Process for manipulating the level of glycan content of a glycoprotein

Abstract

The present invention provides a method for manipulating the fucosylated glycan content on a recombinant protein.

1 . A method for increasing the fucosylated glycan content on a recombinant protein comprising
 inoculating a bioreactor with host cells expressing the recombinant protein,   culturing the host cells in a serum free, chemically defined cell culture medium; increasing at least one of the following:   the level of copper,   the level of manganese, or   the pH,   harvesting the recombinant protein produced by the host cell.   
     
     
         2 . The method of  claim 1 , further comprising an increase in the level of β-galactosylation on the recombinant protein. 
     
     
         3 . The method according to  claim 1 , wherein the concentration of copper is increased to 100 ppb. 
     
     
         4 . The method according to  claim 1 , wherein the concentration of manganese is increased to 1000 nM. 
     
     
         5 . The method according to  claim 1 , wherein the fucosylated glycan content is increased to influence the effector function of the recombinant protein. 
     
     
         6 . The method according to  claim 1 , further comprising a temperature shift. 
     
     
         7 . The method according to  claim 6 , wherein the temperature shift is from 36° C. to 31° C. 
     
     
         8 . The method according to  claim 6 , wherein the temperature shift occurs at the transition between the growth phase and production phase. 
     
     
         9 . The method according to  claim 6 , wherein the temperature shift occurs during the production phase. 
     
     
         10 . The method according  claim 1  wherein the host cell expressing the recombinant protein is cultured in a batch culture, fed-batch culture, perfusion culture, or combinations thereof. 
     
     
         11 . The method according to  claim 10 , wherein the culture is a perfusion culture. 
     
     
         12 . The method according to  claim 11 , wherein perfusion comprises continuous perfusion. 
     
     
         13 . The method according to  claim 11 , wherein the rate of perfusion is constant. 
     
     
         14 . The method according to  claim 11 , wherein the perfusion is performed at a rate of less than or equal to 1.0 working volumes per day. 
     
     
         15 . The method according to  claim 11 , wherein the perfusion is accomplished by alternating tangential flow. 
     
     
         16 . The method according to  claim 1 , wherein the bioreactor has a capacity of at least 500 L. 
     
     
         17 . The method according to  claim 1  wherein the bioreactor has a capacity of at least 500 L to 2000 L. 
     
     
         18 . The method according to  claim 1  wherein the bioreactor has a capacity of at least 1000 L to 2000 L. 
     
     
         19 . The method according to  claim 1 , wherein the bioreactor is inoculated with at least 0.5 × 10 6  cells/mL. 
     
     
         20 . The method according to  claim 1 , wherein the serum-free chemically defined cell culture medium is a perfusion cell culture medium. 
     
     
         21 . The method according to  claim 1 , wherein the host cells are mammalian cells. 
     
     
         22 . The method according to  claim 1 , wherein the host cells are Chinese Hamster Ovary (CHO) cells. 
     
     
         23 . The method according to  claim 1 , wherein the recombinant protein is a glycoprotein. 
     
     
         24 . The method according to  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. 
     
     
         25 - 28 . (canceled) 
     
     
         29 . The method according to  claim 1 , wherein the pH is increased to 7.0.