CPC C07K 14/71 (2013.01) [A61K 38/1866 (2013.01); C07K 1/113 (2013.01); C07K 1/16 (2013.01); C07K 1/18 (2013.01); C07K 1/22 (2013.01); C07K 1/36 (2013.01); C07K 16/22 (2013.01); C07K 16/283 (2013.01); C12N 5/0018 (2013.01); C12N 5/0031 (2013.01); C12N 15/66 (2013.01); C12P 21/02 (2013.01); C12P 21/06 (2013.01); G01N 30/80 (2013.01); C07K 2317/622 (2013.01); C07K 2319/30 (2013.01); C07K 2319/33 (2013.01); C12N 2800/10 (2013.01); G01N 2030/027 (2013.01)] | 30 Claims |
1. A method of producing aflibercept from a host cell cultured in a chemically defined medium (CDM), comprising:
(a) culturing said host cell in said CDM under suitable conditions in which said host cell expresses aflibercept wherein (i) the cumulative concentration of nickel in said CDM is less than or equal to 0.4 (ii) the cumulative concentration of iron in said CDM is less than or equal to 55.0 (iii) the cumulative concentration of copper in said CDM is less than or equal to 0.8 (iv) the cumulative concentration of zinc in said CDM is less than or equal to 56.0 (v) the cumulative concentration of cysteine in said CDM is less than or equal to 10.0 mM, or (vi) the cumulative concentration of an antioxidant in said CDM is 0.001 mM to 10.0 mM for any single anti-oxidant and the cumulative concentration of all anti-oxidants is less than or equal to 30.0 mM;
(b) binding aflibercept from said clarified harvest to a Protein A resin;
(c) eluting said aflibercept of step (b) forming an affinity eluate, and subjecting said eluate comprising aflibercept to anion exchange chromatography (AEX); and
(d) collecting a flowthrough fraction, wherein said aflibercept of step d) has a glycosylation profile based on molar percentage selected from the group consisting of 40% to 50% total fucosylated glycans, 30% to 55% total sialylated glycans, 2% to 15% mannose-5, and 60% to 79% galactosylated glycans.
|