Cell culture medium comprising transition metals or trace elements

1 . A composition for use in growing cells, said composition comprising trace compounds wherein a sum of concentrations of said trace compounds not including Zn is greater than or greater than: about 4×10 −7 M or not including copper about 4×10 −6 M.

2 . The composition according to claim 1 wherein the trace compounds are selected from the group consisting of Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, As, Se, Br, Al, Si, P, Y, Zr, Nb, Mo, Tc, Ru, Rh, Rb, Ce, Ag, Pd, Ag, Cd, In, Sn, Sb, F, Te, Au, Pt, Bi, Ir, Os, Re, W, Ta and Hf.

3 . The composition according to claim 1 wherein the trace compounds are selected from the group consisting of Al, Cd, Rb, Zr, Co, Sn, Cr, Ni, F, Cu, Mn, Mo, Ge, V, Br, I, Ba, Ag, Ti, Se, Cu and Zn.

4 . The composition according to claim 2 comprising at least three of said trace compounds.

5 . The composition according to claim 2 comprising at least five of said trace compounds.

6 . The composition according to claim 2 comprising at least seven of said trace compounds.

7 . The composition according to claim 3 comprising each of said trace compounds.

8 . The composition according to claim 1 comprising an excess of zinc or copper, said excess of zinc or copper being at least or at least about 0.2, 0.5, 1.0. 1.5 or 2.0 times the sum of the concentrations of the other trace compounds.

9 . The composition according to claim 1 made by adding trace compounds other than one of Ag or Ni in at least a five fold excess to the Ag or the Ni.

10 . A composition comprising a cell selected from the group consisting of CD34 + hematopoietic cells and cells of myeloid lineage, 293 embryonic kidney cells, A-549, Jurkat, Namalwa, Hela, 293BHK cells, HeLa cervical epithelial cells and PER-C6 retinal cells, aka PER.C6, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS 180 cells, LS 174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I cells, LLC-MK 2 cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK 1 cells, PK(15) cells, GH 1 cells, GH 3 cells, L2 cells, LLC-RC 256 cells, MH 1 C 1 cells, XC cells, MDOK cells, VSW cells, TH-I, B1 cells, or derivatives thereof, fibroblast cells from any tissue or organ (including but not limited to heart, liver, kidney, colon, intestines, esophagus, stomach, neural tissue (brain, spinal cord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow, and blood), spleen, fibroblast and fibroblast-like cell lines), TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl 1 cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C 3 H/IOTI/2 cells, HSDM 1 C 3 cells, KLN205 cells, McCoy cells, Mouse L cells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L) cells, L-MTK (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, NS0, NS1, Swiss/3T3 cells, Indian muntjac cells, SIRC cells, C II cells, Jensen cells, COS cells and Sp2/0 cells, Mimic cells and/or derivatives thereof in serum-free culture;

zinc at a concentration is greater than or greater than about 0.0005, 0.001, 0.002, 0.004, 0.005, 0.007, 0.008, 0.01, 0.012, 0.015 or 0.02 mM; and

a cell culture medium that otherwise provides for the physical and chemical needs of the cell.

11 . The composition according to claim 1 wherein the zinc concentration is about or is greater than about 0.5, 1, 2, 3, 4, 5, 7, 8, 10, 12, 15, 16, 18, 20, 22, 25 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100×10 −7 M.

12 . The composition according to claim 10 wherein the zinc concentration is about or is greater than about 0.5, 1, 2, 3, 4, 5, 7, 8, 10, 12, 15, 16, 18, 20, 22, 25 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100×10 −7 M.

13 . The composition according to claim 8 , wherein the excess is not greater than or not greater than about 2, 5 or 10× the sum of the concentrations of the other trace compounds.

14 . The composition according to claim 10 wherein the composition is insulin free.

15 . The composition according to claim 2 comprising at least ten of said trace compounds.

16 . The composition according to claim 2 comprising at least twelve of said trace compounds.

17 . The composition according to claim 2 comprising at least sixteen of said trace compounds.

18 . The composition according to claim 2 comprising at least twenty of said trace compounds.

19 . The composition according to claim 1 made by adding trace compounds excluding Zn and Ni in at least a 5, 10, 20, 35, 30, 40, 50, 75, 80, 100, 150, 200, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 1200, 1250, 1500, 1800, or 2000 fold excess to added Ni.

