PROCESS FOR REDUCING UNWANTED CULTURE BYPRODUCTS IN CELL CULTURE MEDIUM

Abstract

Provided are an improved culture process for reducing unwanted culture byproducts when a target protein is produced in a glutamine-free cell culture medium, an improved culture process for producing a target protein in a glutamine-free cell culture medium, or an improved culture process for producing a target protein in a glutamine-free cell culture medium.

Claims

1. An improved culture process for reducing an unwanted culture by-product when a target protein is produced in a glutamine-free cell culture medium, the improved culture process comprising: culturing a mammalian cell in the glutamine-free cell culture medium under conditions allowing survival or proliferation of the cell, wherein the cell culture medium comprises hypoxanthine at a concentration of about 20 μM to about 1,000 μM and thymidine at a concentration of about 2 μM to about 1,000 μM.

2. The improved culture process of claim 1, wherein the unwanted culture by-product is ammonia.

3. The improved culture process of claim 2, wherein an amount of the ammonia is reduced by about 10% or more, as compared to that in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

4. The improved culture process of claim 3, wherein the amount of the ammonia is reduced by about 20% or more, as compared to that in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

5. The improved culture process of claim 4, wherein the amount of the ammonia is reduced by about 30% to about 40%, as compared to that in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

6. The improved culture process of any one of claims 1 to 5, wherein the cell maintained in the process conditions exhibits increased glucose consumption, as compared to that in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

7. The improved culture process of claim 6, wherein the glucose consumption is increased by about 10% or more, as compared to glucose consumption in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

8. The improved culture process of claim 7, wherein the glucose consumption is increased by about 15% or more, as compared to glucose consumption in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

9. The improved culture process of claim 8, wherein the glucose consumption is increased by about 15% to about 30%, as compared to glucose consumption in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

10. The improved culture process of any one of claims 1 to 9, wherein the cell maintained in the process conditions exhibits increased cell viability, as compared to that in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

11. The improved culture process of claim 10, wherein the cell viability is increased by about 3% or more, as compared to cell viability in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

12. The improved culture process of claim 11, wherein the cell viability is increased by about 5% or more, as compared to cell viability in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

13. The improved culture process of claim 12, wherein the cell viability is increased by about 5% to about 10%, as compared to cell viability in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

14. The improved culture process of any one of claims 1 to 13, wherein the cell maintained in the process conditions exhibits an increased expression level (titer) of a polypeptide, as compared to that in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

15. The improved culture process of claim 14, wherein the polypeptide expression level (titer) is increased by about 10% or more, as compared to a polypeptide expression level in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

16. The improved culture process of claim 15, wherein the polypeptide expression level (titer) is increased by about 20% or more, as compared to a polypeptide expression level in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

17. The improved culture process of claim 16, wherein the polypeptide expression level (titer) is increased by about 30% to about 40%, as compared to a polypeptide expression level in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

18. The improved culture process of any one of claims 1 to 17, wherein the cell culture medium comprises hypoxanthine at a concentration of about 100 μM to about 1,000 μM.

19. The improved culture process of claim 18, wherein the cell culture medium comprises hypoxanthine at a concentration of about 100 μM to about 500 μM.

20. The improved culture process of claim 19, wherein the cell culture medium comprises hypoxanthine at a concentration of about 150 μM to about 300 μM.

21. The improved culture process of any one of claims 1 to 20, wherein the cell culture medium comprises thymidine at a concentration of about 16 μM to about 500 μM.

22. The improved culture process of claim 21, wherein the cell culture medium comprises thymidine at a concentration of about 16 μM to about 300 μM.

23. The improved culture process of claim 22, wherein the cell culture medium comprises thymidine at a concentration of about 25 μM to about 150 μM.

24. An improved culture process for reducing an unwanted culture by-product when a target protein is produced in a glutamine-free cell culture medium, the improved culture method comprising: culturing a mammalian cell in a glutamine-free cell culture medium under conditions allowing survival or proliferation of the cell, wherein the cell culture medium comprises hypoxanthine at a concentration of about 150 μM to about 300 μM and thymidine at a concentration of about 25 μM to about 150 μM, and wherein the cell maintained under the process conditions exhibit at least one of a decrease in an amount of ammonia as an unwanted culture by-product, an increase in glucose consumption of the cell, an increase in cell viability, and an increase in an expression level of a polypeptide of the cell, as compared to those in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

25. The improved culture process of claim 24, wherein the cell exhibits an about 10% or more decrease in the amount of ammonia, an about 10% or more increase in glucose consumption of the cell, an about 3% or more increase in cell viability, an about 10% or more increase in an expression level of a polypeptide of the cell, or a combination thereof.

