Methods for increasing mannose content of recombinant proteins

Abstract

The present invention relates to methods of modulating the mannose content of recombinant proteins.

1 . A method for modulating mannose 5 on a recombinant protein during a mammalian cell culture process comprising limiting the amount of glucose in the cell culture medium, wherein the concentration of the glucose is from about 0 to 6 g/L, and supplementing the cell culture medium with galactose or sucrose, wherein the concentration of galactose is from 6-13 g/L or the concentration of sucrose is from about 16-24 g/L. 
     
     
         2 . The method according to  claim 1 , wherein the glucose concentration in the cell culture medium is sufficient to result in a concentration of glucose in the spent medium at about 0 g/L. 
     
     
         3 . (canceled) 
     
     
         4 . A method according the  claim 1 , wherein the concentration of glucose in the cell culture medium is from 4 to 6 g/L. 
     
     
         5 . The method according to  claim 1 , wherein the concentration of glucose in the cell culture medium is from 1 to 3 g/L. 
     
     
         6 . The method according to  claim 1 , wherein the concentration of glucose in the cell culture medium is from 2 to 3 g/L. 
     
     
         7 . The method according the  claim 1 , wherein the concentration of glucose in the cell culture medium is 2.5 g/L. 
     
     
         8 . The method according the  claim 1 , wherein the concentration of glucose in the cell culture medium is 0 g/L. 
     
     
         9 . (canceled) 
     
     
         10 . The method according to  claim 1 , wherein the concentration of galactose is from 10 to 13 g/L. 
     
     
         11 . The method according to  claim 1 , wherein the concentration of galactose in the cell culture medium is from 10 to 12 g/L. 
     
     
         12 . The method according to  claim 1 , wherein the concentration of galactose in the cell culture medium is 11.5 g/L. 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . The method according to  claim 1 , wherein the limiting amount of glucose is added during a production phase. 
     
     
         16 . (canceled) 
     
     
         17 . The method according the  claim 1 , wherein the cell culture process is a perfusion process. 
     
     
         18 .- 63 . (canceled) 
     
     
         64 . The method according to  claim 1 , wherein the mammalian cells are Chinese Hamster Ovary (CHO) cells. 
     
     
         65 . The method of  claim 1 , wherein the recombinant protein is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a recombinant fusion protein, or a cytokine. 
     
     
         66 . The method according to  claim 1 , further comprising a step of harvesting the recombinant protein produced by the cell culture. 
     
     
         67 . The method of  claim 1 , wherein the recombinant protein produced by the cell culture is purified and formulated in a pharmaceutically acceptable formulation. 
     
     
         68 . The method of  claim 1 , wherein recombinant protein production in the high mannose glycan species are increased compared to a culture where the cells are not subjected to limited glucose in combination with galactose.