20 . The composition according to claim 1 made by adding trace compounds excluding Zn and Cu in at least or at least about a 1.2, 1.5, 1.8, 2, 4, 5, or 10 fold excess to added Cu.

21 . A composition for use in growing cells, said composition comprising trace compounds wherein a sum of concentrations of said trace compounds not including Zn is greater than about 10 −7 , 2×10 −7 , 3×10 −7 , 5×10 −7 , 7×10 −7 , 8×10 −7 , 9×10 −7 , 10×10 −7 or 12×10 −7 M and Ni is not an added trace compound.

22 . A method of improving cell culture medium comprising:

adding trace compounds to said medium to produce a decreased ratio of a transition metal selected from the group consisting of Ni, Cd, Hg and Pb to a total concentration of trace compounds.

23 . The method according to claim 22 wherein the ratio is decreased by at least or at least about 2, 5, 10, 12, 15, 18, 20, 25, 40, 50, 60, 75, 80, 100, 150, 200, 500, or 1000.

24 . The composition according to claim 1 , wherein said sum does not exceed 10 −6 , 2×10 −6 , 3×10 −6 , 4×10 −6 , 5×10 −6 , 6×10 −6 , 7×10 −6 , 8×10 −6 , 9×10 −6 or 10×10 −6 M.

25 . A composition for use in growing cells, said composition comprising trace compounds wherein a sum of concentrations of said trace compounds not including Zn is less than or less than about 3×10 −7 M or less than or less than about 6×10 −7 M including Zn.

26 . A method of growing cells in culture comprising providing a trace compound according to the present invention to a cell in a culture container and culturing said cell.

27 . A method for inhibiting apoptosis of cells grown in culture comprising culturing said cells in a composition or in a presence of a medium according to the present invention.

28 . A method of preventing or reducing lag in cells in culture comprising culturing cells in a composition or in the presence of a medium according to the present invention.

29 . The method according to claim 28 wherein the cells are 293 cells or derivatives thereof.

30 . A method of culturing cells to a density of at least or at least about 4×10 6 comprising culturing said cells in a composition or in a medium according to the present invention.

31 . The method according to claim 30 wherein said cells are cultured to at least or at least about 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , or 10 7 cells/ml.

32 . The method according to claim 31 wherein said cells are selected from the group consisting of Per.C6, 293, hybridoma and Jurkat.

33 . Method of producing protein in cells at a concentration about or at least about 1, 2, 4, 8, 10 or 12 mg/ml comprising culturing said cells according to a method of any one of claims 26 - 30 .

34 . A supplement that when added to a basal medium produces a medium according to the present invention.

35 . A method of culturing cells in a base cell culture medium, the improvement being adding trace compounds to said medium to reduce a ratio of a metal selected from the group consisting of Ni, Cd and Pb to total trace compounds by about or about at least 200, 500, 800, 100, 1200, 1500, 2000, or 2500 times.

36 . The composition according to claim 1 further comprising cells selected from the group consisting of PER.C6 cells, 293 cells and HeLa cells.

37 . The composition according to claim 36 comprising 293 cells.

38 . The composition according to claim 36 comprising PER.C6 cells.

39 . The composition according to claim 10 , wherein said cells are cultured to a concentration selected from the group consisting of at least or at least about 1.8×10 6 , 2.0×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 and 9×10 6 cells per ml.

40 . A composition of cells cultured to a concentration of at least 10 7 said cells selected from the group consisting of CD34 + hematopoietic cells and cells of myeloid lineage, 293 embryonic kidney cells, A-549, Jurkat, Namalwa, Hela, 293, BHK cells, HeLa cervical epithelial cells and PER-C6 retinal cells, aka PER.C6, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS 180 cells, LS 174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I cells, LLC-MK 2 cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK 1 cells, PK(15) cells, GH 1 cells, GH 3 cells, L2 cells, LLC-RC 256 cells, MH 1 C 1 cells, XC cells, MDOK cells, VSW cells, TH-I, B1 cells, or derivatives thereof, fibroblast cells from any tissue or organ (including but not limited to heart, liver, kidney, colon, intestines, esophagus, stomach, neural tissue (brain, spinal cord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow, and blood), spleen, fibroblast and fibroblast-like cell lines), TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl 1 cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells, NS0, NS1, NOR-10 cells, C 3 H/IOTI/2 cells, HSDM 1 C 3 cells, KLN205 cells, McCoy cells, Mouse L cells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L) cells, L-MTK (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indian muntjac cells, SIRC cells, C II cells, Jensen cells, COS cells and Sp2/0 cells, Mimic cells, and/or derivatives thereof in serum-free culture.

