Anti-vegf protein compositions and methods for producing the same
AssigneeRegeneron Pharmaceuticals, Inc.
InventorsAndrew Tustian, Ankit Vartak, Thomas Daly, Erica Pyles, Nisha Palackal, Shunhai Wang, Ning Li, Matthew Franklin
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Abstract
The present disclosure pertains compositions comprising anti-VEGF proteins and methods for producing such compositions.
Claims30
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 about 40% to about 60% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 0% to about 20% mannose-5, and about 70% to about 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.
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 about 40% to about 60% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 0% to about 20% mannose-5, and about 70% to about 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;
- g) formulating said MiniTrap of step f) into a drug product.
An aflibercept MiniTrap preparation, comprising aflibercept MiniTrap having about 40% to about 60% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 0% to about 20% mannose-5, and about 70% to about 95% galactosylated glycans,
- wherein glycan analysis in carried out using hydrophilic interaction liquid chromatography with post-column fluorescence detection, and
- wherein labeled 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.
An aflibercept MiniTrap composition comprising aflibercept MiniTrap with a glycosylation profile including one or more glycan(s) selected from the group consisting of about 40% to about 60% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 0% to about 20% mannose-5, and about 70% to about 95% galactosylated glycans.
Dependencies are inferred from the claim text.