Mammallian cell culture process for protein production

Abstract

The present invention describes methods and processes for the production of proteins, particularly glycoproteins, by animal cell or mammalian cell culture, preferably, but not limited to, fed-batch cell cultures. In one aspect, the methods comprise at least two temperature shifts performed during the culturing period, in which the temperature is lower at the end of the culturing period than at the time of initial cell culture. Throughout their duration, the culturing processes of the invention involving two or more downward shifts in temperature sustain a high viability of the cultured cells, and can yield an increased end titer of protein product, and a high quality of protein product, as determined, e.g., by sialic acid content of the produced protein. In another aspect, the methods comprise the delayed addition of polyanionic compound during the culturing period. The delayed addition of polyanionic compound sustains a high viability of the cultured cells, and can extend the growth phase, delay the onset of the death phase, and arrest the death phase.

1 . A cell culturing process comprising:
 a) culturing host cells which produce a protein of interest; and   b) adding polyanionic compound to the cell culture at a time after innoculation.   
     
     
         2 . The process according to  claim 1 , wherein the polyanionic compound is selected from the group consisting of: dextran sulfate, heparin, heparan sulfate, mannan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, hyaluronate, poly(vinyl sulfate), kappa-carrageenan, and suramin. 
     
     
         3 . The process according to  claim 2 , wherein the polyanionic compound is a polysulfated compound. 
     
     
         4 . The process according to  claim 3 , wherein the polysulfated compound is dextran sulfate. 
     
     
         5 . The process according to  claim 1 , wherein the polyanionic compound is added on day 1 of the culture or later. 
     
     
         6 . The process according to  claim 1 , wherein the polyanionic compound is added to bring the concentration in the culture to 1-1000 mg/L. 
     
     
         7 . The process according to  claim 1 , wherein the protein of interest is a glycoprotein. 
     
     
         8 . The process according to  claim 1 , wherein the cells are mammalian cells. 
     
     
         9 . The process according to  claim 8 , wherein the mammalian cells are CHO cells. 
     
     
         10 . The process according to  claim 1 , wherein the protein of interest is a soluble CTLA4 molecule. 
     
     
         11 . The process according to  claim 10 , wherein the soluble CTLA4 molecule is a CTLA4 fusion protein. 
     
     
         12 . The process according to  claim 11 , wherein the soluble CTLA4 fusion protein is a CTLA4Ig. 
     
     
         13 . The process according to  claim 12 , wherein the soluble CTLA4 fusion protein is CTLA4Ig comprising amino acids −1 to 357 or +1 to 357 as shown in  FIG. 3 . 
     
     
         14 . The process according to  claim 1 , wherein the protein of interest is a soluble CTLA4 mutant molecule. 
     
     
         15 . The process according to  claim 14 , wherein the soluble CTLA4 mutant molecule is L104EA29YIg comprising amino acids −1 to 357 or +1 to 357 as shown in  FIG. 4 . 
     
     
         16 . The process according to  claim 1  wherein the polyanionic compound is added at a time after innoculation that is before the beginning of the initial death phase. 
     
     
         17 . The process according to  claim 16  wherein the polyanionic compound is added at a time after innoculation that is during the initial growth phase. 
     
     
         18 . The process according to  claim 17 , wherein the polyanionic compound is added during the second half of the initial growth phase. 
     
     
         19 . The process according to  claim 18 , wherein the polyanionic compound is added on or about the end of the initial growth phase. 
     
     
         20 . The process according to  claim 1 , wherein the polyanionic compound is added at a time after innoculation that is during the initial death phase. 
     
     
         21 . The process according to  claim 16 , wherein the growth phase is extended. 
     
     
         22 . The process according to  claim 21 , wherein the peak viable cell density achieved during the extended growth phase is higher than the peak viable cell density achieved during the initial growth phase. 
     
     
         23 . The process according to  claim 16 , where the onset of the death phase is delayed. 
     
     
         24 . The process according to  claim 20 , wherein the death phase is arrested. 
     
     
         25 . The process according to  claim 1 , wherein cell viability is increased. 
     
     
         26 . A cell culturing process comprising:
 a) culturing CHO cells which produce a soluble CTLA4 molecule; and   b) adding dextran sulfate to the cell culture at a time after innoculation.   
     
     
         27 . The process according to  claim 26 , wherein the onset of the death phase is delayed. 
     
     
         28 . The process according to  claim 1 , further comprising:
 c) culturing the host cells at a temperature at or near 37° C. under conditions and for a time period that allow for cell growth;   d) then culturing the cells at a second temperature at or near 34° C.; and   e) then culturing the cells at a third temperature at or near 32° C.   
     
     
         29 . The process according to  claim 25 , further comprising:
 c) culturing the CHO cells at a temperature at or near 37° C. under conditions and for a time period that allow for cell growth;   d) culturing the CHO cells at a second temperature at or near 34° C. starting about day 6 of the culture;   e) culturing the CHO cells at a third temperature at or near 32° C. starting about day 10 of the culture.