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1. (US20170305999) CELL CULTURE MEDIUM
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Claims

1. A medium for culturing mammalian cells comprising:
(a) (i) a basal medium or (ii) a feed medium; and
(b) one or more sources of lithium ions.
2. The medium of claim 1, wherein the cells have been genetically engineered to produce one or more recombinant polypeptides.
3. The medium of claim 1, wherein the medium further comprises (c) ethanol; and/or (d) one or more fatty acids.
4. The medium of claim 3, wherein said one or more fatty acids is selected from the group consisting of oleic acid, linoleic acid, linolenic acid, myristic acid, palmitic acid stearic acid, thymol, cholesteryl acetate, methyl octanoate, 1-octanoyl-rac-glycerol, cholesterol, butyric (C4), valeric (C5), caproic (C6), enanthic (C7), caprylic (C8), pelargonic (C9), capric (C10), undecylic (C11), lauric (C12), tridecylic (C13), myristic (C14), pentadecanoic (C15), margaric (C17), nonadecylic (C19), arachidic (C20), heneicosylic (C21), behenic (C22), tricosylic (C23), lignoceric (C24), pentacosylic (C25), cerotic (C26), heptacosylic (C27), montanic (C28), nonacosylic (C29), melissic (C30), hentriacontylic (C31), lacceroic (C32), psyllic (C33), geddic (C34), ceroplastic (C35), hexatriacontylic (C36), heptatriacontanoic (C37), and octatriacontanoic (C38) acids.
5. The medium of claim 1, wherein said one or more sources of lithium ions is selected from the group of one or more of lithium acetate, lithium chloride, lithium carbonate, lithium oxybutyrate, lithium orotate, lithium bromide, lithium citrate, lithium fluoride, lithium iodide, lithium nitrate, and lithium sulfate.
6. The medium of claim 1, wherein said lithium ions are present in a concentration from about 0.1 μM to about 25 mM.
7. The medium of claim 2, wherein the titer of said one or more recombinant polypeptides is increased compared to the titer of recombinant polypeptides produced by mammalian cells that are not cultured in said medium.
8. The medium of claim 2, wherein the amount of high molecular weight species of said one or more recombinant polypeptides produced by said cells is modulated compared to recombinant polypeptides produced by mammalian cells that are not cultured in said medium.
9. The medium of claim 2, wherein the amount of low molecular weight species of said one or more recombinant polypeptides produced by said cells is modulated compared to recombinant polypeptides produced by mammalian cells that are not cultured in said medium.
10. The medium of claim 2, wherein the glycosylation profile of said one or more recombinant polypeptides produced by said cells is modulated compared to recombinant polypeptides produced by mammalian cells that are not cultured in said medium.
11. The medium of claim 10, wherein said modulated glycosylation comprises modulated terminal mannose glycan species.
12. The medium of claim 10, wherein said modulated glycosylation comprises modulation of one or more glycan species selected from mannose-5-N-acetylglycosamine-2 (Man5), mannose-6-N-acetylglycosamine-2 (Man6), mannose-3-N-acetylglucosamine-4 (G0), mannose-3-N-acetylglucosamine-4-fucose (G0F), mannose-3-N-acetylglucosamine-4-galactose-1-fucose (G1F), or mannose-3-N-acetylglucosamine-4-galactose-2-fucose (G2F).
13. The medium of claim 2, wherein the amount of acidic or basic charge variants of said one or more recombinant polypeptides produced by said cells is modulated compared to recombinant polypeptides produced by mammalian cells that are not cultured in said medium.
14. The medium of claim 3, wherein ethanol is present at a concentration from about 0.001% to about 4% (v/v).
15. The medium of claim 3, wherein the one or more fatty acids are present at a concentration of about 1 μM to about 4 mM.
16. A medium for culturing mammalian cells comprising:
(a) (i) a basal medium or (ii) a feed medium; and
(b) ethanol.
