Serum-free cell culture medium

Abstract

The specification describes an improved serum-free animal cell culture medium, which can used for the production of a protein of interest. Ornithine, or a combination of ornithine and putrescine can be added to serum-free media or chemically defined media to improve viable cell density, to reduce cell doubling time, and to increase the production of a protein of interest.

What is claimed is: 
     
         1 . A method for producing aflibercept comprising:
 (a) culturing Chinese hamster ovary (CHO) cells expressing aflibercept in a cell culture medium, wherein said cell culture medium is serum-free,   (b) culturing said CHO cells in said cell culture medium at a temperature of 35° C. to 38° C. during a growth phase; and   (c) culturing said CHO cells in said cell culture medium at a decreased temperature of 29° C. to 37° C. during a production phase when the cell density of the growth phase reaches between 1.6×10 6  and 12.6×10 6  viable cells per mL.   
     
     
         2 . The method of  claim 1 , wherein step (c) comprises culturing said CHO cells at a decreased temperature of 30° C. to 34° C. during the production phase. 
     
     
         3 . The method of  claim 1 , wherein the decreased temperature of step (c) of the production phase begins between day 3 and day 7 when the cell density of the growth phase reaches between 1.6×10 6  and 12.6×10 6  viable cells per mL. 
     
     
         4 . The method of  claim 2 , wherein the decreased temperature of step (c) of the production phase begins between day 5 and day 7 when the cell density of the growth phase reaches between 2.0×10 6  and 3.6×10 6  viable cells per mL. 
     
     
         5 . The method of  claim 1 , wherein said cell culture medium comprises ≥0.09 mM 0.014 mM ornithine and decreasing the temperature at step (c) of the production phase between day 3 and day 7 when the cell density of the growth phase reaches between 5.1×10 6  and 12.6×10 6  viable cells per mL. 
     
     
         6 . The method of  claim 2 , wherein said cell culture medium comprises ≥0.09 mM 0.014 mM ornithine and decreasing the temperature at step (c) of the production phase between day 3 and day 7 when the cell density of the growth phase reaches between 5.1×10 6  and 12.6×10 6  viable cells per mL. 
     
     
         7 . The method of  claim 1 , wherein said cell culture medium comprises ≥0.09 mM±0.014 mM ornithine and decreasing the temperature at step (c) of the production phase between day 5 and day 7 when the cell density of the growth phase reaches between 5.9×10 6  and 12.6×10 6  viable cells per mL. 
     
     
         8 . The method of  claim 1 , wherein the cell culture medium comprises ornithine at a concentration ranging from 0.09±0.014 mM to 0.9±0.14 mM and decreasing the temperature at step (c) of the production phase between day 3 and day 7 when the cell density of the growth phase reaches between 5.1×10 6  and 12.6×10 6  viable cells per mL. 
     
     
         9 . The method of  claim 1 , wherein the cell culture medium comprises ornithine at a concentration ranging from 0.09±0.014 mM to 0.9±0.14 mM and decreasing the temperature at step (c) of the production phase between day 5 and day 7 when the cell density of the growth phase reaches between 5.9×10 6  and 12.6×10 6  viable cells per mL. 
     
     
         10 . The method of  claim 1 , wherein the cell culture medium comprises 0.6±0.09 mM ornithine and 0.714±0.11 mM putrescine and decreasing the temperature at step (c) of the production phase between day 3 and day 7 when the cell density of the growth phase reaches between 5.0×10 6  and 12.4×10 6  viable cells per mL. 
     
     
         11 . The method of  claim 2 , wherein the cell culture medium comprises 0.6±0.09 mM ornithine and 0.714±0.11 mM putrescine and decreasing the temperature at step (c) of the production phase between day 5 and day 7 when the cell density of the growth phase reaches between 5.8×10 6  and 12.4×10 6  viable cells per mL. 
     
     
         12 . The method of  claim 1 , wherein the cell culture medium comprises 0.6±0.09 mM ornithine and 0.714±0.11 mM putrescine and decreasing the temperature at step (c) of the production phase between day 5 and day 7 when the cell density of the growth phase reaches between 5.8×10 6  and 12.4×10 6  viable cells per mL. 
     
     
         13 . The method of  claim 2 , wherein the cell culture medium comprises 0.6±0.09 mM ornithine and 0.714±0.11 mM putrescine and decreasing the temperature at step (c) of the production phase between day 3 and day 7 when the cell density of the growth phase reaches between 5.0×10 6  and 12.4×10 6  viable cells per mL. 
     
