Methods of culturing a mammalian cell

1. A method of culturing a Chinese hamster ovary (CHO) cell in a liquid medium to achieve a viable cell density of greater than 90×10 6 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 35×10 6 cells/mL to less than 60×10 6 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 60×10 6 cells/mL to about 90×10 6 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 90×10 6 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).

2. The method of claim 1 , wherein perfusion culturing comprises:

providing a vessel comprising CHO cells disposed in a first liquid medium;

incubating the vessel with agitation and for a culturing period of at least about 7 days at a temperature of about 32° C. to about 40° C.; and

continuously or periodically after the first 48 to 96 hours of the culturing period removing a first volume of the first liquid medium and adding to the first liquid medium a second volume of a second liquid medium, wherein the first and second volumes are about equal.

3. The method of claim 2 , wherein the second liquid medium comprises poloxamer-188 at greater concentration than that in the first liquid medium.

4. The method of claim 2 , wherein the first liquid medium comprises poloxamer-188 at a concentration of 1.8 g/L or at a concentration less than 1.8 g/L, and the second liquid medium comprises poloxamer-188 at a greater concentration greater than 1.8 g/L.

5. The method of claim 2 further comprising:

collecting recombinant protein from the CHO cells, from the first liquid medium, or the second liquid medium, or any combination thereof; and

formulating the collected recombinant protein into a pharmaceutical composition.

6. The method of claim 2 , wherein one or both of the first liquid medium and the second liquid medium is selected from the group consisting of: a chemically-defined liquid culture medium, a serum-free liquid culture medium, a serum-containing liquid culture medium, an animal-derived component free liquid culture medium, and a protein-free medium.

7. The method of claim 1 , wherein the recombinant protein is an immunoglobulin, an enzyme, a growth factor, a protein fragment, or an engineered protein.

8. The method of claim 7 , wherein the enzyme is a galactosidase.

9. The method of claim 8 , wherein the galactosidase is an α-galactosidase.

10. The method of claim 9 , wherein the α-galactosidase is α-galactosidase-A.

11. The method of claim 1 , wherein the perfusion culturing in (a) is performed at a rate of 4 reactor volumes per day.

12. The method of claim 1 , wherein the perfusion culturing in (b) is performed at a rate of 6 reactor volumes per day.

13. The method of claim 1 , wherein the perfusion culturing in (c) is performed at a rate of 7 reactor volumes per day.