17- 28. (canceled)
29. A medium for culturing mammalian cells comprising:
(a) (i) a basal medium or (ii) a feed medium; and
(b) one or more fatty acids.
30- 39. (canceled)
40. A method for producing one or more recombinant polypeptides from an engineered mammalian cell, the method comprising:
(a) culturing said engineered mammalian cell in the medium of claim 1 under suitable conditions for the production of said one or more recombinant polypeptides; and
(b) producing said one or more recombinant polypeptides.
41. The method of claim 40, wherein the method further comprises (c) isolating said one or more recombinant polypeptides.
42. The method of claim 40, wherein the medium is (a) a basal medium; or (b) a feed medium.
43. (canceled)
44. The method of claim 40 wherein said one or more recombinant polypeptides is an antibody or fragment thereof.
45. The method of claim 44, wherein said antibody is a monoclonal antibody.
46. The method of claim 45, wherein the monoclonal antibody inhibits the growth of a proliferating cell.
47. The method of claim 44, wherein said antibody or fragment thereof binds to HER2, TNF-α, VEGF-A, α4-integrin, CD20, CD52, CD25, CD11a, EGFR, respiratory syncytial virus (RSV), glycoprotein IIb/IIIa, IgG1, IgE, complement component 5 (C5), B-cell activating factor (BAFF), CD19, CD30, interleukin-1 beta (IL1β), prostate specific membrane antigen (PSMA), CD38, RANKL, GD2, SLAMF7 (CD319), proprotein convertase subtilisin/kexin type 9 (PCSK9), dabigatran, cytotoxic T-lymphocyte-associated protein 4 (CTLA4), interleukin-5 (IL-5), programmed cell death protein (PD-1), VEGFR2 (KDR), protective antigen (PA) of B. anthracis, interleukin-17 (IL-17), interleukin-6 (IL-6), interleukin-6 receptor (IL6R), interleukin-12 (IL-12), interleukin 23 (IL-23), sclerostin (SOST), myostatin (GDF-8), activin receptor-like kinase 1, delta like ligand 4 (DLL4), angiopoietin 3, VEGFR1, selectin, oxidized low-density lipoprotein (oxLDL), platelet-derived growth factor receptor beta, neuropilin 1, Von Willebrand factor (vWF), integrin α vβ 3, neural apoptosis-regulated proteinase 1, integrin α IIbβ 3, beta-amyloid, reticulon 4 (RTN4)/Neurite Outgrowth Inhibitor (NOGO-A), nerve growth factor (NGF), LINGO-1, myelin-associated glycoprotein, or integrin α4β7.
48. The method of claim 45, wherein said monoclonal antibody is trastuzumab, pertuzumab, infliximab, adalimumab, bevacizumab, ranibizumab, natalizumab, rituximab, alemtuzumab, daclizumab, efalizumab, golimumab, certolizumab, cetuximab, panitumumab, palivizumab, abciximab, basiliximab, ibritumomab, omalizumab, eculizumab, abciximab, alirocumab, basiliximab, belimumab, blinatumomab, brentuximab, canakinumab, capromab, daratumumab, denosumab, dinutuximab, eculizumab, elotuzumab, evolocumab, idarucizumab, ipilimumab, mepolizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, palivizumab, pembrolizumab, ramucirumab, raxibacumab, ecukinumab, siltuximab, tocilizumab, ustekinumab, alacizumab, denosumab, blosozumab, romosozumab, stamulumab, alirocumab, ascrinvacumab, enoticumab, evinacumab, evolocumab, icrucumab, inclacumab, nesvacumab, orticumab, ramucirumab, rinucumab, vesencumab, bococizumab, caplacizumab, demcizumab, etaracizumab, idarucizumab, ralpancizumab, tadocizumab, aducanumab, atinumab, fasinumab, fulranumab, gantenerumab, opicinumab, bapineuzumab, crenezumab, ozanezumab, ponezumab, refanezumab, solanezumab, tanezumab, and vedolizumab.
