Methods of culturing a mammalian cell

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.