US 12,077,570 B2
Anti-VEGF protein compositions and methods for producing the same
Andrew Tustian, Millwood, NY (US); Ankit Vartak, Elmsford, NY (US); Thomas Daly, New City, NY (US); Erica Pyles, New City, NY (US); and Nisha Palackal, White Plains, NY (US)
Assigned to Regeneron Pharmaceuticals, Inc., Tarrytown, NY (US)
Filed by Regeneron Pharmaceuticals, Inc., Tarrytown, NY (US)
Filed on Mar. 7, 2023, as Appl. No. 18/118,558.
Application 18/118,558 is a continuation of application No. 17/575,585, filed on Jan. 13, 2022, granted, now 11,649,273.
Application 17/575,585 is a continuation of application No. 17/211,333, filed on Mar. 24, 2021, granted, now 11,286,290, issued on Mar. 29, 2022.
Application 17/211,333 is a continuation of application No. 16/996,042, filed on Aug. 18, 2020, granted, now 11,098,112, issued on Aug. 24, 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 2023/0272044 A1, Aug. 31, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C07K 1/36 (2006.01); A61K 38/18 (2006.01); C07K 1/113 (2006.01); C07K 1/16 (2006.01); C07K 1/18 (2006.01); C07K 1/22 (2006.01); C07K 14/71 (2006.01); C07K 16/22 (2006.01); C07K 16/28 (2006.01); C12N 5/00 (2006.01); C12N 15/66 (2006.01); C12P 21/02 (2006.01); C12P 21/06 (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 preparing a prefilled syringe comprising aflibercept, comprising:
a) producing a clarified harvest of cells cultured in a chemically defined medium (CDM);
b) binding aflibercept from said clarified harvest to an affinity chromatography column comprising a polypeptide capable of binding to or interacting with said aflibercept, wherein said polypeptide is an antibody, a fusion protein, a ScFv or a fragment thereof;
c) eluting said aflibercept of step (b) forming an affinity eluate;
d) subjecting said eluate comprising aflibercept of step (c) to anion exchange chromatography (AEX) column;
e) collecting a flowthrough fraction, wherein the flowthrough fraction comprises said aflibercept, and wherein said aflibercept includes oxidized variants that have at least one oxidized amino acid residue selected from the group consisting of tryptophan, histidine, and a combination thereof; and
f) preparing a syringe using said aflibercept of step (e) to form said prefilled syringe.