Cell culture media and methods of antibody production

Abstract

Cell culture media are provided herein as are methods of using the media for cell culture and antibody production from cells. Compositions comprising antibodies and fragments thereof, produced by the methods herein are also provided.

1 . A method of producing bevacizumab, or a fragment thereof, comprising the step of culturing a Chinese hamster ovary (CHO) cell comprising a nucleic acid encoding bevacizumab or fragment thereof in a cell culture medium, wherein the cell culture medium comprises two or more components selected from the group consisting of copper, insulin, and cystine, wherein the cell culture medium further comprises a plant-derived hydrolysate and an animal-derived hydrolysate, and wherein the cell produces bevacizumab, or a fragment thereof. 
     
     
         2 . The method of  claim 1 , wherein the cell culture medium comprises copper and insulin. 
     
     
         3 . The method of  claim 1 , wherein the cell culture medium comprises copper and cystine. 
     
     
         4 . The method of  claim 1 , wherein the cell culture medium comprises insulin and cystine. 
     
     
         5 . The method of  claim 1 , wherein the cell culture medium comprises copper, insulin, and cystine. 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein the cell culture medium comprises insulin at a concentration of from about 1.0 mg/L to about 100.0 mg/L. 
     
     
         8 . The method of  claim 1 , wherein the cell culture medium comprises insulin at a concentration of from about 10.0 mg/L to about 100.0 mg/L. 
     
     
         9 . The method of  claim 1 , wherein the cell culture medium comprises insulin at a concentration of from about 10.0 mg/L to about 50.0 mg/L. 
     
     
         10 . The method of  claim 1 , wherein the cell culture medium comprises insulin at a concentration of from about 10.0 mg/L to about 35.0 mg/L. 
     
     
         11 . The method of  claim 1 , wherein the cell culture medium comprises insulin at a concentration of from about 10.0 mg/L to about 25.0 mg/L. 
     
     
         12 . The method of  claim 1 , wherein the cell culture medium comprises insulin at a concentration of about 25 mg/L. 
     
     
         13 . The method of  claim 1 , wherein the cell culture medium comprises copper at a concentration of from about 69 nM to about 1,000 nM. 
     
     
         14 . The method of  claim 1 , wherein the cell culture medium comprises copper at a concentration of from about 325 nM to about 375 nM. 
     
     
         15 . The method of  claim 1 , wherein the cell culture medium comprises copper at a concentration of from about 325 nM to about 350 nM. 
     
     
         16 . The method of  claim 1 , wherein the cell culture medium comprises copper at a concentration of about any one of 330 nM, 335 nM, 339 nM, 340 nM, 345 nM or 350 nM. 
     
     
         17 . The method of  claim 1 , wherein the cell culture medium comprises copper at a concentration of about 339 nM. 
     
     
         18 . The method of  claim 1 , wherein the cell culture medium comprises cystine at a concentration of from about 0.7 mM to about 2.0 mM. 
     
     
         19 . The method of  claim 1 , wherein the cell culture medium comprises cystine at a concentration of from about 1.0 mM to about 1.6 mM. 
     
     
         20 . The method of  claim 1 , wherein the cell culture medium comprises cystine at a concentration of from about 1.2 mM to about 1.4 mM 
     
     
         21 . The method of  claim 1 , wherein the cell culture medium comprises cystine at a concentration of about 1.3 mM. 
     
     
         22 . The method of  claim 1 , wherein the cell culture medium comprises the animal-derived hydrolysate at a concentration of from about 6.0 g/L to about 20.0 g/L. 
     
     
         23 . The method of  claim 1 , wherein the cell culture medium comprises the animal-derived hydrolysate at a concentration of from about 8.0 g/L to about 12.0 g/L. 
     
     
         24 . The method of  claim 1 , wherein the cell culture medium comprises the animal-derived hydrolysate at a concentration of from about 9.0 g/L to about 11.0 g/L. 
     