26. The improved culture process of any one of claims 1 to 25, further comprising supplementing the cell culture medium with uridine.

27. The improved culture process of claim 26, wherein an amount of uridine in the cell culture medium is in a range of about 0.01 mM to about 100 mM.

28. The improved culture process of claim 27, wherein the amount of uridine in the cell culture medium is in a range of about 1 mM to about 50 mM.

29. The improved culture process of claim 28, wherein the amount of uridine in the cell culture medium is in a range of about 1 mM to about 25 mM.

30. The improved culture process of any one of claims 1 to 25, further comprising supplementing the cell culture medium with magnesium.

31. The improved culture process of claim 30, wherein the magnesium is magnesium ions or a magnesium salt.

32. The improved culture process of claim 30 or 31, wherein an amount of magnesium in the cell culture medium is in a range of about 0.01 mM to about 100 mM.

33. The improved culture process of claim 32, wherein the amount of magnesium in the cell culture medium is in a range of about 0.01 mM to about 50 mM.

34. The improved culture process of claim 33, wherein the amount of magnesium in the cell culture medium is in a range of about 0.5 mM to about 25 mM.

35. The improved culture process of any one of claims 14 to 34, wherein the polypeptide is a target protein.

36. The improved culture process of any one of claims 1 to 35, wherein the target protein is selected from the group consisting of an antibody or an antigen-binding fragment thereof, immunoadhesin, transforming growth factor (TGF)-β superfamily signaling molecule, blood coagulation factor, anti-tumor necrosis factor receptor (TNFR) antibody, anti-human epidermal growth factor receptor 2 (HER2) antibody, and TNFR:Fc.

37. The improved culture process of any one of claims 1 to 36, wherein the target protein is selected from the group consisting of abagovomab, abciximab, adalimumab, adecatumumab, alemtuzumab, altumomab, altumomab pentetate, anatumomab, anatumomab mafenatox, arcitumomab, atlizumab, basiliximab, bectumomab, ectumomab, belimumab, benralizumab, bevacizumab, brentuximab, canakinumab, capromab, capromab pendetide, catumaxomab, certolizumab, clivatuzumab tetraxetan, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, etaracizumab, ertumaxomab, fanolesomab, fontolizumab, gemtuzumab, girentuximab, golimumab, ibritumomab, igovomab, infliximab, ipilimumab, labetuzumab, mepolizumab, muromonab, muromonab-CD3, natalizumab, necitumumab, nimotuzumab, ofatumumab, omalizumab, oregovomab, palivizumab, panitumumab, ranibizumab, rituximab, satumomab, sulesomab, ibritumomab, ibritumomab tiuxetan, tocilizumab, tositumomab, trastuzumab, ustekinumab, visilizumab, votumumab, zalutumumab, brodalumab, anrukinzumab, bapineuzumab, dalotuzumab, demcizumab, ganitumab, inotuzumab, mavrilimumab, moxetumomab pasudotox, rilotumumab, sifalimumab, tanezumab, tralokinumab, tremelimumab, urelumab, adornase alfa, Rebif, becaplermin, alteplase, laronidase, alefacept, aflibercept, Raxibacumab, darbepoetin alfa, becaplermin concentrate, interferon beta)-1b, Botulinum toxin type A, rasburicase, asparaginase, epoetin alfa, etanercept, agalsidase beta, interferon alfacon-1, interferon alfa-2a, anakinra, Botulinum toxin type B, pegfilgrastim, oprelvekin, filgrastim, denileukin diftitox, peginterferon alfa-2a, aldesleukin, dornase alfa, interferon beta-1a, becaplermin, reteplase, interferon alfa-2, tenecteplase, drotrecogin alfa, rilonacept, romiplostim, methoxypolyethylene glycol-epoetin beta, a C1 esterase inhibitor, idursulfase, alglucosidase alfa, abatacept, galsulfase, palifermin, and interferon gamma-1b.

