Cell culture compositions with antioxidants and methods for polypeptide production

Abstract

Cell culture media comprising antioxidants are provided herein as are methods of using the media for cell culturing and polypeptide production from cells. Compositions comprising polypeptides, such as therapeutic polypeptides, produced by the methods herein are also provided.

1 . A method of culturing a cell comprising a nucleic acid encoding a polypeptide, wherein the method comprises the step of contacting the cell with a cell culture medium comprising hypotaurine or an analog or precursor thereof, wherein the cell culture medium comprising the hypotaurine or an analog of precursor thereof reduces the color intensity of a composition comprising the polypeptide produced by the cell as compared to the color intensity of a composition comprising the polypeptide produced by the cell cultured in a cell culture medium that does not comprise the hypotaurine or an analog or precursor thereof. 
     
     
         2 . The method of  claim 1 , wherein the cell culture medium comprising the hypotaurine or an analog or precursor thereof reduces the color intensity of a composition comprising the polypeptide produced by the cell by at least about 0.1% as compared to a composition comprising the polypeptide produced by the cell cultured in a cell culture medium that does not comprise the hypotaurine or an analog or precursor thereof. 
     
     
         3 . The method of  claim 1 , wherein the cell culture medium comprising the hypotaurine or an analog or precursor thereof reduces the color intensity of a composition comprising the polypeptide produced by the cell by about 5% to about 50% as compared to a composition comprising the polypeptide produced by the cell cultured in a cell culture medium that does not comprise the hypotaurine or an analog or precursor thereof. 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein the cell culture medium comprises the hypotaurine or an analog or precursor thereof at a concentration of at least about 0.0001 mM. 
     
     
         5 . The method of  claim 4 , wherein the cell culture medium comprises the hypotaurine or an analog or precursor thereof at a concentration from about 0.0001 mM to about 500.0 mM. 
     
     
         6 . The method of  claim 4 , wherein the cell culture medium comprises the hypotaurine or an analog or precursor thereof at a concentration from about 1.0 mM to about 40.0 mM. 
     
     
         7 . The method of  claim 4 , wherein the cell culture medium comprises the hypotaurine or an analog or precursor thereof at a concentration from about 1.0 mM to about 10.0 mM. 
     
     
         8 . The method of any one of  claims 1 - 7 , wherein the hypotaurine or an analog or precursor thereof is selected from the group consisting of hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulphinic acid, and taurine. 
     
     
         9 . The method of any one of  claims 1 - 8 , wherein the cell culture medium comprising the hypotaurine or an analog or precursor thereof is a chemically defined cell culture medium. 
     
     
         10 . The method of any one of  claims 1 - 8 , wherein the cell culture medium comprising the hypotaurine or an analog or precursor thereof is a chemically undefined cell culture medium. 
     
     
         11 . The method of any one of  claims 1 - 10 , wherein the cell culture medium comprising the hypotaurine or an analog or precursor thereof is a basal cell culture medium. 
     
     
         12 . The method of any one of  claims 1 - 10 , wherein the cell culture medium comprising the hypotaurine or an analog or precursor thereof is a feed cell culture medium. 
     
     
         13 . The method of any one of  claims 1 - 10 , wherein the cell is contacted with the cell culture medium comprising the hypotaurine or an analog or precursor thereof during the cell's growth phase. 
     
     
         14 . The method of any one of  claims 1 - 10 , wherein the cell is contacted with the cell culture medium comprising the hypotaurine or an analog or precursor thereof during the cell's production phase. 
     
     
         15 . The method of any one of  claims 1 - 14 , wherein the hypotaurine or an analog or precursor thereof is added to the cell culture medium on at least one day of a cell culture cycle. 
     
     
         16 . The method of any one of  claims 1 - 14 , wherein the hypotaurine or an analog or precursor thereof is added to the cell culture medium on day 0 of a 14 day cell culture cycle. 
     
     
         17 . The method of any one of  claims 1 - 16 , wherein the cell is a mammalian cell. 
     
     
         18 . The method of  claim 17 , wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell. 
     
     
         19 . The method of any one of  claims 1 - 18 , wherein the polypeptide is an antibody or fragment thereof. 
     
