US 11,702,628 B2
Perfusion medium for culturing mammalian cells and reducing cell bleed in a perfusion cell culture
Samantha Wang, San Francisco, CA (US); and Henry Lin, Westfield, NJ (US)
Assigned to Boehringer Ingelheim International GmbH, Ingelheim am Rhein (DE)
Appl. No. 16/496,527
Filed by Boehringer Ingelheim International GmbH, Ingelheim am Rhein (DE)
PCT Filed Mar. 27, 2018, PCT No. PCT/EP2018/057757
§ 371(c)(1), (2) Date Sep. 23, 2019,
PCT Pub. No. WO2018/178069, PCT Pub. Date Oct. 4, 2018.
Claims priority of provisional application 62/479,422, filed on Mar. 31, 2017.
Prior Publication US 2020/0377849 A1, Dec. 3, 2020
Int. Cl. C12N 5/00 (2006.01); C12P 21/02 (2006.01)
CPC C12N 5/0037 (2013.01) [C12P 21/02 (2013.01); C12N 2500/12 (2013.01)] 11 Claims
 
1. A method of culturing mammalian cells expressing a heterologous protein in a perfusion cell culture comprising:
(a) inoculating mammalian cells expressing a heterologous protein in a serum-free culture medium;
(b) culturing the mammalian cells during growth phase by perfusion with a serum-free perfusion medium; and
(c) culturing the mammalian cells during production phase by perfusion with a serum-free perfusion medium comprising potassium ions at a concentration of 30 mM to 250 mM and a molar ratio of sodium to potassium ions of less than 1,
wherein the osmolarity of the serum-free perfusion media is between 300 and 500 mOsmol/kg,
wherein step (b) is optional,
and wherein step (c) further comprises maintaining a cell density by cell bleeding, wherein cell bleeding is reduced compared to a perfusion cell culture using the same serum-free perfusion medium comprising potassium ions at a concentration of less than 30 mM and a molar ratio of sodium to potassium of more than 2 and cultured under the same conditions.