38. An improved culture process for producing a target protein in a glutamine-free cell culture medium, the improved culture process comprising: culturing a mammalian cell in a glutamine-free cell culture medium under conditions allowing survival or proliferation of the cell, wherein the cell culture medium comprises uridine at a concentration of about 0.01 mM to about 100 mM.

39. The improved culture process of claim 38, wherein the cell culture medium comprises uridine at a concentration of about 1 mM to about 50 mM.

40. The improved culture process of claim 39, wherein the cell culture medium comprises uridine at a concentration of about 1 mM to about 25 mM.

41. The improved culture process of any one of claims 38 to 40, wherein the cell maintained in the cell culture medium exhibits increased cell viability, as compared to that in a glutamine-free culture medium that does not comprise uridine.

42. The improved cultured process of claim 41, wherein the cell viability is increased by about 3% or more, as compared to cell viability in a glutamine-free culture medium that does not comprise uridine.

43. The improved cultured process of claim 42, wherein the cell viability is increased by about 5% or more, as compared to cell viability in a glutamine-free culture medium that does not comprise uridine.

44. The improved cultured process of claim 43, wherein the cell viability is increased by about 5% to about 10%, as compared to cell viability in a glutamine-free culture medium that does not comprise uridine.

45. The improved cultured process of any one of claims 38 to 44, wherein the cell maintained in the cell culture medium exhibit an increased viable cell density, as compared to that in a glutamine-free culture medium that does not comprise uridine.

46. The improved cultured process of claim 45, wherein the viable cell density is increased by about 5% or more, as compared to a viable cell density in a glutamine-free culture medium that dost not comprise uridine.

47. The improved cultured process of claim 46, wherein the viable cell density is increased by about 10% or more, as compared to a viable cell density in a glutamine-free culture medium that dost not comprise uridine.

48. The improved cultured process of claim 47, wherein the viable cell density is increased by about 10% to about 30%, as compared to a viable cell density in a glutamine-free culture medium that dost not comprise uridine.

49. The improved cultured process of any one of claims 38 to 48, wherein the cell culture medium further comprises hypoxanthine at a concentration of about 20 μM to about 1,000 μM and thymidine at a concentration of about 2 μM to about 1,000 μM, and an amount of ammonia as an unwanted culture by-product of the cell culture medium is reduced, as compared to that in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

50. The improved culture process of claim 49, wherein the amount of ammonia is reduced by about 10% or more, as compared to that in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

51. The improved culture process of claim 50, wherein the amount of ammonia is reduced by about 20% or more, as compared to that in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

52. The improved culture process of claim 51, wherein the amount of ammonia is reduced by about 30% to about 40%, as compared to that in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

53. An improved culture process for producing a target protein in a glutamine-free cell culture medium, the improved culture process comprising: culturing a mammalian cell in a glutamine-free cell culture medium under conditions allowing survival or proliferation of the cell, wherein the cell culture medium comprises magnesium at a concentration of about 0.01 mM to about 100 mM.

54. The improved culture process of claim 53, wherein the cell culture medium comprises magnesium at a concentration of about 0.01 mM to about 50 mM.

55. The improved culture process of claim 54, wherein the cell culture medium comprises magnesium at a concentration of about 0.5 mM to about 25 mM.

56. The improved culture process of any one of claims 53 to 55, wherein the cell maintained in the cell culture medium exhibits increased cell viability, as compared to that in a glutamine-free culture medium that does not comprise magnesium.

57. The improved culture process of claim 56, wherein the cell viability is increased by about 1% or more, as compared to cell viability in a glutamine-free culture medium that does not comprise magnesium.

58. The improved culture process of claim 57, wherein the cell viability is increased by about 1.5% or more, as compared to cell viability in a glutamine-free culture medium that does not comprise magnesium.

59. The improved culture process of claim 57, wherein the cell viability is increased by about 1% to about 10%, as compared to cell viability in a glutamine-free culture medium that does not comprise magnesium.

60. The improved culture process of any one of claims 53 to 59, wherein the cell maintained in the cell culture medium exhibits an increased viable cell density, as compared to that in a glutamine-free culture medium that does not comprise magnesium.

61. The improved culture process of claim 60, wherein the viable cell density is increased by about 5% or more, as compared to a viable cell density in a glutamine-free culture medium that does not comprise magnesium.

62. The improved culture process of claim 61, wherein the viable cell density is increased by about 10% or more, as compared to a viable cell density in a glutamine-free culture medium that does not comprise magnesium.