49. A method for modulating the glycosylation profile of one or more recombinant polypeptides produced by a genetically engineered mammalian cell, the method comprising:
(a) culturing said mammalian cell in the medium of claim 1 under suitable conditions for the production of said one or more recombinant polypeptides; and
(b) producing said one or more recombinant polypeptides, wherein said one or more recombinant polypeptides has a modulated glycosylation profile compared to recombinant polypeptides produced by mammalian cells that are not cultured in the medium of claim 1.
50. The method of claim 49, wherein said modulated glycosylation profile comprises modulated terminal mannose glycan species.
51. The method of claim 49, wherein said modulated glycosylation comprises modulation of one or more glycan species selected from mannose-5-N-acetylglycosamine-2 (Man5), mannose-6-N-acetylglycosamine-2 (Man6), mannose-3-N-acetylglucosamine-4 (G0), mannose-3-N-acetylglucosamine-4-fucose (G0F), mannose-3-N-acetylglucosamine-4-galactose-1-fucose (G1F), and/or mannose-3-N-acetylglucosamine-4-galactose-2-fucose (G2F).
52. The method of claim 49, wherein the ratio of the terminal mannose glycan species to the total sum of glycan species is modulated by about 40% to about 50%.
53. The method of claim 49, wherein said one or more recombinant polypeptides is an antibody or fragment thereof.
54. The method of claim 53, wherein said antibody is a monoclonal antibody.
55. The method of claim 54, wherein the monoclonal antibody inhibits the growth of a proliferating cell.
56. The method of claim 53, wherein said antibody or fragment thereof binds to HER2, TNF-α, VEGF-A, α4-integrin, CD20, CD52, CD25, CD11a, EGFR, respiratory syncytial virus (RSV), glycoprotein IIb/IIa, IgG1, IgE, complement component 5 (C5), B-cell activating factor (BAFF), CD19, CD30, interleukin-1 beta (IL1β), prostate specific membrane antigen (PSMA), CD38, RANKL, GD2, SLAMF7 (CD319), proprotein convertase subtilisin/kexin type 9 (PCSK9), dabigatran, cytotoxic T-lymphocyte-associated protein 4 (CTLA4), interleukin-5 (IL-5), programmed cell death protein (PD-1), VEGFR2 (KDR), protective antigen (PA) of B. anthracis, interleukin-17 (IL-17), interleukin-6 (IL-6), interleukin-6 receptor (IL6R), interleukin-12 (IL-12), interleukin 23 (IL-23), sclerostin (SOST), myostatin (GDF-8), activin receptor-like kinase 1, delta like ligand 4 (DLL4), angiopoietin 3, VEGFR1, selectin, oxidized low-density lipoprotein (oxLDL), platelet-derived growth factor receptor beta, neuropilin 1, Von Willebrand factor (vWF), integrin α vβ 3, neural apoptosis-regulated proteinase 1, integrin α IIbβ 3, beta-amyloid, reticulon 4 (RTN4)/Neurite Outgrowth Inhibitor (NOGO-A), nerve growth factor (NGF), LINGO-1, myelin-associated glycoprotein, or integrin α4β7.