     
         25 . The method of  claim 1 , wherein the cell culture medium comprises the animal-derived hydrolysate at a concentration of about 13 g/L. 
     
     
         26 . The method of  claim 1 , wherein the cell culture medium comprises the plant-derived hydrolysate at a concentration of from about 1.0 g/L to about 10.0 g/L. 
     
     
         27 . The method of  claim 1 , wherein the cell culture medium comprises the plant-derived hydrolysate at a concentration of from about 2.0 g/L to about 3.0 g/L. 
     
     
         28 . The method of  claim 1 , wherein the cell culture medium comprises the plant-derived hydrolysate at a concentration of from about 2.25 g/L to about 2.75 g/L 
     
     
         29 . The method of  claim 1 , wherein the cell culture medium comprises the plant-derived hydrolysate at a concentration of about 3.1 g/L. 
     
     
         30 . (canceled) 
     
     
         31 . The method of  claim 1 , wherein the cell culture medium comprises insulin and the method further comprises the step of adding an additional amount of insulin to the cell culture medium. 
     
     
         32 . The method of  claim 31 , wherein the additional amount of insulin is added to the cell culture medium once during the cell culture cycle. 
     
     
         33 . The method of  claim 31 , wherein the additional amount of insulin is added to the cell culture medium at least three times during the cell culture cycle. 
     
     
         34 . The method of  claim 31 , wherein the additional amount of insulin is added to the cell culture medium at least six times during the cell culture cycle. 
     
     
         35 . The method of  claim 31 , wherein the additional amount of insulin is added in an amount to provide insulin in the cell culture medium at a concentration of from about 1.0 mg/L to about 100.0 mg/L. 
     
     
         36 . The method of  claim 31 , wherein the additional amount of insulin is added in an amount to provide insulin in the cell culture medium at a concentration of from about 10.0 mg/L to about 100.0 mg/L. 
     
     
         37 . The method of  claim 31 , wherein the additional amount of insulin is added in an amount to provide insulin in the cell culture medium at a concentration of from about 10.0 mg/L to about 50.0 mg/L. 
     
     
         38 . The method of  claim 31 , wherein the additional amount of insulin is added in an amount to provide insulin in the cell culture medium at a concentration of from about 10.0 mg/L to about 35.0 mg/L. 
     
     
         39 . The method of  claim 31 , wherein the additional amount of insulin is added in an amount to provide insulin in the cell culture medium at a concentration of from about 10.0 mg/L to about 25.0 mg/L. 
     
     
         40 . The method of  claim 31 , wherein the additional amount of insulin is added in an amount to provide insulin in the cell culture medium at a concentration of about 15 mg/L. 
     
     
         41 . The method of  claim 1 , wherein the method further comprises the step of adding cysteine to the cell culture medium. 
     
     
         42 . The method of  claim 41 , wherein cysteine is added in an amount to provide from about 0.5 to about 2.0 mM cysteine in the cell culture medium. 
     
     
         43 . The method of  claim 41 , wherein cysteine is added in an amount to provide about 0.8 mM cysteine in the cell culture medium. 
     
     
         44 . The method of  claim 1 , wherein the method further comprises the step of adding cystine to the cell culture medium. 
     
     
         45 . The method of  claim 44 , wherein cystine is added in an amount to provide from about 0.1 to about 1.5 mM cystine in the cell culture medium. 
     
     
         46 . The method of  claim 44 , wherein cystine is added in an amount to provide about 0.2 mM cystine in the cell culture medium. 
     
     
         47 . The method of  claim 1 , wherein the cell is cultured at a temperature ranging from about 28° C. to about 37° C. 
     
     
         48 . The method of  claim 47 , wherein the cell is cultured at a temperature ranging from about 31° C. to about 35° C. 
     
     
         49 . The method of  claim 1 , wherein the cell is cultured at a first temperature of about 35° C. for a first period of time, is cultured at a second temperature of about 33° C. for a second period of time, and is cultured at a third temperature of about 31° C. for a third period of time. 
     
