Methods for increasing mannose content of recombinant proteins

1. A method for increasing a high mannose glycoform content of a recombinant protein during a cell culture process comprising:

establishing a cell culture comprising mammalian cells in a bioreactor with a cell culture media that does not contain mannose;

growing said mammalian cells in a growth phrase with a cell culture media that does not contain mannose;

initiating and maintaining a production phase in said cell culture by perfusion with a serum-free perfusion media comprising mannose to produce said recombinant protein, wherein a mannose to total hexose ratio in said serum-free perfusion media is greater than 0 but less than 1.0, wherein said high mannose glycoform content of said recombinant protein is increased compared to a cell culture wherein said cell culture is not subjected to a serum-free perfusion media comprising mannose with a mannose to total hexose ratio greater than 0 and less than 1.0.

2. The method of claim 1 , wherein said perfusion begins on day 3 to on day 9 of said cell culture.

3. The method of claim 1 , wherein aid perfusion begins when said cell culture has reached a production phase.

4. The method of claim 1 , wherein said perfusion comprises continuous perfusion.

5. The method of claim 1 , wherein said perfusion is performed at a constant rate.

6. The method of claim 1 , wherein said perfusion is performed at a rate of less than or equal to 1.0 working volumes per day.

7. The method of claim 1 , wherein a perfusion is accomplished by alternating tangential flow.

8. The method of 7 , wherein said alternating tangential flow comprises an ultrafilter or a microfilter.

9. The method of claim 1 , further comprising a temperature shift that occurs at a transition between said growth phase and said production phase.

10. The method of claim 1 , further comprising a temperature shift that occurs during said production phase.

11. The method of claim 1 , wherein said bioreactor has a capacity of at least 500 L.

12. The method of claim 1 , wherein said mammalian cells are Chinese Hamster Ovary (CHO) cells.

13. The method of claim 1 , wherein said recombinant protein is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a recombinant fusion protein, and a cytokine.

14. The method of claim 1 , further comprising a step of harvesting and purifying said recombinant protein produced by the cell culture.

15. The method of claim 1 , further comprising purifying said recombinant protein from said cell culture and formulating said recombinant protein into a pharmaceutically acceptable formulation.