57. The method of claim 54, wherein said monoclonal antibody is trastuzumab, pertuzumab, infliximab, adalimumab, bevacizumab, ranibizumab, natalizumab, rituximab, alemtuzumab, daclizumab, efalizumab, golimumab, certolizumab, cetuximab, panitumumab, palivizumab, abciximab, basiliximab, ibritumomab, omalizumab, eculizumab, abciximab, alirocumab, basiliximab, belimumab, blinatumomab, brentuximab, canakinumab, capromab, daratumumab, denosumab, dinutuximab, eculizumab, elotuzumab, evolocumab, idarucizumab, ipilimumab, mepolizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, palivizumab, pembrolizumab, ramucirumab, raxibacumab, ecukinumab, siltuximab, tocilizumab, ustekinumab, alacizumab, denosumab, blosozumab, romosozumab, stamulumab, alirocumab, ascrinvacumab, enoticumab, evinacumab, evolocumab, icrucumab, inclacumab, nesvacumab, orticumab, ramucirumab, rinucumab, vesencumab, bococizumab, caplacizumab, demcizumab, etaracizumab, idarucizumab, ralpancizumab, tadocizumab, aducanumab, atinumab, fasinumab, fulranumab, gantenerumab, opicinumab, bapineuzumab, crenezumab, ozanezumab, ponezumab, refanezumab, solanezumab, tanezumab, and vedolizumab.
58. A method for modulating the amount of high or low molecular weight species of one or more recombinant polypeptides produced by an engineered mammalian cell, the method comprising:
(a) culturing said mammalian cell in the medium of claim 1 under suitable conditions for the production of said one or more recombinant polypeptides; and
(b) producing said one or more recombinant polypeptides, wherein said one or more recombinant polypeptides have reduced amounts of high or low molecular weight species compared to recombinant polypeptides produced by mammalian cells that are not cultured in the medium of claim 1.
59. The method of claim 58, wherein said one or more recombinant polypeptides has reduced amounts of (a) high molecular weight species; or (b) low molecular weight species.
60. (canceled)
61. The method of claim 59, wherein said low molecular weight species comprise polypeptide fragments that are not completely assembled and/or folded.
62. The method of claim 59, wherein said high molecular weight species comprise more than one subunit of a recombinant polypeptide.
63. The method of claim 61, wherein the percent specific ratio of low molecular weight species to the sum of all (1) non-aggregated; (2) low molecular weight species; and (3) high molecular weight species is modulated relative to the percent specific ratio compared to recombinant polypeptides produced by mammalian cells that are not cultured in the medium of claim 1.
64. The method of claim 62, wherein the percent specific ratio of high molecular weight species to the sum of all (1) non-aggregated; (2) low molecular weight species; and (3) high molecular weight species is modulated relative to the percent specific ratio compared to recombinant polypeptides produced by mammalian cells that are not cultured in the medium of claim 1.
65. The method of claim 58, wherein said one or more recombinant polypeptides is an antibody or fragment thereof.
66. The method of claim 65, wherein said antibody is a monoclonal antibody.
67. The method of claim 66, wherein the monoclonal antibody inhibits the growth of a proliferating cell.
68. The method of claim 65, wherein said antibody or fragment thereof binds to HER2, TNF-α, VEGF-A, α4-integrin, CD20, CD52, CD25, CD11a, EGFR, respiratory syncytial virus (RSV), glycoprotein IIb/IIIa, IgG1, IgE, complement component 5 (C5), B-cell activating factor (BAFF), CD19, CD30, interleukin-1 beta (IL1β), prostate specific membrane antigen (PSMA), CD38, RANKL, GD2, SLAMF7 (CD319), proprotein convertase subtilisin/kexin type 9 (PCSK9), dabigatran, cytotoxic T-lymphocyte-associated protein 4 (CTLA4), interleukin-5 (IL-5), programmed cell death protein (PD-1), VEGFR2 (KDR), protective antigen (PA) of B. anthracis, interleukin-17 (IL-17), interleukin-6 (IL-6), interleukin-6 receptor (IL6R), interleukin-12 (IL-12), interleukin 23 (IL-23), sclerostin (SOST), myostatin (GDF-8), activin receptor-like kinase 1, delta like ligand 4 (DLL4), angiopoietin 3, VEGFR1, selectin, oxidized low-density lipoprotein (oxLDL), platelet-derived growth factor receptor beta, neuropilin 1, Von Willebrand factor (vWF), integrin α vβ 3, neural apoptosis-regulated proteinase 1, integrin α IIbβ 3, beta-amyloid, reticulon 4 (RTN4)/Neurite Outgrowth Inhibitor (NOGO-A), nerve growth factor (NGF), LINGO-1, myelin-associated glycoprotein, or integrin α4β7.
