Product quality enhancement in mammalian cell culture processes 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, illustratively, but not limited to, fed-batch cell cultures. The methods comprise feeding the cells with D-galactose, preferably with feed medium containing D-galactose, preferably daily, to sustain a sialylation effective level of D-galactose in the culture for its duration, thus increasing sialylation of the produced proteins. The methods can also 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. The cell culture processes of the invention involving two or more temperature shifts sustain a high cell viability, and can allow for an extended protein production phase. The methods can also comprise the delayed addition of polyanionic compound at a time after inoculation. Supplementation of the cultures with D-galactose, preferably in a feed medium, to sustain galactose at sialylation effective levels in the cultures until the end of a culture run reverses a decline in sialylation that accompanies culture scale up, and is advantageous for large scale culturing processes.

1 . A cell culture process for the production of a soluble CTLA4 molecule, comprising: 
 a) culturing host cells which produce a soluble CTLA4 molecule in cell culture under conditions that allow for protein production; and    b) adding polyanionic compound to the cell culture at a time after inoculation.    
     
     
         2 . The process according to  claim 1 , wherein the polyanionic addition step b) occurs at the end of the initial growth phase.  
     
     
         3 . The process according to  claim 2 , wherein the polyanionic addition step b) occurs on about day 6.  
     
     
         4 . The process according to  claim 1 , wherein the polyanionic addition step b) occurs during the death phase.  
     
     
         5 . The process according to  claim 4 , wherein the polyanionic addition step b) occurs on or after day 7.  
     
     
         6 . The process according to  claim 1 , wherein the polyanionic compound is added to a culture concentration of from 1 to 1000 mg/L, from 1 to 200 mg/L, from 1 to 100 mg/L, or from 25 to 75 mg/L.  
     
     
         7 . The process according to  claim 4 , wherein the polyanionic compound is added to a culture concentration of 50 mg/L.  
     
     
         8 . The process according to  claim 1 , wherein the polyanionic compound is selected from the group consisting of dextran sulfate, heparin, heparan sulfate, pentosan sulfate, xylofuranan sulfate, curdlan sulfate, curdlan galactose sulfate, curdlan arabinose sulfate, mannan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, poly(vinyl sulfate), kappa-carrageenan, suramin; and salts thereof.  
     
     
         9 . The process according to  claim 8 , wherein the polyanionic compound is dextran sulfate.  
     
     
         10 . The process according to  claim 1 , wherein the cells are mammalian cells.  
     
     
         11 . The process according to  claim 10 , wherein the mammalian cells are CHO cells.  
     
     
         12 . The process according to  claim 1 , wherein the cell culture is a large scale cell culture.  
     
     
         13 . The process according to  claim 12 , wherein the large scale cell culture is greater than about 50 L.  
     
     
         14 . The process according to  claim 1 , wherein cell viability is increased.  
     
     
         15 . The process according to  claim 1 , wherein the soluble CTLA4 molecule is a CTLA4 fusion protein.  
     
     
         16 . The process according to  claim 17 , wherein the soluble CTLA4 fusion protein is a CTLA4Ig.  
     
     
         17 . The process according to  claim 18 , wherein the soluble CTLA4 fusion protein is CTLA4Ig comprising amino acids −1 to 357 or +1 to 357 as shown in SEQ ID NO: 2.  
     
     
         18 . The process according to  claim 17 , wherein the soluble CTLA4 molecule is a soluble CTLA4 mutant molecule.  
     
     
         19 . The process according to claim  20 , wherein the soluble CTLA4 mutant molecule is L104EA29YIg comprising amino acids −1 to 357 or +1 to 357 as shown in SEQ ID NO: 4.