	US 12,325,737 B2
	Total afucosylated glycoforms of antibodies produced in cell culture
Prince Bhebe, Thousand Oaks, CA (US); Matthew Jerums, Thousand Oaks, CA (US); Irene Liu, Thousand Oaks, CA (US); and Kurt Kunas, Thousand Oaks, CA (US)
Assigned to AMGEN INC., Thousand Oaks, CA (US)
Appl. No. 17/040,422
Filed by AMGEN INC., Thousand Oaks, CA (US)
PCT Filed Mar. 26, 2019, PCT No. PCT/US2019/024154 § 371(c)(1), (2) Date Sep. 22, 2020, PCT Pub. No. WO2019/191150, PCT Pub. Date Oct. 3, 2019.
Claims priority of provisional application 62/648,308, filed on Mar. 26, 2018.
Prior Publication US 2021/0079065 A1, Mar. 18, 2021
Int. Cl. C07K 16/00 (2006.01); C07K 16/30 (2006.01); C12P 21/00 (2006.01)

CPC C07K 16/00 (2013.01) [C07K 16/30 (2013.01); C12P 21/005 (2013.01); C07K 2317/14 (2013.01); C07K 2317/41 (2013.01); C07K 2317/51 (2013.01); C07K 2317/515 (2013.01); C07K 2317/732 (2013.01)]

15 Claims

1. A method of producing an antibody composition, wherein the level of total afucosylated (TAF) glycans in the antibody composition is less than 10%, the method comprising maintaining glycosylation-competent cells comprising a nucleic acid encoding an antibody of the antibody composition in a cell culture medium wherein;

a. the cell culture medium comprises fucose at a concentration of 0.17 g/L to 1.0 g/L or 0.1 g/L to 1.0 g/L and a pH of the cell culture medium is 6.85 to 7.05, or greater than 7.05 and less than 7.20, and

b. the glycosylation-competent cells are not genetically modified to alter activity of an enzyme of the de novo pathway or the salvage pathway, and

c. the method further comprises adding glucose to the cell culture medium according to a glucose feeding schedule, wherein the glucose feeding schedule is initiated at 4 to 6 days post-cell culture inoculation to achieve an average glucose concentration of 10 g/L or less in the cell culture medium or based on the concentration of fucose in the cell culture medium as calculated by Formula I:

T=3.354−1.388F+0.111G+[F−0.4375]×[1.9527(F−0.4375)] (Formula I)

wherein T is the targeted % total afucosylated (TAF) glycans in the antibody composition and is 2.5% to 6%, 2.75% to 5.5%, or 3% to 5%, F is the concentration (g/L) of fucose in the medium, and G is the average glucose concentration (g/L) in the medium.