US 11,732,025 B2
Anti-VEGF protein compositions and methods for producing the same
Shunhai Wang, Scarsdale, NY (US); Ning Li, New Canaan, CT (US); Hunter Chen, New York, NY (US); Amardeep Singh Bhupender Bhalla, Montvale, NJ (US); Shawn Lawrence, Nyack, NY (US); Amy Johnson, Briarcliff Manor, NY (US); Meghan Casey, Ringwood, NJ (US); and Jaimie Grapel, Waldwick, NJ (US)
Assigned to Regeneron Pharmaceuticals, Inc., Tarrytown, NY (US)
Filed by Regeneran Pharmaceuticals, Inc., Tarrytown, NY (US)
Filed on Nov. 25, 2022, as Appl. No. 17/994,223.
Application 17/994,223 is a continuation of application No. 17/841,349, filed on Jun. 15, 2022, granted, now 11,548,932.
Application 17/841,349 is a continuation of application No. 17/711,723, filed on Apr. 1, 2022, granted, now 11,505,594, issued on Nov. 22, 2022.
Application 17/711,723 is a continuation of application No. 17/492,440, filed on Oct. 1, 2021, granted, now 11,407,813, issued on Aug. 9, 2022.
Application 17/492,440 is a continuation of application No. 16/996,127, filed on Aug. 18, 2020, granted, now 11,180,540, issued on Nov. 23, 2021.
Claims priority of provisional application 63/065,012, filed on Aug. 13, 2020.
Claims priority of provisional application 62/944,635, filed on Dec. 6, 2019.
Prior Publication US 2023/0119662 A1, Apr. 20, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C07K 1/16 (2006.01); C07K 1/18 (2006.01); C07K 1/36 (2006.01); C07K 14/71 (2006.01); C12P 21/06 (2006.01); C07K 1/113 (2006.01); C12N 5/00 (2006.01); C07K 1/22 (2006.01); C12P 21/02 (2006.01); A61K 38/18 (2006.01); C07K 16/28 (2006.01); C12N 15/66 (2006.01); C07K 16/22 (2006.01); G01N 30/80 (2006.01); G01N 30/02 (2006.01)
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.