Mammalian cell culture

Abstract

The invention provides a method for culturing mammalian cells. The method provides greater control over cell o growth to achieve high product titer cell cultures.

What is claimed is: 
     
         1 . A method of culturing Chinese Hamster Ovary (CHO) cells expressing a monoclonal antibody, comprising; 
 (a) establishing a CHO cell culture in a serum-free culture medium in a bioreactor by inoculating the bioreactor with at least 0.5 x 10 6  to 3.0 x 10 6  cells/ml in a serum-free culture medium;   (b) growing the CHO cells during a growth phase and supplementing the culture medium with bolus feeds of a serum-free feed medium;   (c) starting perfusion when viable cell density (VCD) is at least 10x10 6  viable cells/mL;   (d) maintaining the CHO cells by perfusion with a serum-free perfusion medium, wherein the VCD of the culture is maintained at between 10x10 6  viable cells/mL and 1 x 10 8  cells/mL; and   (e) harvesting the monoclonal antibody produced by the CHO cells.   
     
     
         2 . The method of  claim 1 , further comprising purifying the monoclonal antibody. 
     
     
         3 . The method of  claim 2 , further comprising formulating said monoclonal antibody into a pharmaceutically acceptable formulation. 
     
     
         4 . The method of  claim 3 , wherein said monoclonal antibody is denosumab. 
     
     
         5 . The method of  claim 3 , wherein said monoclonal antibody is panitumumab. 
     
     
         6 . The method of  claim 1 , wherein perfusion begins at a time that is between day 5 and day 9 of the cell culture. 
     
     
         7 . The method of  claim 1 , wherein perfusion begins when the cells have reached a production phase. 
     
     
         8 . The method of  claim 1 , wherein perfusion takes place prior to a production phase. 
     
     
         9 . The method of  claim 1 , wherein in step (d), said perfusion medium comprises L-asparagine at a concentration of 5 mM or less. 
     
     
         10 . The method of  claim 1 , further comprising a temperature shift wherein temperature of the culture is lowered. 
     
     
         11 . The method of  claim 1 , further comprising a temperature shift, wherein the temperature is lowered from between 35° C. and 38° C. to between 30° C. and 34° C. 
     
     
         12 . The method of  claim 1 , further comprising a temperature shift, wherein the growth phase occurs at a first temperature that is between 35° C. and 38° C., and the production phase occurs at a second temperature that is between 30° C. and 34° C. 
     
     
         13 . A method of culturing Chinese Hamster Ovary (CHO) cells expressing a monoclonal antibody, wherein said monoclonal antibody is denosumab, comprising; 
 (a) establishing a CHO cell culture in a serum-free culture medium in a bioreactor by inoculating the bioreactor with at least 0.5 x 10 6  to 3.0 x 10 6  cells/ml in a serum-free culture medium;   (b) growing the CHO cells during a growth phase and supplementing the culture medium with bolus feeds of a serum-free feed medium;   (c) starting perfusion between day 5 and day 9 of the cell culture;   (d) maintaining the CHO cells by perfusion with a serum-free perfusion medium, wherein the VCD of the culture is maintained at between 10x10 6  viable cells/mL and 80 x 10 6  cells/mL; and   (e) harvesting the monoclonal antibody produced by the CHO cells.   
     
     
         14 . The method of  claim 13 , further comprising purifying the monoclonal antibody. 
     
     
         15 . The method of  claim 14 , further comprising formulating said monoclonal antibody into a pharmaceutically acceptable formulation. 
     
     
         16 . The method of  claim 13 , wherein perfusion begins when the cells have reached a production phase. 
     
     
         17 . The method of  claim 13 , wherein perfusion takes place prior to a production phase. 
     
     
         18 . The method of  claim 13 , wherein in step (d), said perfusion medium comprises L-asparagine at a concentration of 5 mM or less. 
     
     
         19 . The method of  claim 13 , further comprising a temperature shift wherein temperature of the culture is lowered. 
     
     
         20 . The method of  claim 13 , further comprising a temperature shift, wherein the temperature is lowered from between 35° C. and 38° C. to between 30° C. and 34° C. 
     
     
         21 . The method of  claim 13 , further comprising a temperature shift, wherein the growth phase occurs at a first temperature that is between 35° C. and 38° C., and the production phase occurs at a second temperature that is between 30° C. and 34° C. 
     
     
         22 . A method of culturing Chinese Hamster Ovary (CHO) cells expressing a monoclonal antibody, wherein said monoclonal antibody is panitumumab, comprising; 
 (a) establishing a CHO cell culture in a serum-free culture medium in a bioreactor by inoculating the bioreactor with at least 0.5 x 10 6  to 3.0 x 10 6  cells/ml in a serum-free culture medium;   (b) growing the CHO cells during a growth phase and supplementing the culture medium with bolus feeds of a serum-free feed medium;   (c) starting perfusion between day 5 and day 9 of the cell culture;   (d) maintaining the CHO cells by perfusion with a serum-free perfusion medium, wherein the VCD of the culture is maintained at between 10x10 6  viable cells/mL and 80 x 10 6  cells/mL; and   (e) harvesting the monoclonal antibody produced by the CHO cells.   
     
     
         23 . The method of  claim 22 , further comprising purifying the monoclonal antibody. 
     
     
         24 . The method of  claim 23 , further comprising formulating said monoclonal antibody into a pharmaceutically acceptable formulation. 
     
     
         25 . The method of  claim 22 , wherein perfusion begins when the cells have reached a production phase. 
     
     
         26 . The method of  claim 22 , wherein perfusion takes place prior to a production phase. 
     
     
         27 . The method of  claim 22 , wherein in step (d), said perfusion medium comprises L-asparagine at a concentration of 5 mM or less. 
     
     
         28 . The method of  claim 22 , further comprising a temperature shift wherein temperature of the culture is lowered. 
     
     
         29 . The method of  claim 22 , further comprising a temperature shift, wherein the temperature is lowered from between 35° C. and 38° C. to between 30° C. and 34° C. 
     
     
         30 . The method of  claim 22 , further comprising a temperature shift, wherein the growth phase occurs at a first temperature that is between 35° C. and 38° C., and the production phase occurs at a second temperature that is between 30° C. and 34° C.