Methods for increasing mannose content of recombinant proteins

1 . A method of modulating the amount of the mannose-5 glycoform of an Immunoglobulin G (IgG) molecule in an IgG composition, wherein said IgG composition is produced by a Chinese Hamster Ovary (CHO) cell culture, comprising:

(a) establishing a cell culture of CHO cells in a bioreactor, wherein the CHO cell produces an IgG molecule;

(b) limiting the amount of glucose in the bioreactor, wherein the concentration of the glucose in the spent medium of the bioreactor is from about 0 to 3 g/L;

(c) supplementing the cell culture with a feeding medium comprising galactose, such that the concentration of galactose in the resulting spent medium of the bioreactor is above 2.5 g/L; and

(d) harvesting and purifying the IgG composition from the cell culture.

2 . The method of claim 1 , wherein said CHO cell culture is maintained for at least 7 days before the IgG composition is harvested.

3 . The method of claim 1 , wherein the concentration of galactose in the resulting spent medium is from 4 to 8 g/L.

4 . The method of claim 1 , wherein the concentration of galactose in the resulting spent medium is above 4 g/L.

5 . The method of claim 1 , wherein in step (c), the glucose concentration in the feeding medium is sufficient to result in a concentration of glucose in the spent medium at about 0 g/L.

6 . The method of claim 1 , wherein in step (c), the feeding medium is fed during a production phase.

7 . The method of claim 1 , wherein said cell culture process comprises a perfusion process.

8 . The method of claim 1 , wherein said cell culture process comprises a bolus feed or continuous feed process.

9 . The method of claim 1 , wherein the amount of mannose-5 glycoform of the IgG molecule in the IgG composition is increased, as compared to that of an IgG composition obtained from a CHO cell culture where the cells are not subjected to limited glucose in combination with galactose.

10 . The method of claim 1 , wherein said IgG composition is formulated in a pharmaceutically acceptable formulation.

11 . A method of producing an immunoglobulin G (IgG) composition by a Chinese Hamster Ovary (CHO) cell culture, comprising:

(a) establishing a cell culture of CHO cells in a bioreactor, wherein the CHO cell produces an IgG molecule;

(b) limiting the amount of glucose in the bioreactor, wherein the concentration of the glucose in the spent medium of the bioreactor is from about 0 to 3 g/L;

(c) supplementing the cell culture with a feeding medium comprising galactose, such that the concentration of galactose in the resulting spent medium of the bioreactor is above 2.5 g/L; and

(d) harvesting and purifying the IgG composition from the cell culture.

12 . The method of claim 11 , wherein said CHO cell culture is maintained for at least 7 days before the IgG composition is harvested.

13 . The method of claim 11 , wherein the concentration of galactose in the resulting spent medium is from 4 to 8 g/L.

14 . The method of claim 11 , wherein the concentration of galactose in the resulting spent medium is above 4 g/L.

15 . The method of claim 11 , wherein in step (c), the glucose concentration in the feeding medium is sufficient to result in a concentration of glucose in the spent medium at about 0 g/L.

16 . The method of claim 11 , wherein in step (c), the feeding medium is fed during a production phase.

17 . The method of claim 11 , wherein said cell culture process comprises a perfusion process.

18 . The method of claim 11 , wherein said cell culture process comprises a bolus feed or continuous feed process.

19 . The method of claim 11 , wherein the amount of mannose-5 glycoform of the IgG molecule in the IgG composition is increased, as compared to that of an IgG composition obtained from a CHO cell culture where the cells are not subjected to limited glucose in combination with galactose.

20 . The method of claim 11 , wherein said IgG composition is formulated in a pharmaceutically acceptable formulation.