US 11,939,559 B2
Method of culturing eukaryotic cells
Dinesh Baskar, Bangalore (IN); Jenny Hsiung, Mountain View, CA (US); Woon-Lam Susan Leung, San Mateo, CA (US); and Inn H. Yuk, Berkeley, CA (US)
Assigned to GENENTECH, INC., South San Francisco, CA (US)
Filed by Genentech, Inc., South San Francisco, CA (US)
Filed on Jan. 13, 2021, as Appl. No. 17/148,190.
Application 17/148,190 is a division of application No. 16/218,323, filed on Dec. 12, 2018, granted, now 10,894,940.
Application 16/218,323 is a continuation of application No. 14/480,366, filed on Sep. 8, 2014, granted, now 10,190,084, issued on Jan. 29, 2019.
Application 14/480,366 is a continuation of application No. 13/383,829, abandoned, previously published as PCT/US2010/041082, filed on Jul. 6, 2010.
Claims priority of provisional application 61/223,313, filed on Jul. 6, 2009.
Prior Publication US 2021/0130759 A1, May 6, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12M 1/00 (2006.01); C12M 1/34 (2006.01); C12M 1/36 (2006.01); C12M 3/06 (2006.01); C12N 5/00 (2006.01)
CPC C12M 23/14 (2013.01) [C12M 23/28 (2013.01); C12M 27/16 (2013.01); C12M 29/10 (2013.01); C12M 29/20 (2013.01); C12M 41/12 (2013.01); C12M 41/26 (2013.01); C12M 41/32 (2013.01); C12M 41/48 (2013.01); C12N 5/0018 (2013.01); C12N 2500/05 (2013.01)] 11 Claims
 
1. A method for culturing Chinese hamster ovary (CHO) cells comprising:
(i) providing a cell culture comprising from about 1.5×106 to about 10×106 CHO cells in about 20 L of a bicarbonate-containing culture liquid with a pH between 6.8 and 7.2 to a vessel, wherein said vessel has a volume of about 50 L and has walls that encapsulate said cell culture and a gas phase head space above said cell culture, and wherein said vessel comprises at least one port that provides an entrance and an egress of gas to and from said head space;
(ii) perfusing fresh culture medium into said vessel and removing spent culture medium from said vessel at a perfusion rate of about 1 volume per day; and
(iii) providing gas to said head space through said port to sweep accumulated CO2 from the head space of the vessel, wherein said gas is supplemented with O2 in an amount of 30% (v/v) of said gas 48 hours after the start of perfusion, thereby modulating pH of the cell culture to maintain the pH of the culture between pH 6.8 and 7.2, and maintaining dissolved O2 to a level greater than 20% of air saturation,
wherein the fresh culture medium has a pH of 7.2, clearance rate of the head space is between 0.002 and 0.1 head space volume per minute (hvm), the vessel is agitated by rocking the vessel at a rock rate between 19 and 25 rocks per minute (rpm) at a rock angle of between 8° and 12°, and wherein the method does not require continuous monitoring and feedback control of pH and dissolved O2.