Methods for increasing mannose content of recombinant proteins

Abstract

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.

What is claimed is: 
     
         1 . A cell culture media containing mannose, wherein the mannose to total hexose ratio in the cell culture media is greater than 0 but less than 1.0. 
     
     
         2 . The cell culture media according to  claim 1 , wherein the cell culture media contains at least 3 g/L mannose. 
     
     
         3 . A method for upregulating the high mannose glycoform content of a recombinant protein during a mammalian cell culture process comprising;
 establishing a mammalian cell culture in a bioreactor with a cell culture media that does not contain mannose; and   maintaining the cell culture with a cell culture media according to  claim 1 , wherein the high mannose glycoform content of a recombinant protein is increased compared to a culture where the cells are not subjected to a cell culture media containing a mannose to total hexose ratio of greater than 0 and less than 1.0.   
     
     
         4 . A method for upregulating the high mannose glycoform content of a recombinant protein during a mammalian cell culture process comprising;
 establishing a mammalian cell culture in a bioreactor with a cell culture media that does not contain mannose;   growing the mammalian cells during a growth phase with a cell culture media that does not contain mannose;   initiating and maintaining a production phase in the cell culture by perfusion with a serum-free perfusion media containing mannose, wherein the mannose to total hexose ratio in the perfusion media is greater than 0 but less than 1.0.   
     
     
         5 . The method according to  claim 4 , wherein the cell culture is maintained by perfusion. 
     
     
         6 . The method according to  claim 4 , wherein perfusion begins on or about day 3 to on or about day 9 of the cell culture. 
     
     
         7 . The method according to  claim 4 , wherein perfusion begins when the cells have reached a production phase. 
     
     
         8 . The method according to  claim 4 , wherein perfusion comprises continuous perfusion. 
     
     
         9 . The method according to  claim 4 , wherein the rate of perfusion is constant. 
     
     
         10 . The method according to  claim 4 , wherein perfusion is performed at a rate of less than or equal to 1.0 working volumes per day. 
     
     
         11 . The method according to 4, wherein the perfusion is accomplished by alternating tangential flow. 
     
     
         12 . The method according to 11, wherein the perfusion is accomplished by alternating tangential flow using an ultrafilter or a microfilter. 
     
     
         13 . The method according to  claim 4 , further comprising a temperature shift that occurs at the transition between the growth phase and the production phase. 
     
     
         14 . The method according to  claim 4 , further comprising a temperature shift that occurs during the production phase. 
     
     
         15 . The method according to  claim 4 , wherein the bioreactor has a capacity of at least 500 L. 
     
     
         16 . The method according to  claim 4 , wherein the mammalian cells are Chinese Hamster Ovary (CHO) cells. 
     
     
         17 . The method according  claim 4 , 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. 
     
     
         18 . The method according to  claim 4 , further comprising a step of harvesting the recombinant protein produced by the cell culture. 
     
     
         19 . The method according to  claim 4 , wherein the recombinant protein produced by the cell culture is purified and formulated in a pharmaceutically acceptable formulation. 
     
     
         20 . The method of  claim 4 , wherein the high mannose glycoform content of a recombinant protein is increased compared to a culture where the cells are not subjected to a cell culture media containing a mannose to total hexose ratio of greater than 0 and less than 1.0.