     
         50 . The method of  claim 1 , wherein bevacizumab, or a fragment thereof, is secreted into the cell culture medium. 
     
     
         51 . The method of  claim 1 , further comprising the step of recovering the bevacizumab, or a fragment thereof, from the cell culture. 
     
     
         52 . Bevacizumab, or fragment thereof, produced by the method of  claim 1 . 
     
     
         53 . A composition comprising: (i) bevacizumab, or a fragment thereof, produced by the method of  claim 1  and (ii) a pharmaceutically acceptable carrier. 
     
     
         54 . A method of culturing a Chinese hamster ovary (CHO) cell comprising a nucleic acid encoding bevacizumab, or a fragment thereof, the method comprising the step of contacting the CHO cell with a cell culture medium comprising two or more components selected from the group consisting of copper, insulin and cystine, wherein the cell culture medium further comprises a plant-derived hydrolysate and an animal-derived hydrolysate. 
     
     
         55 . The method of  claim 54 , wherein the cell culture medium comprises copper and insulin. 
     
     
         56 . The method of  claim 54 , wherein the cell culture medium comprises copper and cystine. 
     
     
         57 . The method of  claim 54 , wherein the cell culture medium comprises insulin and cystine. 
     
     
         58 . The method of  claim 54 , wherein the cell culture medium comprises copper, insulin, and cystine. 
     
     
         59 . (canceled) 
     
     
         60 . The method of  claim 54 , wherein the cell culture medium comprises insulin at a concentration of from about 1.0 mg/L to about 100.0 mg/L. 
     
     
         61 . The method of  claim 54 , wherein the cell culture medium comprises insulin at a concentration of from about 10.0 mg/L to about 100.0 mg/L. 
     
     
         62 . The method of  claim 54 , wherein the cell culture medium comprises insulin at a concentration of from about 10.0 mg/L to about 50.0 mg/L. 
     
     
         63 . The method of  claim 54 , wherein the cell culture medium comprises insulin at a concentration of from about 10.0 mg/L to about 35.0 mg/L. 
     
     
         64 . The method of  claim 54 , wherein the cell culture medium comprises insulin at a concentration of from about 10.0 mg/L to about 25.0 mg/L. 
     
     
         65 . The method of  claim 54 , wherein the cell culture medium comprises insulin at a concentration of about 25 mg/L. 
     
     
         66 . The method of  claim 54 , wherein the cell culture medium comprises copper at a concentration of from about 69 nM to about 1,000 nM. 
     
     
         67 . The method of  claim 54 , wherein the cell culture medium comprises copper at a concentration of from about 325 nM to about 375 nM. 
     
     
         68 . The method of  claim 54 , wherein the cell culture medium comprises copper at a concentration of from about 325 nM to about 350 nM. 
     
     
         69 . The method of  claim 54 , wherein the cell culture medium comprises copper at a concentration of about any one of 330 nM, 335 nM, 339 nM, 340 nM, 345 nM or 350 nM. 
     
     
         70 . The method of  claim 54 , wherein the cell culture medium comprises copper at a concentration of about 339 nM. 
     
     
         71 . The method of  claim 54 , wherein the cell culture medium comprises cystine at a concentration of from about 0.7 mM to about 2.0 mM. 
     
     
         72 . The method of  claim 54 , wherein the cell culture medium comprises cystine at a concentration of from about 1.0 mM to about 1.6 mM. 
     
     
         73 . The method of  claim 54 , wherein the cell culture medium comprises cystine at a concentration of from about 1.2 mM to about 1.4 mM 
     
     
         74 . The method of  claim 54 , wherein the cell culture medium comprises cystine at a concentration of about 1.3 mM. 
     
     
         75 . The method of  claim 54 , wherein the cell culture medium comprises the animal-derived hydrolysate at a concentration of from about 6.0 g/L to about 20.0 g/L. 
     