     
         20 . A method of culturing a cell comprising a nucleic acid encoding a polypeptide, wherein the method comprises the step of contacting the cell with a cell culture medium, wherein the cell culture medium comprises one or more of components (a)-(h):
 (a) hypotaurine;   (b) s-carboxymethylcysteine;   (c) carnosine;   (d) anserine;   (e) butylated hydroxyanisole;   (f) lipoic acid;   (g) quercitrin hydrate; and   (h) aminoguanidine;   and wherein the cell culture medium comprising one or more of components (a)-(h) reduces the color intensity of a composition comprising the polypeptide produced by the cell as compared to a composition comprising the polypeptide produced by the cell cultured in a cell culture medium that does not comprise the one or more of components (a)-(h).   
     
     
         21 . The method of  claim 20 , wherein the cell culture medium comprising one or more of components (a)-(h) reduces the color intensity of a composition comprising the polypeptide produced by the cells by at least about 0.1% as compared to a composition comprising the polypeptide produced by the cell cultured in a cell culture medium that does not comprise the one or more of components (a)-(h). 
     
     
         22 . The method of  claim 20 , wherein the cell culture medium comprising one or more of components (a)-(h) reduces the color intensity of a composition comprising the polypeptide produced by the cells by about 5% to about 75% as compared to a composition comprising the polypeptide produced by the cell cultured in a cell culture medium that does not comprise the one or more of components (a)-(h). 
     
     
         23 . The method of any one of  claims 20 - 22 , wherein the cell culture medium comprising one or more of components (a)-(h) comprises the one or more components (a)-(h) in an amount selected from:
 (a) hypotaurine at a concentration from at least about 0.0001 mM;   (b) s-carboxymethylcysteine at a concentration from at least about 0.0001 mM;   (c) carnosine at a concentration from at least about 0.0001 mM;   (d) anserine at a concentration from at least about 0.0001 mM;   (e) butylated hydroxyanisole at a concentration from at least about 0.0001 mM;   (f) lipoic acid at a concentration from at least about 0.0001 mM;   (g) quercitrin hydrate at a concentration from at least about 0.0001 mM; and   (h) aminoguanidine at a concentration from at least about 0.0003 mM.   
     
     
         24 . The method of  claim 23 , wherein the cell culture medium comprises hypotaurine at a concentration from about 2.0 mM to about 50.0 mM. 
     
     
         25 . The method of  claim 23  or  24 , wherein the cell culture medium comprises s-carboxymethylcysteine at a concentration from about 8.0 mM to about 12.0 mM. 
     
     
         26 . The method of any one of  claims 23 - 25 , wherein the cell culture medium comprises carnosine at a concentration from about 8.0 mM to about 12.0 mM. 
     
     
         27 . The method of any one of  claims 23 - 26 , wherein the cell culture medium comprises anserine at a concentration from about 3.0 mM to about 5.0 mM. 
     
     
         28 . The method of any one of  claims 23 - 27 , wherein the cell culture medium comprises butylated hydroxyanisole at a concentration from about 0.025 mM to about 0.040 mM. 
     
     
         29 . The method of any one of  claims 23 - 28 , wherein the cell culture medium comprises lipoic acid at a concentration from about 0.040 mM to about 0.060 mM. 
     
     
         30 . The method of any one of  claims 23 - 28 , wherein the cell culture medium comprises quercitrin hydrate at a concentration from about 0.010 mM to about 0.020 mM. 
     
     
         31 . The method of any one of  claims 23 - 30 , wherein the cell culture medium comprises aminoguanidine at a concentration from about 0.0003 mM to about 10 mM. 
     
     
         32 . The method of any one of  claims 20 - 31 , wherein the cell culture medium is a chemically defined cell culture medium. 
     
     
         33 . The method of any one of  claims 20 - 31 , wherein the cell culture medium is a chemically undefined cell culture medium. 
     
     
         34 . The method of any one of  claims 20 - 33 , wherein the cell culture medium is a basal cell culture medium. 
     
     
         35 . The method of any one of  claims 20 - 33 , wherein the cell culture medium is a feed cell culture medium. 
     
     
         36 . The method of any one of  claims 20 - 33 , wherein the cell is contacted with the cell culture medium during the cell's growth phase. 
     
     
         37 . The method of any one of  claims 20 - 33 , wherein the cell is contacted with the cell culture medium during the cell's production phase. 
     
     
         38 . The method of any one of  claims 20 - 37 , wherein the one or more of components (a)-(h) is added to the cell culture medium on at least one day of a cell culture cycle. 
     
