US 12,486,524 B2
Methods of culturing a mammalian cell
Christopher Hwang, Cambridge, MA (US); Timothy Johnson, Cambridge, MA (US); Jason Walther, Cambridge, MA (US); Cheng Cheng, Cambridge, MA (US); Jonathan Wang, Cambridge, MA (US); Neha Shah, Cambridge, MA (US); and Seul-A Bae, Cambridge, MA (US)
Assigned to Genzyme Corporation, Cambridge, MA (US)
Filed by Genzyme Corporation, Cambridge, MA (US)
Filed on Jan. 11, 2022, as Appl. No. 17/572,978.
Application 17/572,978 is a division of application No. 14/976,486, filed on Dec. 21, 2015, granted, now 11,306,341.
Claims priority of provisional application 62/095,734, filed on Dec. 22, 2014.
Prior Publication US 2022/0145348 A1, May 12, 2022
Int. Cl. C12P 21/00 (2006.01); C07K 16/00 (2006.01); C12N 5/00 (2006.01)
CPC C12P 21/00 (2013.01) [C07K 16/00 (2013.01); C12N 5/0043 (2013.01); C12N 5/005 (2013.01); C07K 2317/14 (2013.01); C12N 2500/50 (2013.01); C12N 2510/02 (2013.01)] 5 Claims
 
1. A method of culturing a CHO cell in a liquid culture medium to achieve a viable cell density of greater than 60×106 cells/mL, the method comprising:
providing a culturing system comprising a vessel, a liquid culture medium disposed within the vessel, and a sparger disposed within the vessel comprising a plurality of pores configured to dispense gas through the sparger into the liquid culture medium; and
perfusion culturing a CHO cell comprising a recombinant protein-encoding nucleic acid in the culturing system comprising the liquid culture medium comprising an amount of poloxamer-188 and antifoam under conditions and a gas flow rate sufficient to produce the recombinant protein in the system, wherein the ratio of antifoam (g/L) to poloxamer-188 (g/L) is about 1.0% to about 3.0% in the liquid culture medium, and wherein:
(i) the plurality of pores are drilled pores with a pore size of 900 μm to 1.1 mm and the poloxamer-188 concentration is 2.5 g/L to 3.1 g/L;
(ii) the plurality of pores are drilled pores with a pore size of 400 μm to 600 μm and the poloxamer-188 concentration is 3.5 g/L to 4.1 g/L;
(iii) the plurality of pores are drilled pores with a pore size of 1 μm to 250 μm and the poloxamer-188 concentration is 4.3 g/L to 8.0 g/L; or
(iv) the plurality of pores are sintered pores with a pore size of 100 μm and the poloxamer-188 concentration is 5.8 g/L, and
wherein the method results in a viable cell density in the liquid culture medium that is greater than 60×106 cells/mL.