Methods of culturing a mammalian cell

A method of culturing a Chinese hamster ovary (CHO) cell in a liquid medium to achieve a viable cell density of greater than 90×106 cells/mL, the method comprising:

• (a) perfusion culturing a CHO cell comprising a nucleic acid encoding a recombinant protein under conditions sufficient to produce the recombinant protein, wherein the perfusion culturing comprises culturing the CHO cells in a liquid medium comprising poloxamer-188 at a concentration of 1.8 g/L and antifoam;
• (b) determining that the viable cell density in the liquid medium is about 35×106 cells/mL to less than 60×106 cells/mL, then increasing the poloxamer-188 concentration in the liquid medium to between about 1.8 g/L and about 2.9 g/L;
• (c) determining that the viable cell density in the liquid medium is 60×106 cells/mL to about 90×106 cells/mL, then increasing the poloxamer-188 concentration in the liquid medium to between about 3.0 g/L and about 5.7 g/L;
• and
• (d) determining that the viable cell density in the liquid medium is greater than 90×106 cells/mL, then increasing the poloxamer-188 concentration in the liquid medium to between about 5.8 g/L and about 8.0 g/L,
• wherein the ratio of antifoam (g/L) to poloxamer-188 (g/L) is about 1.0% to about 3.0% in the liquid medium during the method steps (a)-(d).