Mammalian cell culture

Abstract

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

What is claimed is: 
     
         1 . A method of arresting cell growth in a mammalian cell culture expressing a recombinant protein comprising:
 establishing a mammalian cell culture in a serum-free culture medium in a bioreactor;   inducing cell growth-arrest by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5 mM or less;   maintaining the mammalian cells in a growth-arrested state by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5 mM or less.   
     
     
         2 . A method of limiting a mammalian cell culture expressing a recombinant protein at a desired packed cell volume (PCV) comprising:
 establishing a mammalian cell culture in a serum-free culture medium in a bioreactor,   inducing cell growth-arrest by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5 mM or less,   
       maintaining the mammalian cells in a growth-arrested state by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5 mM or less. 
     
     
         3 . The method of  claim 2 , further comprising a temperature shift, wherein the temperature is lowered from between 35° C. and 38° C. to between 30° C. and 34° C. 
     
     
         4 . The method of  claim 2 , wherein the temperature shift occurs at the transition between a growth phase and a production phase. 
     
     
         5 . The method of  claim 2 , wherein the temperature shift occurs during a production phase. 
     
     
         6 . The method of  claim 2 , further comprising a temperature shift, wherein the growth phase occurs at a temperature from about 35° C. to about 38° C. and the production phase occurs at a temperature from about 30° C. to about 34° C. 
     
     
         7 . The method of  claim 2 , wherein the desired PCV is 35% or less. 
     
     
         8 . A method of culturing mammalian cells expressing a recombinant protein comprising:
 establishing a mammalian cell culture in a serum-free culture medium in a bioreactor;   growing the mammalian cells during a growth phase and supplementing the culture medium with bolus feeds of a serum-free feed medium, and   maintaining the mammalian cells during a production phase by perfusion with a serum-free perfusion medium, wherein the packed cell volume during the production phase is less than or equal to 35%.   
     
     
         9 . The method of  claim 8 , wherein perfusion begins when the cells have reached a production phase. 
     
     
         10 . The method of  claim 8 , further comprising inducing cell growth-arrest by perfusion with a serum-free perfusion medium having an L-asparagine concentration of 5 mM or less. 
     
     
         11 . The method of  claim 8 , wherein the packed cell volume is less than or equal to 30%. 
     
     
         12 . The method of  claim 8 , wherein the viable cell density of the mammalian cell culture at a packed cell volume less than or equal to 35% is 10×10 6  viable cells/ml to 80×10 6  viable cells/ml. 
     
     
         13 . The method of  claim 8 , further comprising a temperature shift, wherein the temperature is lowered from between 35° C. and 38° C. to between 30° C. and 34° C. 
     
     
         14 . The method of  claim 8 , wherein the temperature shift occurs at the transition between a growth phase and a production phase. 
     
     
         15 . The method of  claim 8 , wherein the temperature shift occurs during a production phase. 
     
     
         16 . The method of  claim 8 , further comprising a temperature shift, wherein the growth phase occurs at a temperature from about 35° C. to about 38° C. and the production phase occurs at a temperature from about 30° C. to about 34° C.