     
         39 . The method of any one of  claims 20 - 37 , wherein the one or more of components (a)-(h) is added to the cell culture medium on day 0 of a 14 day cell culture cycle. 
     
     
         40 . The method of any one of  claims 20 - 39 , wherein the cell is a mammalian cell. 
     
     
         41 . The method of  claim 40 , wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell. 
     
     
         42 . The method of any one of  claims 20 - 41 , wherein the polypeptide is an antibody or fragment thereof. 
     
     
         43 . A method of producing a polypeptide comprising the step of culturing a cell comprising a nucleic acid encoding the polypeptide in a cell culture medium comprising hypotaurine or an analog or precursor thereof, and wherein the cell culture medium comprising the hypotaurine or an analog or precursor thereof reduces the color intensity of a composition comprising the polypeptide produced by the cell as compared to the color intensity of a composition comprising the polypeptide produced by the cell cultured in a cell culture medium that does not comprise the hypotaurine or an analog or precursor thereof 
     
     
         44 . The method of  claim 43 , wherein the cell culture medium comprising the hypotaurine or an analog or precursor thereof reduces the color intensity of a composition comprising the polypeptide produced by the cell by at least about 0.1% as compared to a composition comprising the polypeptide produced by the cell cultured in a cell culture medium that does not comprise the hypotaurine or an analog or precursor thereof 
     
     
         45 . The method of  claim 43 , wherein the cell culture medium comprising the hypotaurine or an analog or precursor thereof reduces the color intensity of a composition comprising the polypeptide produced by the cell by about 5% to about 50% as compared to a composition comprising the polypeptide produced by the cell cultured in a cell culture medium that does not comprise the hypotaurine or an analog or precursor thereof 
     
     
         46 . The method of any one of  claims 43 - 45 , wherein the cell culture medium comprises the hypotaurine or an analog or precursor thereof at a concentration from at least about 0.0001 mM. 
     
     
         47 . The method of  claim 46 , wherein the cell culture medium comprising comprises the hypotaurine or an analog or precursor thereof at a concentration from about 0.0001 mM to about 500.0 mM. 
     
     
         48 . The method of  claim 46 , wherein the cell culture medium comprises the hypotaurine or an analog or precursor thereof at a concentration from about 1.0 mM to about 40.0 mM. 
     
     
         49 . The method of  claim 46 , wherein the cell culture medium comprises the hypotaurine or an analog or precursor thereof at a concentration from about 1.0 mM to about 10.0 mM. 
     
     
         50 . The method of any one of  claims 43 - 49 , wherein the hypotaurine or an analog or precursor thereof is selected from the group consisting of hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulphinic acid, and taurine. 
     
     
         51 . The method of any one of  claims 43 - 50 , wherein the cell culture medium is a chemically defined cell culture medium. 
     
     
         52 . The method of any one of  claims 43 - 51 , wherein the cell culture medium is a chemically undefined cell culture medium. 
     
     
         53 . The method of any one of  claims 43 - 52 , wherein the cell culture medium is a basal cell culture medium. 
     
     
         54 . The method of any one of  claims 43 - 52 , wherein the cell culture medium is a feed cell culture medium. 
     
     
         55 . The method of any one of  claims 43 - 54 , wherein the hypotaurine or an analog or precursor thereof is added to the cell culture medium on at least one day of a cell culture cycle. 
     
     
         56 . The method of any one of  claims 43 - 54 , wherein the hypotaurine or an analog or precursor thereof is added to the cell culture medium on day 0 of a 14 day cell culture cycle. 
     
     
         57 . The method of any one of  claims 43 - 56 , wherein the cell is a mammalian cell. 
     
     
         58 . The method of  claim 57 , wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell. 
     
     
         59 . The method of any one of  claims 43 - 58 , wherein the polypeptide is an antibody. 
     
     
         60 . The method of  claim 59 , wherein the antibody is an IgG1 antibody. 
     
     
         61 . The method of  claim 59  or  60 , wherein the antibody is secreted into the cell culture medium comprising the hypotaurine or an analog or precursor thereof 
     
     
         62 . The method of any one of  claims 43 - 61 , further comprising the step of recovering the polypeptide from the cell culture medium comprising the hypotaurine or an analog or precursor thereof 
     
     
         63 . The method of  claim 62 , wherein a composition comprising the recovered polypeptide is a liquid composition or a non-liquid composition. 
     