63. The improved culture process of claim 62, wherein the viable cell density is increased by about 10% to about 30%, as compared to a viable cell density in a glutamine-free culture medium that does not comprise magnesium.

64. The improved culture process of any one of claims 53 to 63, wherein the cell culture medium further comprises hypoxanthine at a concentration of about 20 μM to about 1,000 μM and thymidine at a concentration of about 2 μM to about 1,000 μM, and an amount of ammonia as an unwanted culture by-product of the cell culture medium is reduced, as compared to that in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

65. The improved culture process of claim 64, wherein the amount of ammonia is reduced by about 10% or more, as compared to that in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

66. The improved culture process of claim 65, wherein the amount of ammonia is reduced by about 20% or more, as compared to that in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

67. The improved culture process of claim 66, wherein the amount of ammonia is reduced by about 30% to about 40%, as compared to that in a glutamine-free culture medium that does not substantially comprise at least one of hypoxanthine and thymidine.

68. The improved culture process of any one of claims 1 to 67, wherein the cell is selected from the group consisting of CHO cell, DG44 cell, HEK cell, NS0 cell, PER.C6 cell, HeLa cell, and MDCK cell.

69. The improved culture process of claim 68, wherein the CHO cell is CHO K1 or CHO DUKK.

70. The improved culture process of any one of claims 38 to 69, wherein the target protein is selected from the group consisting of an antibody or an antigen-binding fragment thereof, immunoadhesin, transforming growth factor (TGF)-β superfamily signaling molecule, blood coagulation factor, anti-tumor necrosis factor receptor (TNFR) antibody, anti-human epidermal growth factor receptor 2 (HER2) antibody, and TNFR:Fc.

71. The improved culture process of any one of claims 38 to 69, wherein the target protein is selected from the group consisting of abagovomab, abciximab, adalimumab, adecatumumab, alemtuzumab, altumomab, altumomab pentetate, anatumomab, anatumomab mafenatox, arcitumomab, atlizumab, basiliximab, bectumomab, ectumomab, belimumab, benralizumab, bevacizumab, brentuximab, canakinumab, capromab, capromab pendetide, catumaxomab, certolizumab, clivatuzumab tetraxetan, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, etaracizumab, ertumaxomab, fanolesomab, fontolizumab, gemtuzumab, girentuximab, golimumab, ibritumomab, igovomab, infliximab, ipilimumab, labetuzumab, mepolizumab, muromonab, muromonab-CD3, natalizumab, necitumumab, nimotuzumab, ofatumumab, omalizumab, oregovomab, palivizumab, panitumumab, ranibizumab, rituximab, satumomab, sulesomab, ibritumomab, ibritumomab tiuxetan, tocilizumab, tositumomab, trastuzumab, ustekinumab, visilizumab, votumumab, zalutumumab, brodalumab, anrukinzumab, bapineuzumab, dalotuzumab, demcizumab, ganitumab, inotuzumab, mavrilimumab, moxetumomab pasudotox, rilotumumab, sifalimumab, tanezumab, tralokinumab, tremelimumab, urelumab, adornase alfa, Rebif, becaplermin, alteplase, laronidase, alefacept, aflibercept, Raxibacumab, darbepoetin alfa, becaplermin concentrate, interferon beta)-1b, Botulinum toxin type A, rasburicase, asparaginase, epoetin alfa, etanercept, agalsidase beta, interferon alfacon-1, interferon alfa-2a, anakinra, Botulinum toxin type B, pegfilgrastim, oprelvekin, filgrastim, denileukin diftitox, peginterferon alfa-2a, aldesleukin, dornase alfa, interferon beta-1a, becaplermin, reteplase, interferon alfa-2, tenecteplase, drotrecogin alfa, rilonacept, romiplostim, methoxypolyethylene glycol-epoetin beta, a C1 esterase inhibitor, idursulfase, alglucosidase alfa, abatacept, galsulfase, palifermin, and interferon gamma-1b.

72. The improved culture process of any one of claims 1 to 71, wherein the culturing is performed at a temperature of about 25□ to about 42° C.

