Process for Cell Culturing by Continuous Perfusion

Abstract

The invention relates to a process for the culturing of cells by continuous perfusion culturing of a cell culture comprising cell culture medium and cells, wherein cell culture medium is added to the cell culture, the cell culture is circulated over a filter module comprising hollow fibers resulting in an outflow of liquid having a lower cell density than the cell culture and the flow within the filter module is an alternating tangential flow. Preferably, culture medium is added at a particular perfusion rate and/or biomass is removed form the culture at least once. The method is especially suitable for the culturing of aggregating cells. The invention also relates to such a process wherein a biological substance, preferably an antibody, is produced by the cells, which biological substance may be further purified in downstream processing.

1 . A method for limiting cell aggregation during cell culture to a level wherein no more than 5% of the animal cells in suspension in the culture comprise aggregates of at least 5 cells, comprising:
 culturing of a cell culture comprising cell culture medium and animal cells that easily or inherently form aggregates during culturing, wherein the culturing comprises:   (a) circulating the cell culture through a filter module comprising hollow fibers, in an alternating tangential flow resulting in an outflow of cell culture liquid through the pores of the filter module having a lower animal cell density per ml than the cell culture prior to circulating through the filter module; and   (b) adding cell culture medium to the cell culture, and   wherein no more than 5% of the animal cells in the culture form aggregates in suspension of at least 5 cells during the culturing, and wherein the culturing is continued until animal cells are present in the cell culture at a density of at least 80×10 6  viable animal cells/ml.   
     
     
         2 . The method of  claim 1 , wherein biomass is removed at least once from the cell culture and additional cell culture medium is added to the cell culture to compensate for the biomass removal. 
     
     
         3 . The method of  claim 2 , wherein the biomass removal is started just before or just after the animal cell density has reached a steady state. 
     
     
         4 . The method of  claim 2 , wherein the volume of biomass removed is between 2% and 40% of the total volume of the cell culture per day. 
     
     
         5 . The method of  claim 1 , wherein a first pump circulates the cell culture within the filter module comprising hollow fibers and a second pump removes the liquid having a lower cell density than the cell culture prior to circulating through the filter module. 
     
     
         6 . The method of  claim 1 , wherein the animal cells are cultured to a cell viability of at least 90%. 
     
     
         7 . The method of  claim 1 , wherein no more than 4% of the animal cells in the culture form aggregates in suspension of at least 5 cells during the culturing. 
     
     
         8 . The method of  claim 1 , wherein the cells are mammalian cells. 
     
     
         9 . The method of  claim 8 , wherein the mammalian cells are human cells. 
     
     
         10 . The method of  claim 1 , wherein the cells produce a biological substance. 
     
     
         11 . The method of  claim 10 , wherein the biological substance is a protein or a polynucleotide. 
     
     
         12 . The method of  claim 10 , wherein the biological substance is further purified from the cell culture in downstream processing. 
     
     
         13 . The method of  claim 3 , wherein the cells are mammalian cells. 
     
     
         14 . The method of  claim 13 , wherein the mammalian cells are human cells. 
     
     
         15 . The method of  claim 13 , wherein the cells produce a biological substance. 
     
     
         16 . The method of  claim 15 , wherein the biological substance is a protein or a polynucleotide. 
     
     
         17 . The method of  claim 15 , wherein the biological substance is further purified from the cell culture in downstream processing. 
     
     
         18 . The method of  claim 14 , wherein the cells produce a biological substance. 
     
     
         19 . The method of  claim 18 , wherein the biological substance is a protein or a polynucleotide. 
     
     
         20 . The method of  claim 18 , wherein the biological substance is further purified from the cell culture in downstream processing.