     
         64 . The method of  claim 63 , wherein the composition comprising the recovered polypeptide appears as a colorless or slightly colored liquid. 
     
     
         65 . A method of producing a polypeptide comprising the step of culturing a cell comprising a nucleic acid encoding the polypeptide in a cell culture medium, wherein the cell culture medium comprises one or more of components (a)-(h):
 (a) hypotaurine;   (b) s-carboxymethylcysteine;   (c) carnosine;   (d) anserine;   (e) butylated hydroxyanisole;   (f) lipoic acid;   (g) quercitrin hydrate; and   (h) aminoguanidine;   and wherein the cell culture medium comprising one or more of components (a)-(h) reduces the color intensity of a composition comprising the polypeptide produced by the cell as compared to a composition comprising the polypeptide produced by the cell cultured in a cell culture medium that does not comprise one or more of components (a)-(h).   
     
     
         66 . The method of  claim 65 , wherein the cell culture medium comprising one or more of components (a)-(h) reduces the color intensity of a composition comprising the polypeptide produced by the cells by at least about 0.1% as compared to a composition comprising the polypeptide produced by the cell cultured in a cell culture medium that does not comprise the one or more of components (a)-(h). 
     
     
         67 . The method of  claim 65 , wherein the cell culture medium comprising one or more of components (a)-(h) reduces the color intensity of a composition comprising the polypeptide produced by the cells by about 5% to about 50% as compared to a composition comprising the polypeptide produced by the cell cultured in a cell culture medium that does not comprise the one or more of components (a)-(h). 
     
     
         68 . The method of any one of  claims 65 - 67 , wherein the cell culture medium comprises the one or more components (a)-(h) in an amount selected from:
 (a) hypotaurine at a concentration from at least about 0.0001 mM;   (b) s-carboxymethylcysteine at a concentration from at least about 0.0001 mM;   (c) carnosine at a concentration from at least about 0.0001 mM;   (d) anserine at a concentration from at least about 0.0001 mM;   (e) butylated hydroxyanisole at a concentration from at least about 0.0001 mM;   (f) lipoic acid at a concentration from at least about 0.0001 mM;   (g) quercitrin hydrate at a concentration from at least about 0.0001 mM; and   (h) aminoguanidine at a concentration from at least about 0.0003 mM.   
     
     
         69 . The method of  claim 68 , wherein the cell culture medium comprises hypotaurine at a concentration from about 2.0 mM to about 50.0 mM. 
     
     
         70 . The method of  claim 68  or  69 , wherein the cell culture medium comprises s-carboxymethylcysteine at a concentration from about 8.0 mM to about 12.0 mM. 
     
     
         71 . The method of any one of  claims 68 - 70 , wherein the cell culture medium comprises carnosine at a concentration from about 8.0 mM to about 12.0 mM. 
     
     
         72 . The method of any one of  claims 68 - 71 , wherein the cell culture medium comprises anserine at a concentration from about 3.0 mM to about 5.0 mM. 
     
     
         73 . The method of any one of  claims 68 - 72 , wherein the cell culture medium comprises butylated hydroxyanisole at a concentration from about 0.025 mM to about 0.040 mM. 
     
     
         74 . The method of any one of  claims 68 - 73 , wherein the cell culture medium comprises lipoic acid at a concentration from about 0.040 mM to about 0.060 mM. 
     
     
         75 . The method of any one of  claims 68 - 74 , wherein the cell culture medium comprises quercitrin hydrate at a concentration from about 0.010 mM to about 0.020 mM. 
     
     
         76 . The method of any one of  claims 68 - 75 , wherein the cell culture medium comprises aminoguanidine at a concentration from about 0.0003 mM to about 10 mM. 
     
     
         77 . The method of any one of  claims 65 - 76 , wherein the cell culture medium is a chemically defined cell culture medium. 
     
     
         78 . The method of any one of  claims 65 - 76 , wherein the cell culture medium is a chemically undefined cell culture medium. 
     
     
         79 . The method of any one of  claims 65 - 78 , wherein the cell culture medium is a basal cell culture medium. 
     
     
         80 . The method of any one of  claims 65 - 78 , wherein the cell culture medium is a feed cell culture medium. 
     