73. The improved culture process of any one of claims 1 to 71, wherein the culturing comprises conditions of at least one temperature change, conditions of at least one pH change, or a combination thereof, wherein the conditions of at least one temperature change comprise the following processes: a cell is grown in a medium at a first temperature for 3 days or more, the temperature is changed to a second temperature, the second temperature being about 1 ° C. to about 8° C. lower than the first temperature, and the cell is maintained at the second temperature for about 2 days or more, and wherein the conditions of at least one pH change comprise the following processes: a cell is grown in a medium at a first pH value for about 2 days or more, the pH is changed to a second pH value, the second pH value being about 0.05 to about 1 lower than the first pH value, and the cell is grown at the second pH value for about 1 day or more.

74. The improved culture process of claim 73, wherein the first temperature is in a range of about 30□ to about 42° C.

75. The improved culture process of claim 73, wherein the second temperature is in a range of about 25° C. to about 41° C.

76. The improved culture process of claim 73, wherein the first pH value is in a range of about pH 6.8 to about pH 7.5.

77. The improved culture process of claim 73, wherein the second pH value is in a range of about pH 6.0 to about pH 7.1.

78. The improved culture process of any one of claims 1 to 77, further comprising adding glucose to the glutamine-free cell culture medium such that a concentration of the glucose is maintained at about 2 g/L or more.

79. The improved culture process of claim 78, wherein the glucose is added to be maintained at a concentration of about 2 g/L to about 7 g/L in the glutamine-free cell culture medium.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0049] FIG. 1 is a graph showing the concentration (mM) of ammonia in a cell culture medium (glutamine-free) with respect to culture time (days) of CHO cells in the presence of hypoxanthine and thymidine (.square-solid.: 0 mM glutamine+2×HT, .circle-solid.: 0 mM glutamine+1×HT).

[0050] FIG. 2 is a graph showing the concentration (g/L) of glucose in a cell culture medium (glutamine-free) with respect to culture time (days) of CHO cells in the presence of hypoxanthine and thymidine (.square-solid.: 0 mM glutamine+2×HT, .circle-solid.: 0 mM glutamine+1×HT).

[0051] FIG. 3 is a graph showing cell viability (%) with respect to culture time (days) of CHO cells in the presence of hypoxanthine and thymidine (.square-solid.: 0 mM glutamine+2×HT, .circle-solid.: 0 mM glutamine+1×HT).

[0052] FIG. 4 is a graph showing a polypeptide expression level (g/L) with respect to culture time (days) of CHO cells in the presence of hypoxanthine and thymidine ( 0 mM glutamine+2×HT, : 0 mM glutamine+1×HT).

[0053] FIG. 5A is a graph showing viable cell density (CHO cells/ml) with respect to culture time (days) when 4 mM uridine was added to a 2×HT medium (.square-solid.: 0 mM glutamine+2×HT+4 mM uridine, .circle-solid.: 0 mM glutamine+2×HT).

[0054] FIG. 5B is a graph showing viable cell density (CHO cells/ml) with respect to culture time (days) when 8 mM galactose was added to a 2×HT medium (.square-solid.: 0 mM glutamine+2×HT+8 mM galactose, .circle-solid.: 0 mM glutamine+2×HT).

[0055] FIG. 6A is a graph showing cell viability (%) with respect to culture time (days) when 4 mM uridine was added to a 2×HT medium (.square-solid.: 0 mM glutamine+2×HT+4 mM uridine, .circle-solid.: 0 mM glutamine+2×HT).

[0056] FIG. 6B is a graph showing cell viability (%) with respect to culture time (day) when 8 mM galactose was added to a 2×HT medium (.square-solid.: 0 mM glutamine+2×HT+8 mM galactose, .circle-solid.: 0 mM glutamine+2×HT).

[0057] FIGS. 7 and 8 are graphs respectively showing viable cell density (CHO cells/ml) and cell viability (%) when 1 mM magnesium was added to a 2×HT medium (.square-solid.: 0 mM glutamine+2×HT+1 mM magnesium, .circle-solid.: 0 mM glutamine+2×HT).

MODE OF DISCLOSURE

[0058] Hereinafter, the present disclosure will be described in further detail with reference to the following examples. However, these examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure.

EXAMPLE 1

Changes in Concentrations of Ammonia and Glucose in Cell Culture Medium according to the Presence of 2×HT

[0059] When cells are cultured, the concentration of ammonia in a cell culture medium is increased, and ammonia has cytotoxicity. In addition, during cell culture, as the number of cells increases, the amount of glucose consumed by the cells increases, and the content of glucose in the cell culture medium decreases. It was examined whether the amount of ammonia produced by cells during cell culture was decreased and the consumption of glucose of the cells was increased, in a case in which 2×HT was added to a cell culture medium.

