Decreasing ornithine production to decrease high mannose glycoform content of recombinant proteins
The present invention relates to a method for manipulating the high mannose glycoform content of recombinant glycoproteins by regulating ornithine metabolism during cell culture.
1. A method for decreasing the high mannose glycoform content of a recombinant protein comprising culturing a host cell expressing the recombinant protein in a cell culture comprising an arginase inhibitor, wherein ornithine production in the host cell is decreased when compared to the host cell expressing the recombinant protein cultured in a cell culture lacking the arginase inhibitor, and
the recombinant protein has a decreased high mannose glycoform content than when the recombinant protein is expressed in the cell culture lacking the arginase inhibitor.
2. The method according to claim 1 , wherein the arginase inhibitor is selected from the group consisting of N G -hydroxy-L-arginine monoacetate, Nω-hydroxy-nor-arginine diacetate, BEC (S-(2-boronoethyl)-1-cysteine) and DL-a-Difluoromethylornithine.
3. The method according to claim 1 , wherein the arginase inhibitor is BEC (S-(2-boronoethyl)-l-cysteine).
4. The method according to claim 1 , wherein the arginase inhibitor is DL-a-Difluoromethylornithine.
5. The method according to claim 1 , wherein the arginase inhibitor is:
(a) N G -hydroxy-L-arginine monoacetate, having a concentration of 1 μM, 10 μM or 20 μM;
(b) Nω-hydroxy-nor-arginine diacetate, having a concentration of 10 μM or 20 μM;
(c) BEC (S-(2-boronoethyl)-l-cysteine), having a concentration of 1 μM, 10 μM, 20 μM or 0.5 mM; or
(d) DL-α-difluoromethylornithine, having a concentration of 1 μM, 10 μM, 20 μM, 1 mM or 2 mM.
6. The method according to claim 5 , wherein concentration of the arginase inhibitor is selected from the group consisting of at least 10 μM, from 10 μM to 2 mM, 0.5 mM to 2 mM, 0.5 mM to 1 mM and 1 mM to 2 mM.
7. The method according to claim 5 , wherein the arginase inhibitor is selected from the group consisting of Nω-hydroxy-nor-arginine diacetate, BEC (S-(2-boronoethyl)-1-cysteine); and DL-α-difluoromethylornithine, wherein the concentration of the arginase inhibitor is 10 μM.
8. The method according to claim 5 , wherein the arginase inhibitor is BEC (S-(2-boronoethyl)-l-cysteine), wherein the concentration of the arginase inhibitor is 0.5 mM.
9. The method according to claim 5 , wherein the arginase inhibitor is DL-α-difluoromethylornithine, wherein the concentration of the arginase inhibitor is 1 mM.
10. The method according to claim 5 , wherein the arginase inhibitor is DL-α-difluoromethylornithine, wherein the concentration of the arginase inhibitor is 2 mM.
11. A method of decreasing high mannose glycoform content of a recombinant protein comprising culturing a host cell expressing the recombinant protein in a cell culture comprising spermine, wherein ornithine production in the host cell is decreased when compared to the host cell expressing the recombinant protein cultured in a cell culture with a greater concentration of spermine and
the recombinant protein has a decreased high mannose glycoform content than the recombinant protein expressed by the cell culture having a greater concentration of spermine.
12. The method according to claim 11 , wherein the concentration of spermine is 35 μM or less.
13. The method according to claim 11 , wherein the concentration of spermine is 7 μM to 35 μM.
14. The method according to claim 11 , wherein the concentration of spermine is 27 μM to 35 μM.
15. The method according to claim 11 , wherein the concentration of spermine is 7 μM to 27 μM.
16. The method according to claim 11 , wherein the concentration of spermine is 35 μM.
17. The method according to claim 11 , wherein the concentration of spermine is 27 μM.