Method of production of recombinant proteins in eukaryote cells

Abstract

The invention provides a method for production of polypeptides in eukaryote microcarrier cell culture, the method comprising the steps of (i) Culturing cells expressing said polypeptide on microcarriers under conditions and at a setpoint temperature appropriate for expression of said polypeptide; (ii) Cooling the culture to a predetermined temperature below said setpoint; (iii) Sedimenting the microcarriers; and (iv) Harvesting all or part of the culture medium.

1 . A method for production of a polypeptide in eukaryote cells, comprising the steps of 
 (i) Culturing cells expressing a Factor VII or Factor VII-related polypeptide on microcarriers under conditions and at a setpoint temperature appropriate for expression of said polypeptide;    (ii) Actively cooling the culture to a predetermined temperature prior to step (iii), wherein the predetermined temperature is about 10° C. below the set-point;    (iii) Sedimenting the microcarriers; and    (iv) Harvesting all or part of the culture medium.    
     
     
         2 . A method according to  claim 1 , further comprising a step of adding fresh medium to the culture after said harvesting.  
     
     
         3 . A method according to  claim 1 , further comprising a step of recovering said polypeptide from the harvested culture medium.  
     
     
         4 . A method according to  claim 1 , wherein the culture is cooled to a temperature from about 25° C. to about 27° C.  
     
     
         5 . A method according to  claim 1 , wherein the eukaryote cells are insect cells.  
     
     
         6 . A method according to  claim 1 , wherein the eukaryote cells are mammalian cells.  
     
     
         7 . A method according to  claim 6 , wherein the mammalian cells are selected from the group consisting of human embryonic kidney (HEK), baby hamster kidney (BHK), and Chinese hamster ovary (CHO) cells.  
     
     
         8 . A method according to  claim 7 , wherein the mammalian cells are CHO cells.  
     
     
         9 . A method according to  claim 1 , wherein the polypeptide is wild-type human factor VII.  
     
     
         10 . A method according to  claim 1 , wherein the polypeptide is a factor VII-related polypeptide selected from the group consisting of: L305V-FVII, L305V/M306D/D309S-FVII, L3051-FVII, L305T-FVII, F374P-FVII, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII, V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII, K157A-FVII, E296V-FVII, E296WM298Q-FVII, V158D/E296V-FVII, V158D/M298K-FVII, S336G-FVII; S52A-Factor VII, S60A-Factor VII; R152E-Factor VII, S344A-Factor VII, Factor VIIa lacking the Gla domain; P11Q/K33E-FVII, T106N-FVII, K143N/N145T-FVII, V253N-FVII, R290N/A292T-FVII, G291N-FVII, R315NN317T-FVII, K143N/N145T/R315NN317T-FVII; and FVII having substitutions, additions or deletions in the amino acid sequence from 233Thr to 240Asn, and FVII having substitutions, additions or deletions in the amino acid sequence from 304Arg to 329Cys.  
     
     
         11 . A method according to  claim 1 , wherein the polypeptide is produced at a level at least about 15 mg/l of culture.  
     
     
         12 . A method according to  claim 1 , wherein the polypeptide is human factor VII, the cells are CHO cells, the carriers are macroporous carriers, the culture set-point is 36° C., and the culture is cooled to about 26 ° C. before allowing the carriers to sediment.