     
         81 . The method of any one of  claims 65 - 78 , wherein the cell is contacted with the cell culture medium during the cell's growth phase. 
     
     
         82 . The method of any one of  claims 65 - 78 , wherein the cell is contacted with the cell culture medium during the cell's production phase. 
     
     
         83 . The method of any one of  claims 65 - 82 , wherein the one or more of components (a)-(h) is added to the cell culture medium on at least one day of a cell culture cycle. 
     
     
         84 . The method of any one of  claims 65 - 82 , wherein the one or more of components (a)-(h) is added to the cell culture medium on day 0 of a 14 day cell culture cycle. 
     
     
         85 . The method of any one of  claims 65 - 84 , wherein the cell is a mammalian cell. 
     
     
         86 . The method of  claim 85 , wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell. 
     
     
         87 . The method of any one of  claims 65 - 86 , wherein the polypeptide is an antibody or fragment thereof 
     
     
         88 . The method of  claim 87 , wherein the antibody is an IgG1 antibody. 
     
     
         89 . The method of  claim 87  or  88 , wherein the antibody is secreted into the cell culture medium. 
     
     
         90 . The method of any one of  claims 65 - 89 , further comprising the step of recovering the polypeptide from the cell culture medium comprising one or more of components (a)-(h). 
     
     
         91 . The method of  claim 90 , wherein a composition comprising the recovered polypeptide is a liquid composition or a non-liquid composition. 
     
     
         92 . The method of  claim 91 , wherein the composition comprising the recovered polypeptide appears as a colorless or slightly colored liquid. 
     
     
         93 . A polypeptide produced by the method of any one of  claims 43 - 92 . 
     
     
         94 . A pharmaceutical composition comprising a polypeptide of  claim 93  and a pharmaceutically acceptable carrier. 
     
     
         95 . A kit for supplementing a cell culture medium with chemically defined constituents, the kit comprising hypotaurine or an analog or precursor thereof at a concentration of at least about 0.0001 mM, and wherein the hypotaurine or an analog or precursor is selected from the group consisting of hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulphinic acid, and taurine. 
     
     
         96 . A kit for supplementing a cell culture medium with chemically defined constituents, the kit comprising one or more of:
 (a) hypotaurine in an amount to provide from at least about 0.0001 mM hypotaurine in the cell culture medium;   (b) s-carboxymethylcysteine in an amount to provide from at least about 0.0001 mM s-carboxymethylcysteine in the cell culture medium;   (c) carnosine in an amount to provide from at least about 0.0001 mM carnosine in the cell culture medium;   (d) anserine in an amount to provide from at least about 0.0001 mM anserine in the cell culture medium;   (e) butylated hydroxyanisole in an amount to provide from at least about 0.0001 mM butylated hydroxyanisole;   (f) lipoic acid in an amount to provide from at least about 0.0001 mM lipoic acid in the cell culture medium;   (g) quercitrin hydrate in an amount to provide from at least about 0.0001 mM quercitrin hydrate in the cell culture medium; and   (h) aminoguanidine in an amount to provide from at least about 0.0003 mM aminoguanidine in the cell culture medium.   
     
     
         97 . A cell culture medium comprising from at least about 0.0001 mM of hypotaurine or an analog or precursor thereof selected from the group consisting of hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulphinic acid, and taurine. 
     
     
         98 . A cell culture medium comprising one or more of components (a)-(h):
 (a) from at least about 0.0001 mM hypotaurine;   (b) from at least about 0.0001 mM s-carboxymethylcysteine;   (c) from at least about 0.0001 mM carnosine;   (d) from at least about 0.0001 mM anserine;   (e) from at least about 0.0001 mM butylated hydroxyanisole;   (f) from at least about 0.0001 mM lipoic acid;   (g) from at least about 0.0001 mM quercitrin hydrate; and   (h) from a least about 0.0003 mM aminoguanidine.   
     
     
         99 . A composition comprising (a) a cell comprising a nucleic acid encoding a polypeptide; and
 (b) a cell culture medium according to  claim 97  or  98 .   
     
     
         100 . A composition comprising: (a) a polypeptide; and (b) a cell culture medium of  claim 97  or 
     
     
         98 . 
     
     
         101 . The composition of  claim 100 , wherein the polypeptide is secreted into the cell culture medium by a cell comprising a nucleic acid encoding the polypeptide.