Method of culturing eukaryotic cells

A method for culturing Chinese hamster ovary (CHO) cells comprising:

• (i) providing a cell culture inoculant comprising CHO cells in about 6 L of a bicarbonate-containing culture liquid to a vessel to achieve a target cell density of about 7.5×105 cells/mL, 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) providing gas to said head space through said port wherein said gas contains CO2 in an amount of 8% (v/v) of said gas on day 1, in an amount of 5% (v/v) of said gas on day 2, and in an amount of 2% (v/v) of said gas thereafter, thereby modulating the pH of said cell culture to maintain the pH of the culture between pH 6.8 and 7.2;
• (iii) providing fresh culture medium to said vessel to achieve a volume of about 20 L;
• and
• (iv) providing gas to said head space through said port, wherein said gas contains CO2 in an amount of 8% (v/v) of said gas on day 1, in an amount of 5% (v/v) of said gas on day 2, and in an amount of 2% (v/v) of said gas thereafter, thereby modulating the pH of said cell culture to maintain the pH of the culture between pH 6.8 and 7.2;
• 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 15 and 30 rocks per minute (rpm) at a rock angle of between 5° and 15°, and wherein the method does not require continuous monitoring and feedback control of pH.