Methods for increasing mannose content of recombinant proteins
The present invention relates to methods of upregulating the high mannose glycoform content of a recombinant protein during a mammalian cell culture by manipulating the mannose to total hexose ratio in the cell culture media formulation.
1. A method for increasing a high mannose glycoform content of a recombinant protein during a mammalian 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;
inducing cell growth-arrest by L-asparagine starvation with a serum-free media that does not contain mannose, followed by perfusion with a serum-free perfusion media that does not contain mannose and having an L-asparagine concentration of 5 mM or less;
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 serum-free media that does not contain mannose for inducing cell growth-arrest by L-asparagine starvation is a serum-free perfusion media.
3. The method of claim 1 , wherein the concentration of L-asparagine in said serum-free perfusion media having an L-asparagine concentration of 5 mM or less is less than or equal to 4 mM.
4. The method of claim 1 , wherein the concentration of L-asparagine in said serum-free perfusion media having an L-asparagine concentration of 5 mM or less is less than or equal to 3 mM.
5. The method of claim 1 , wherein the concentration of L-asparagine in said serum-free perfusion media having an L-asparagine concentration of 5 mM or less is less than or equal to 2 mM.
6. The method of claim 1 , wherein the concentration of L-asparagine in said serum-free perfusion media having an L-asparagine concentration of 5 mM or less is less than or equal to 1 mM.
7. The method of claim 1 , wherein the concentration of L-asparagine in said serum-free perfusion media having an L-asparagine concentration of 5 mM or less is less than or equal to 0 mM.
8. The method of claim 1 , wherein the concentration of L-asparagine in said cell culture media is monitored prior L-asparagine starvation.
9. The method of claim 1 , wherein the concentration of L-asparagine in said cell culture media is monitored prior L-asparagine starvation.
10. A method for increasing a high mannose glycoform content of a recombinant protein during a mammalian 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;
inducing cell growth-arrest by L-asparagine starvation with a serum-free perfusion media that does not contain mannose, followed by perfusion with a serum-free perfusion media that does not contain mannose and having an L-asparagine concentration of 5 mM or less;
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.
11. The method of claim 10 , wherein the concentration of L-asparagine in said serum-free perfusion media having an L-asparagine concentration of 5 mM or less is less than or equal to 4 mM.
12. The method of claim 10 , wherein the concentration of L-asparagine in said serum-free perfusion media having an L-asparagine concentration of 5 mM or less is less than or equal to 3 mM.
13. The method of claim 10 , wherein the concentration of L-asparagine in said serum-free perfusion media having an L-asparagine concentration of 5 mM or less is less than or equal to 2 mM.
14. The method of claim 10 , wherein the concentration of L-asparagine in said serum-free perfusion media having an L-asparagine concentration of 5 mM or less is less than or equal to 1 mM.
15. The method of claim 10 , wherein the concentration of L-asparagine in said serum-free perfusion media having an L-asparagine concentration of 5 mM or less is less than or equal to 0 mM.
16. The method of claim 10 , wherein the concentration of L-asparagine in said cell culture media is monitored prior L-asparagine starvation.
17. The method of claim 10 , wherein the concentration of L-asparagine in said cell culture media is monitored prior L-asparagine starvation.