX column of step e) to obtain a second flowthrough fraction having MiniTrap, wherein said MiniTrap has an amount of glycans selected from the group consisting of 40% to 60% total fucosylated glycans, 30% to 55% total sialylated glycans, 0% to 20% mannose-5, and 70% to 95% galactosylated glycans,
wherein the amount of glycans are determined using hydrophilic interaction liquid chromatography with post-column fluorescence detection, and wherein the glycans are separated and eluted using acetonitrile and aqueous 50 mM ammonium formate (pH 4.4) and detected using a fluorescence detector with an excitation wavelength of 265 nm and an emission wavelength of 425 nm.
2. The method of claim 1 , wherein said MiniTrap of step f) has 40% to 50% total fucosylated glycans.
3. The method of claim 1 , wherein said MiniTrap of step f) has 30% to 55% total sialylated glycans.
4. The method of claim 1 , wherein said MiniTrap of step f) has 6% to 15% mannose-5.
5. The method of claim 1 , wherein said MiniTrap of step f) has 70% to 79% galactosylated glycans.
6. The method of claim 1 , wherein said MiniTrap of step f) has 40% to 50% total fucosylated glycans, 30% to 55% total sialylated glycans, 6% to 15% mannose-5, and 70% to 79% galactosylated glycans.
7. The method of claim 1 , wherein MiniTrap of step f) 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.
8. The method of claim 1 , wherein said MiniTrap of step f) is xylosylated.
9. The method of claim 1 , wherein said MiniTrap of step f) comprises a trisulfide bridge.
10. The method of claim 1 , wherein said MiniTrap of step f) comprises a intrachain disulfide bridge.
11. A method of producing aflibercept MiniTrap preparation, comprising:
a) producing aflibercept using cells cultured in a chemically defined medium (CDM);
b) binding aflibercept to a first capture chromatography;
c) eluting said aflibercept of step b) and subjecting said aflibercept to enzymatic cleavage to remove its Fc domain thereby forming MiniTrap;
d) subjecting MiniTrap of step c) to a second capture chromatography, wherein said second capture chromatography step is subjected to one or more washes to obtain a first flowthrough fraction;
e) subjecting said first flowthrough fraction of step d) to an anion exchange chromatography (AEX) using an AEX column;
f) washing said AEX column of step e) to obtain a second flowthrough fraction having MiniTrap, wherein said MiniTrap has an amount of glycans selected from the group consisting of 40% to 60% total fucosylated glycans, 30% to 55% total sialylated glycans, 0% to 20% mannose-5, and 70% to 95% galactosylated glycans,
wherein the amount of glycans are determined using hydrophilic interaction liquid chromatography with post-column fluorescence detection, and
wherein the glycans are separated and eluted using acetonitrile and aqueous 50 mM ammonium formate (pH 4.4) and detected using a fluorescence detector with an excitation wavelength of 265 nm and an emission wavelength of 425 nm; and
g) formulating said MiniTrap of step f) into a drug product.
12. The method of claim 11 , wherein said MiniTrap of step g) has 40% to 50% total fucosylated glycans.
13. The method of claim 11 , wherein said MiniTrap of step g) has 30% to 55% total sialylated glycans.
14. The method of claim 11 , wherein said MiniTrap of step g) has 6% to 15% mannose-5.
15. The method of claim 11 , wherein said MiniTrap of step g) has 70% to 79% galactosylated glycans.
16. The method of claim 11 , wherein said MiniTrap of step g) has 40% to 50% total fucosylated glycans, 30% to 55% total sialylated glycans, 6% to 15% mannose-5, and 70% to 79% galactosylated glycans.
17. The method of claim 11 , wherein MiniTrap of step g) 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.
18. The method of claim 11 , wherein said MiniTrap of step g) is xylosylated.
19. The method of claim 11 , wherein said MiniTrap of step g) comprises a trisulfide bridge.
20. The method of claim 11 , wherein said MiniTrap of step g) comprises a intrachain disulfide bridge.