     
         14 . The method of  claim 1 , wherein the aflibercept is produced at a titer of at least 100 mg/L. 
     
     
         15 . The method of  claim 1 , wherein the aflibercept is produced at a titer of at least 1 g/L. 
     
     
         16 . The method of  claim 1 , wherein the CHO cell is selected from the group of CHO K1 cells, CHO DUX B-11 cells, Veggie-CHO cells, GS-CHO cells, D-CHO cells, and CHO lec mutant cells. 
     
     
         17 . The method of  claim 1 , wherein the culture medium comprises less than or equal to 7.5 g/L soy hydrolysate. 
     
     
         18 . The method of  claim 1 , wherein the cell culture medium comprises:
 (a) ≥40±6 mM of a mixture of amino acids or salts thereof;   (b) one or more fatty acids;   (c) a mixture of nucleosides; or   (d) one or more divalent cations.   
     
     
         19 . A method for producing aflibercept comprising:
 (a) culturing cells capable of producing aflibercept at a temperature range of 35° C. to 38° C.; and then   (b) culturing said cells at a decreased temperature of 29° C. to 34° C. when the cell density reaches between 1.6×10 6  and 12.6×10 6  viable cells per mL, wherein said cells are cultured in a serum-free cell culture medium.   
     
     
         20 . The method of  claim 19 , wherein the temperature of step (b) is decreased between day 3 and day 7 when the cell density of the growth phase reaches between 1.6×10 6  and 12.6×10 6  viable cells per mL. 
     
     
         21 . The method of  claim 19 , wherein said cell culture medium comprises ≥0.09 mM 0.014 mM ornithine and the temperature at step (b) is decreased between day 3 and day 7 when the cell density reaches between 5.1×10 6  and 12.6×10 6  viable cells per mL. 
     
     
         22 . The method of  claim 19 , wherein said cell culture medium comprises ≥0.09 mM 0.014 mM omithine and the temperature at step (b) is decreased between day 5 and day 7 when the cell density reaches between 5.9×10 6  and 12.6×10 6  viable cells per mL. 
     
     
         23 . The method of  claim 21 , wherein the cell culture medium comprises ornithine at a concentration ranging from 0.09±0.014 mM to 0.9±0.14 mM omithine and the temperature at step (b) is decreased when between day 3 and day 7 when the cell density reaches between 5.1×10 6  and 12.6×10 6  viable cells per mL. 
     
     
         24 . The method of  claim 19 , wherein said cells capable of producing aflibercept are selected from the group of CHO K1 cells, CHO DUX B-11 cells, Veggie-CHO cells, GS-CHO cells, D-CHO cells, and CHO lec mutant cells. 
     
     
         25 . A method for producing aflibercept comprising:
 (a) culturing Chinese hamster ovary (CHO) cells capable of producing aflibercept at a temperature range of 35° C. to 38° C., wherein said cell culture medium is serum-free and comprises hydrolysates and polyamines; and then (b) culturing said CHO cells at a decreased temperature of 29° C. to 34° C. when the cell density reaches between 1.6×10 6  and 12.6×10 6  viable cells per mL.   
     
     
         26 . The method of  claim 25 , wherein said CHO cells capable of producing aflibercept are selected from the group of CHO K1 cells, CHO DUX B-11 cells, Veggie-CHO cells, GS-CHO cells, D-CHO cells, and CHO lec mutant cells. 
     
     
         27 . The method of  claim 25 , wherein the decreased temperature of step (b) begins between day 5 and day 7 when the cell density of the growth phase reaches between 2.0×10 6  and 3.6×10 6  viable cells per mL. 
     
     
         28 . The method of  claim 27 , wherein the aflibercept protein is produced at a titer of at least 100 mg/L. 
     
     
         29 . The method of  claim 28 , wherein the cell culture medium comprises:
 (a) ≥40±6 mM of a mixture of amino acids or salts thereof;   (b) one or more fatty acids;   (c) a mixture of nucleosides; or   (d) one or more divalent cations.   
     
     
         30 . The method of  claim 26 , wherein said culture medium comprises one or more supplements selected form the group of:
 (i) about 29.8 mM NaHCO 3 ,   (ii) about 2mM glutamine,   (iii) about 0.86 μM insulin,   (iv) about 11.1 mM glucose,   (v) about 6.54 μM zinc sulfate,   (vi) copper sulfate,   (vii) ferric chloride,   (viii) nickel sulfate,   (ix) about 85 μM EDTA, and   (x) about 50 μM citrate.