     
         76 . The method of  claim 54 , wherein the cell culture medium comprises the animal-derived hydrolysate at a concentration of from about 8.0 g/L to about 12.0 g/L. 
     
     
         77 . The method of  claim 54 , wherein the cell culture medium comprises the animal-derived hydrolysate at a concentration of from about 9.0 g/L to about 11.0 g/L. 
     
     
         78 . The method of  claim 54 , wherein the cell culture medium comprises the animal-derived hydrolysate at a concentration of about 13 g/L. 
     
     
         79 . The method of  claim 54 , wherein the cell culture medium comprises the plant-derived hydrolysate at a concentration of from about 1.0 g/L to about 10.0 g/L. 
     
     
         80 . The method of  claim 54 , wherein the cell culture medium comprises the plant-derived hydrolysate at a concentration of from about 2.0 g/L to about 3.0 g/L. 
     
     
         81 . The method of  claim 54 , wherein the cell culture medium comprises the plant-derived hydrolysate at a concentration of from about 2.25 g/L to about 2.75 g/L 
     
     
         82 . The method of  claim 54 , wherein the cell culture medium comprises the plant-derived hydrolysate at a concentration of about 3.1 g/L. 
     
     
         83 . (canceled) 
     
     
         84 . The method of  claim 54 , wherein the cell culture medium comprises insulin and the method further comprises the step of adding an additional amount of insulin to the cell culture medium. 
     
     
         85 . The method of  claim 84 , wherein the additional amount of insulin is added to the cell culture medium once during the cell culture cycle. 
     
     
         86 . The method of  claim 84 , wherein the additional amount of insulin is added to the cell culture medium at least three times during the cell culture cycle. 
     
     
         87 . The method of  claim 84 , wherein the additional amount of insulin is added to the cell culture medium at least six times during the cell culture cycle. 
     
     
         88 . The method of  claim 84 , wherein the additional amount of insulin is added in an amount to provide insulin in the cell culture medium at a concentration of from about 1.0 mg/L to about 100.0 mg/L. 
     
     
         89 . The method of  claim 84 , wherein the additional amount of insulin is added in an amount to provide insulin in the cell culture medium at a concentration of from about 10.0 mg/L to about 100.0 mg/L. 
     
     
         90 . The method of  claim 84 , wherein the additional amount of insulin is added in an amount to provide insulin in the cell culture medium at a concentration of from about 10.0 mg/L to about 50.0 mg/L. 
     
     
         91 . The method of  claim 84 , wherein the additional amount of insulin is added in an amount to provide insulin in the cell culture medium at a concentration of from about 10.0 mg/L to about 35.0 mg/L. 
     
     
         92 . The method of  claim 84 , wherein the additional amount of insulin is added in an amount to provide insulin in the cell culture medium at a concentration of from about 10.0 mg/L to about 25.0 mg/L. 
     
     
         93 . The method of  claim 84 , wherein the additional amount of insulin is added in an amount to provide insulin in the cell culture medium at a concentration of about 15 mg/L. 
     
     
         94 . The method of  claim 54 , wherein the method further comprises the step of adding cysteine to the cell culture medium. 
     
     
         95 . The method of  claim 94 , wherein cysteine is added in an amount to provide from about 0.5 to about 2.0 mM cysteine in the cell culture medium. 
     
     
         96 . The method of  claim 94 , wherein cysteine is added in an amount to provide about 0.8 mM cysteine in the cell culture medium. 
     
     
         97 . The method of  claim 54 , wherein the method further comprises the step of adding cystine to the cell culture medium. 
     
     
         98 . The method of  claim 97 , wherein cystine is added in an amount to provide from about 0.1 to about 1.5 mM cystine in the cell culture medium. 
     
     
         99 . The method of  claim 97 , wherein cystine is added in an amount to provide about 0.2 mM cystine in the cell culture medium. 
     
