US2003096366A1PendingUtilityA1

Method for production of recombinant proteins in eukaryote cells

40
Priority: Oct 2, 2001Filed: Sep 25, 2002Published: May 22, 2003
Est. expiryOct 2, 2021(expired)· nominal 20-yr term from priority
Inventors:Ida Mølgaard Knudsen
C12P 21/02C12N 9/647C12N 2510/02C12N 2531/00C12N 9/6437C12N 5/0075C12Y 304/21021C12N 11/04C12N 5/00
40
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Claims

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.

Claims

exact text as granted — not AI-modified
1 . A method for production of a polypeptide in eukaryote cells, 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.    
     
     
         2 . A method according to  claim 1 , further comprising a step of adding fresh medium to the culture vessel 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 predetermined temperature is between about 5° C. and 30° C. below the setpoint.  
     
     
         5 . A method according to  claim 4 , wherein the predetermined temperature is between about 5° C. and 20° C. below the setpoint.  
     
     
         6 . A method according to  claim 5 , wherein the predetermined temperature is between about 5° C. and 15° C. below the setpoint.  
     
     
         7 . A method according to  claim 6 , wherein the predetermined temperature is about 10° C. below the setpoint.  
     
     
         8 . A method according to  claim 1 , wherein the culture is cooled to a temperature between about 18° C. and about 32° C. before the sedimentation.  
     
     
         9 . A method according to  claim 8 , wherein the culture is cooled to a temperature between about 20° C. and about 30° C.  
     
     
         10 . A method according to  claim 9 , wherein the culture is cooled to a temperature between about 22° C. and about 28° C.  
     
     
         11 . A method according to  claim 10 , wherein the culture is cooled to a temperature between about 24° C. and about 28° C.  
     
     
         12 . A method according to  claim 11 , wherein the culture is cooled to a temperature between about 25° C. and about 27° C.  
     
     
         13 . A method according to  claim 1 , wherein the eukaryote cells are insect cells.  
     
     
         14 . A method according to  claim 1 , wherein the eukaryote cells are mammalian cells.  
     
     
         15 . A method according to  claim 14 , wherein the mammalian cells are selected from the group consisting of HEK, BHK, and CHO cells.  
     
     
         16 . A method according to  claim 15 , wherein the mammalian cells are CHO cells.  
     
     
         17 . A method according to  claim 1 , wherein the polypeptide is factor VII or a factor VII-related polypeptide.  
     
     
         18 . A method according to  claim 17 , wherein the polypeptide is wild-type human factor VII.  
     
     
         19 . A method according to  claim 17 , wherein the polypeptide is a factor VII-related polypeptide selected from the group consisting of: L305V-FVII, L305V/M306D/D309S-FVII, L305I-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, E296V/M298Q-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, R315N/V317T-FVII, K143N/N145T/R315N/V317T-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.  
     
     
         20 . A method according to  claim 1 , wherein the polypeptide is produced at a level at least about 15 mg/l of culture.  
     
     
         21 . 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.  
     
     
         22 . A method for cultivation of eukaryote cells, comprising the steps of 
 (i) Culturing cells on microcarriers under conditions and at a setpoint temperature appropriate for maintaining the culture;    (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.    
     
     
         23 . A method according to  claim 22 , further comprising a step of adding fresh medium to the culture vessel after said harvesting.  
     
     
         24 . A method according to  claim 22 , wherein the predetermined temperature is between about 5° C. and 30° C. below the setpoint.  
     
     
         25 . A method according to  claim 24 , wherein the predetermined temperature is between about 5° C. and 20° C. below the setpoint.  
     
     
         26 . A method according to  claim 25 , wherein the predetermined temperature is between about 5° C. and 15° C. below the setpoint.  
     
     
         27 . A method according to  claim 26 , wherein the predetermined temperature is about 10° C. below the setpoint.  
     
     
         28 . A method according to  claim 22 , where the culture is cooled to a temperature between about 18° C. and about 32° C.  
     
     
         29 . A method according to  claim 28 , wherein the culture is cooled to a temperature between about 20° C. and about 30° C.  
     
     
         30 . A method according to  claim 29 , wherein the culture is cooled to a temperature between about 22° C. and about 28° C.  
     
     
         31 . A method according to  claim 30 , wherein the culture is cooled to a temperature between about 24° C. and about 28° C.  
     
     
         32 . A method according to  claim 31 , wherein the culture is cooled to a temperature between about 25° C. and about 27° C.  
     
     
         33 . A method according to  claim 22 , wherein the eukaryote cells are insect cells.  
     
     
         34 . A method according to  claim 22 , wherein the eukaryote cells are mammalian cells.  
     
     
         35 . A method according to  claim 34 , wherein the mammalian cells are selected from the group consisting of HEK, BHK and CHO cells.  
     
     
         36 . A method according to  claim 35 , wherein the mammalian cells are CHO cells.  
     
     
         37 . A method according to  claim 22 , wherein said cells produce a desired factor VII or factor VII-related polypeptide.  
     
     
         38 . A method according to  claim 37 , wherein the desired polypeptide is wild-type human factor VII.  
     
     
         39 . A method according to  claim 38 , wherein the desired polypeptide is a factor VII-related polypeptide selected from the group consisting of: L305V-FVII, L305V/M306D/D309S-FVII, L305I-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, E296V/M298Q-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; and P11Q/K33E-FVII, T106N-FVII, K143N/N145T-FVII, V253N-FVII, R290N/A292T-FVII, G291N-FVII, R315N/V317T-FVII, K143N/N145T/R315N/V317T-FVII; and FVII having substitutions, additions or deletions in the amino acid sequence from 233Thr to 240Asn, FVII having substitutions, additions or deletions in the amino acid sequence from 304Arg to 329Cys.  
     
     
         40 . A method according to  claim 22 , wherein the desired polypeptide is produced at a level at least about 15 mg/l of culture.  
     
     
         41 . A method for harvesting polypeptides produced by eukaryotic cells growing in microcarrier culture, said method comprising (i) cooling the culture to a predetermined temperature below the setpoint of the cultivation, followed by (ii) sedimenting the microcarriers.

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