41 . A Method of producing a biomolecule, virus particle or virus of interest comprising culturing a cell engineered to produce said biomolecule of interest in a composition according to the present invention, said molecule produced in said culture at a concentration exceeding 4, 5, 6, 7, 8, 9, 10 11, 12 14, 15, 18, 20 or 25×10 −4 g/ml or about 3, 4 or 5 mg/ml.

42 . A method of reducing variability of cell growth and/or bioproduction in cell culture comprising providing a culture medium with a supplement according to claim 34 and culturing cells in same.

43 . The method according to claim 42 comprising adding said supplement to said culture medium before said culture medium is contacted with said cells.

44 . The method according to claim 42 comprising adding said supplement to said culture medium while said culture medium is in contact with said cells.

45 . The composition according to claim 1 , wherein a concentration of each trace compound present in said composition is selected from the values or ranges from table 1.

46 . The composition according to claim 45 wherein the composition comprises Zn.

47 . The composition according to claim 45 wherein the composition comprises Cu.

48 . The composition according to claim 45 wherein the composition comprises Zn and Cu.

46 . The composition according to claim 45 wherein the composition comprises all compounds of the table except at least one compound selected from the group consisting of Zn and Cu.

47 . The composition According to claim 1 further comprising CHO cells or myeloma cells in culture and Zn and/or copper at a concentration of at maximum or at maximum about 1.84×10 −7 , more preferably, 4.6×10 −7 , and still more preferably 9.2×10 −7 , for zinc and 2.05×10 −8 , more preferably 5.15×10 −8 , and still more preferably 1.03×10 −7 , for copper.

48 . The composition according to claim 1 further comprising CHO cells or myeloma cells in culture and Zn and/or copper at a concentration of at least or at least about 1.84×10 −5 , more preferably, 7.4×10 −6 , and still more preferably 3.7×10 −6 , for zinc and 2.05×10 −6 , more preferably 8.2×10 −7 , and still more preferably 4.1×10 −7 , for copper.

49 . A method for decreasing detrimental effect of trace components comprising adding additional trace components.

50 . The method according to claim 49 wherein the additional trace components added include at least one trace component already present in a cell culture medium.

51 . The method according to claim 49 wherein the additional trace components added include at least one trace component not present in a cell culture medium.

52 . The method according to one of claims 50 or 51 wherein the at least one trace component includes at least one selected from the group consisting of Al, Cd, Rb, Zr, Co, Sn, Cr, Ni, F, Cu, Mn, Mo, Ge, V, Br, I, Ba, Ag, Ti, Se, Cu and Zn.

53 . The method according to claim 52 wherein the at least one trace component includes at least one selected from the group consisting of Cu and Zn.

54 . The method according to claim 49 , wherein the trace components whose detrimental effect is to be decreased were not intentionally present.

55 . The method according to claim 49 , wherein the trace components whose detrimental effect is to be decreased were present as a byproduct of at least one ingredient intentionally added.

56 . The method according to claim 49 , wherein the trace components whose detrimental effect is to be decreased were present as a result of manufacturing of handling a cell cure medium.

57 . A method for reducing batch-to-batch variability in cell culture comprising adding additional trace components.

58 . The method according to claim 57 wherein the additional trace components added include at least one trace component already present in a cell culture medium.

59 . The method according to claim 57 wherein the additional trace components added include at least one trace component not present in a cell culture medium.

60 . The method according to one of claims 58 or 59 wherein the at least one trace component includes at least one selected from the group consisting of Al, Cd, Rb, Zr, Co, Sn, Cr, Ni, F, Cu, Mn, Mo, Ge, V, Br, I, Ba, Ag, Ti, Se, Cu and Zn.

61 . The method according to claim 60 wherein the at least one trace component includes at least one selected from the group consisting of Cu and Zn.

62 . The method according to claim 57 , wherein the trace components whose detrimental effect is to be decreased were not intentionally present.

63 . The method according to claim 57 , wherein the trace components whose detrimental effect is to be decreased were present as a byproduct of at least one ingredient intentionally added.

64 . The method according to claim 57 , wherein the trace components whose detrimental effect is to be decreased were present as a result of manufacturing of handling a cell cure medium.

65 . The composition according to claim 1 , wherein Fe is not included in the sum of concentrations of trace elements.