[0060] First, a cell line expressing a fusion protein (CHO cell line) was prepared (the cell line was prepared by transfecting a CHO cell line with a vector expressing the fusion protein), and the cells were cultured in a glutamine-free medium. Cells at a density of 9×10.sup.5 cells/ml were inoculated in a cell culture dish, and a glutamine-free medium was added thereto. The medium was supplemented with a 100× stock (catalog number: 11067030, Gibco) of 10 mM sodium hypoxanthine and 1.6 mM thymidine so that the final concentrations of hypoxanthine and thymidine in the medium reached 100 μM and 16 μM, respectively (hereinafter, referred to as “1×HT”), or 200 μM and 32 μM, respectively (hereinafter, referred to as “2×HT”). The cells were cultured at 37° C. and 5% CO.sub.2, and the medium was replaced with a fresh medium every two days.

[0061] While the cells were cultured for about 15 days, the concentrations of ammonia (mM) and glucose (g/L) in the medium according to culture time (day) were measured using an ammonium ion-selective electrode and a glucose membrane kit (catalog No.: 24458, Nova Biomedical) of a Bioprofile analyzer (Model No.: BioProfile 400, Nova Biomedical). The results thereof are illustrated in FIGS. 1 and 2, respectively.

[0062] As illustrated in FIG. 1, when 2×HT was added, the concentration of ammonia in the cell culture medium was significantly decreased over cell culture time, as compared to when 1×HT was added. For example, 14 days after the culture, the concentration of ammonia in the medium was 3.31 mM when 1×HT was added, whereas the concentration of ammonia in the medium was decreased to 2.21 mM when 2×HT was added.

[0063] In addition, as illustrated in FIG. 2, when 2×HT was added to the glutamine-free medium, the concentration of glucose in the cell culture medium was significantly decreased over cell culture time, as compared to when 1×HT was added to the glutamine-free medium. For example, 14 days after the culture, the concentration of glucose in the medium was 14.00 g/L when 1×HT was added, whereas the concentration of glucose in the medium was decreased to 11.12 g/L when 2×HT was added.

[0064] Thus, it was confirmed that, when cells were cultured in a glutamine-free and 2×HT-containing medium, the production of ammonia by the cells was reduced, and cytotoxicity due to the produced ammonia was reduced, and accordingly, glucose consumption of the cells was increased, as compared to a case in which cells were cultured in a glutamine-free and 1×HT-containing medium.

EXAMPLE 2

Changes in Cell Viability and Polypeptide Expression Level of Cells according to Presence of 2×HT

[0065] It was examined whether there were differences in cell viability and polypeptide expression level (titer) of the cultured cells when the cells were cultured in a glutamine-free and 2×HT-containing medium, as compared to when the cells were cultured in a glutamine-free and 1×HT-containing medium.

[0066] The cells were cultured in a glutamine-free and 2×HT-containing medium or a glutamine-free and 1×HT-containing medium using the same method as that used in Example 1. Cells cultured in a glutamine-free medium were used as a negative control.

[0067] The relative cell viability (%) and polypeptide expression level (g/L) of the cells with respect to the negative control were measured using a Cedex HiRes analyzer (Model No.: 05650216001, Roche) and HPLC (Model No.: 720000370EN, Waters), respectively through a Trypan blue exclusion method and a UV280 measurement method. The results thereof are illustrated in FIGS. 3 and 4, respectively.

[0068] As illustrated in FIGS. 3 and 4, when cells were cultured in a glutamine-free and 2×HT-containing medium, the cell viability and polypeptide expression level of the cells were significantly increased, as compared to when cells were cultured in a glutamine-free and 1×HT-containing medium. For example, 14 days after the culture, cell viability was 81.4% when 1×HT was added, whereas the concentration of glucose in the medium was increased to 88.1% when 2×HT was added. In addition, 14 days after the culture, the polypeptide expression level of the cells was 1.29 g/L when 1×HT was added, whereas the concentration of glucose in the medium was increased to 1.76 g/L when 2×HT was added.

