Cell culture processes

Abstract

Culturing heterologous protein-secreting mammalian cells, such as CHO or BHK cells, at 35.1-36.5° C. and/or at pH 7.15-7.20 and/or at a dissolved CO 2 concentration of 10% or lower. Preferred heterologous proteins are Factor VIII, ADAMTS-13, furin or Factor VII.

1 - 51 . (canceled) 
     
     
         52 . A method comprising:
 expressing a heterologous protein in a mammalian cell in continuous cell culture, wherein the continuous cell culture has a dissolved carbon dioxide concentration of from 1% to 10% and is maintained at a cell culture density of from 1.4×10 6  to 2.8×10 6  cells/ml.   
     
     
         53 . The method of  claim 52 , wherein the continuous cell culture has a dissolved carbon dioxide concentration of from 4% to 9%. 
     
     
         54 . The method of  claim 52 , wherein the continuous cell culture has a dissolved carbon dioxide concentration of from 5.5% to 8.5%. 
     
     
         55 . The method of  claim 52 , wherein the continuous cell culture is maintained at a temperature of from 34.2° C. to 36.9° C. 
     
     
         56 . The method of  claim 52 , wherein the continuous cell culture is maintained at a temperature of 36±0.9° C. 
     
     
         57 . The method of  claim 54 , wherein the continuous cell culture is maintained at a temperature of 36±0.9° C. 
     
     
         58 . The method of  claim 52 , wherein the continuous cell culture is maintained at a pH of from 7.10 to 7.25. 
     
     
         59 . The method of  claim 52 , wherein the continuous cell culture is maintained at a pH of 7.20±0.05. 
     
     
         60 . The method of  claim 54 , wherein the continuous cell culture is maintained at a pH of 7.20±0.05. 
     
     
         61 . The method of  claim 56 , wherein the continuous cell culture is maintained at a pH of 7.20±0.05. 
     
     
         62 . The method of  claim 57 , wherein the continuous cell culture is maintained at a pH of 7.20±0.05. 
     
     
         63 . The method of  claim 52 , wherein the continuous cell culture is buffered with bicarbonate. 
     
     
         64 . The method of  claim 52 , wherein the dissolved CO 2  concentration of the continuous cell culture is maintained by sparging the continuous mammalian cell culture with air. 
     
     
         65 . The method of  claim 52 , wherein the heterologous protein is a blood protein. 
     
     
         66 . The method of  claim 52 , wherein the heterologous protein is Factor VIII. 
     
     
         67 . The method of  claim 66 , wherein the Factor VIII is co-expressed with von Willebrand Factor. 
     
     
         68 . The method of  claim 52 , wherein the heterologous protein is ADAMTS-13, Furin, or Factor VII. 
     
     
         69 . The method of  claim 52 , wherein the continuous cell culture comprises copper at a concentration of at least 5 parts per billion (ppb). 
     
     
         70 . The method of  claim 52 , wherein the continuous cell culture comprises copper at a concentration of at least 7 parts per billion (ppb). 
     
     
         71 . The method of  claim 52 , wherein the mammalian cell is a rodent cell. 
     
     
         72 . The method of  claim 52 , wherein the mammalian cell is a CHO cell. 
     
     
         73 . The method of  claim 52 , wherein the mammalian cell is a BHK cell. 
     
     
         74 . The method of  claim 52 , wherein the continuous cell culture is operated under chemostatic conditions. 
     
     
         75 . The method of  claim 52 , wherein the continuous cell culture is operated under turbostatic conditions.