     
         100 . (canceled) 
     
     
         101 . The method of  claim 54 , wherein the cell is cultured at a temperature ranging from about 28° C. to about 37° C. 
     
     
         102 . The method of  claim 101 , wherein the cell is cultured at a temperature ranging from about 28° C. to about 35° C. 
     
     
         103 . The method of  claim 54 , wherein the cell is cultured at a first temperature of about 35° C. for a first period of time, is cultured at a second temperature of about 33° C. for a second period of time, and is cultured at a third temperature of about 31° C. for a third period of time. 
     
     
         104 . The method of  claim 54 , wherein bevacizumab, or a fragment thereof, is secreted into the cell culture medium. 
     
     
         105 . The method of  claim 54 , wherein the CHO cell is contacted with the cell culture medium during the cell's growth phase. 
     
     
         106 . The method of  claim 54 , wherein the CHO cell is contacted with the cell culture medium during the cell's production phase. 
     
     
         107 . A kit for supplementing a cell culture medium for use in culturing a Chinese hamster ovary (CHO) cell comprising a nucleic acid encoding bevacizumab, or a fragment thereof, the kit comprising at least two of components (i)-(iii): (i) insulin in an amount to provide from about 1.0 mg/L to about 100.0 mg/L insulin in the cell culture medium; (ii) cystine in an amount to provide from about 0.7 mM to about 2.0 mM cystine in the cell culture medium; (iii) and copper in an amount to provide from about 69.0 nM to about 1,000.0 nM copper in the cell culture medium, wherein the cell culture medium further comprises a plant-derived hydrolysate and an animal-derived hydrolysate. 
     
     
         108 - 111 . (canceled) 
     
     
         112 . A cell culture medium for use in culturing a Chinese hamster ovary (CHO) cell comprising a nucleic acid encoding bevacizumab, or a fragment thereof, the cell culture medium comprising at least two of components (i)-(iii): (i) from about 1.0 mg/L to about 100.0 mg/L insulin; (ii) from about 69.0 nM to about 1,000.0 nM copper; and (iii) from about 0.7 mM to about 2.0 mM cystine, wherein the cell culture medium further comprises a plant-derived hydrolysate and an animal-derived hydrolysate. 
     
     
         113 - 120 . (canceled) 
     
     
         121 . A composition comprising (a) a Chinese hamster ovary (CHO) cell comprising a nucleic acid encoding bevacizumab, or a fragment thereof; and (b) a cell culture medium according to  claim 112 . 
     
     
         122 . A composition comprising: (a) bevacizumab, or a fragment thereof; and (b) a cell culture medium according to  claim 112 . 
     
     
         123 . (canceled) 
     
     
         124 . A method of enhancing the amount of bevacizumab, or a fragment thereof, produced from a Chinese hamster ovary (CHO) cell comprising a nucleic acid encoding bevacizumab, or a fragment thereof, the method comprising the step of culturing the CHO cell in a cell culture medium comprising at least two of insulin, copper and cystine, wherein the cell culture medium further comprises a plant-derived hydrolysate and an animal-derived hydrolysate, and wherein the amount of bevacizumab, or a fragment thereof, produced from the CHO cell is enhanced relative to culturing the CHO cell in a cell culture medium without at least two of insulin, copper and cystine. 
     
     
         125 - 174 . (canceled) 
     
     
         175 . A method of culturing a Chinese hamster ovary (CHO) cell comprising a nucleic acid encoding bevacizumab, or a fragment thereof, in a cell culture medium comprising at least two of insulin, copper and cystine, wherein the cell culture medium further comprises a plant-derived hydrolysate and an animal-derived hydrolysate, and wherein the amount of bevacizumab, or a fragment thereof, produced from the CHO cell is enhanced relative to culturing the CHO cell in a cell culture medium without at least two of insulin, copper and cystine. 
     
     
         176 - 225 . (canceled)