EXAMPLE 3

Effect of Uridine Addition on Viable Cell Density and Cell Viability

[0069] It was examined whether the addition of uridine affected viable cell density (VCD) and cell viability, when cells were cultured in a medium prepared by adding uridine to 2×HT.

[0070] Cells were prepared using the same method as that used in Example 1, and a glutamine-free and 2×HT-containing medium was added to the prepared cells. 4 mM of uridine (catalog No.: U3003, Sigma) or 8 mM of galactose (Catalog No.: G0750, Sigma) was added to the medium. The cells were cultured at 37° C. and 5% CO.sub.2, and the medium was replaced with a fresh medium every two days. Cells cultured in a glutamine-free medium were used as a negative control.

[0071] After about 14 days, viable cell density and cell viability were measured using a Cedex HiRes analyzer (Model No.: 05650216001, Roche) through a scanner-based imaging method and a Trypan blue exclusion method. The viable cell density (CHO cells/ml) of cells cultured in the presence of uridine and in the presence of galactose is illustrated in FIGS. 5A and 5B, respectively. In addition, the cell viability (%) of cells cultured in the presence of uridine and in the presence of galactose is illustrated in FIGS. 6A and 6B, respectively.

[0072] As illustrated in FIG. 5A, when uridine was added to a glutamine-free and 2×HT-containing medium, the viable cell density of the cultured cells was significantly increased, as compared to a case in which uridine was not added. For example, 14 days after the culture, the viable cell density was 67.22 cells/mL in the case in which the medium did not contain uridine, whereas the viable cell density was increased to 79.37 cells/mL in the case in which the medium contained uridine.

[0073] In contrast, as illustrated in FIG. 5B, there was no significant difference in viable cell density between a case in which galactose was added to a glutamine-free and 2×HT-containing medium and a case in which galactose was not added to the medium.

[0074] As illustrated in FIG. 6A, the viability of the cultured cells was significantly increased in a case in which uridine was added to a glutamine-free and 2×HT-containing medium, as compared to a case in which uridine was not added to the medium. For example, 14 days after the culture, the cell viability was 89.6% in the case in which the medium did not contain uridine, whereas the cell viability was increased to 94.6% in the case in which the medium contained uridine.

[0075] In contrast, as illustrated in FIG. 6B, there was no significant difference in viable cell density of the cultured cells between a case in which galactose was added to a glutamine-free and 2×HT-containing medium and a case in which galactose was not added to the medium.

[0076] Thus, it was confirmed that, when cells were cultured in a 2×HT- and uridine-containing medium, viable cell density and cell viability were significantly increased.

EXAMPLE 4

Effect of Magnesium Addition on Viable Cell Density and Cell Viability

[0077] Similar to Example 3, it was examined whether the addition of magnesium affected viable cell density and cell viability, when cells were cultured in a medium prepared by adding magnesium to 2×HT.

[0078] Cells were prepared using the same method as that used in Example 1, and a glutamine-free and 2×HT-containing medium was added to the prepared cells. 1 mM of magnesium (Catalog No.: M4880, Sigma) was added to the medium. The cells were cultured at 37° C. and 5% CO.sub.2, and the medium was replaced with a fresh medium every two days. Cells cultured in a glutamine-free medium were used as a negative control.

[0079] After about 14 days, viable cell density and cell viability were measured using a Cedex HiRes analyzer (Model No.: 05650216001, Roche) through a scanner-based imaging method and a Trypan blue exclusion method, respectively. The viable cell density (CHO cells/ml) and cell viability of the cells cultured in the presence of magnesium are illustrated in FIGS. 7 and 8, respectively.

[0080] As illustrated in FIGS. 7 and 8, the viable cell density of the cultured cells was significantly increased and the cell viability was significantly increased, in the case in which magnesium was added to the glutamine-free and 2×HT-containing medium, as compared to the case in which magnesium was not added to the medium. For example, 14 days after the culture, the viable cell density was 67.22 cells/mL in the case in which the medium did not contain magnesium, whereas the viable cell density was increased to 76.35 cells/mL in the case in which the medium contained magnesium. In addition, 14 days after the culture, the cell viability was 89.6% in the case in which the medium did not contain magnesium, whereas the cell viability was increased to 91.5% in the case in which the medium contained magnesium.

[0081] Thus, it was confirmed that the viable cell density and the cell viability were significantly increased in a case in which cells were cultured in a 2×HT- and magnesium-containing medium.