69. The method of claim 66, wherein said monoclonal antibody is trastuzumab, pertuzumab, infliximab, adalimumab, bevacizumab, ranibizumab, natalizumab, rituximab, alemtuzumab, daclizumab, efalizumab, golimumab, certolizumab, cetuximab, panitumumab, palivizumab, abciximab, basiliximab, ibritumomab, omalizumab, eculizumab, abciximab, alirocumab, basiliximab, belimumab, blinatumomab, brentuximab, canakinumab, capromab, daratumumab, denosumab, dinutuximab, eculizumab, elotuzumab, evolocumab, idarucizumab, ipilimumab, mepolizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, palivizumab, pembrolizumab, ramucirumab, raxibacumab, ecukinumab, siltuximab, tocilizumab, ustekinumab, alacizumab, denosumab, blosozumab, romosozumab, stamulumab, alirocumab, ascrinvacumab, enoticumab, evinacumab, evolocumab, icrucumab, inclacumab, nesvacumab, orticumab, ramucirumab, rinucumab, vesencumab, bococizumab, caplacizumab, demcizumab, etaracizumab, idarucizumab, ralpancizumab, tadocizumab, aducanumab, atinumab, fasinumab, fulranumab, gantenerumab, opicinumab, bapineuzumab, crenezumab, ozanezumab, ponezumab, refanezumab, solanezumab, tanezumab, and vedolizumab.
70. A method for modulating the amount of acidic or basic charge species of one or more recombinant polypeptides produced by an engineered mammalian cell, the method comprising:
(a) culturing said mammalian cell in the medium of claim 1 under suitable conditions for the production of said one or more recombinant polypeptides; and
(b) producing said one or more recombinant polypeptides, wherein said one or more recombinant polypeptides have reduced amounts of acidic charge species compared to recombinant polypeptides produced by mammalian cells that are not cultured in the medium of claim 1.
71. The method of claim 70, wherein the percent specific ratio of acidic or basic charge species to the total sum of all (1) acidic species; (2) main species; and (3) basic charge species is reduced relative to recombinant polypeptides produced by mammalian cells that are not cultured in the medium of claim 1.
72. The method claim 70, wherein said one or more recombinant polypeptides is an antibody or fragment thereof.
73. The method of claim 70, wherein said antibody is a monoclonal antibody.
74. The method of claim 72, wherein said antibody or fragment thereof binds to HER2, TNF-α, VEGF-A, α4-integrin, CD20, CD52, CD25, CD11a, EGFR, respiratory syncytial virus (RSV), glycoprotein IIb/IIIa, IgG1, IgE, complement component 5 (C5), B-cell activating factor (BAFF), CD19, CD30, interleukin-1 beta (IL1β), prostate specific membrane antigen (PSMA), CD38, RANKL, GD2, SLAMF7 (CD319), proprotein convertase subtilisin/kexin type 9 (PCSK9), dabigatran, cytotoxic T-lymphocyte-associated protein 4 (CTLA4), interleukin-5 (IL-5), programmed cell death protein (PD-1), VEGFR2 (KDR), protective antigen (PA) of B. anthracis, interleukin-17 (IL-17), interleukin-6 (IL-6), interleukin-6 receptor (IL6R), interleukin-12 (IL-12), interleukin 23 (IL-23), sclerostin (SOST), myostatin (GDF-8), activin receptor-like kinase 1, delta like ligand 4 (DLL4), angiopoietin 3, VEGFR1, selectin, oxidized low-density lipoprotein (oxLDL), platelet-derived growth factor receptor beta, neuropilin 1, Von Willebrand factor (vWF), integrin α vβ 3, neural apoptosis-regulated proteinase 1, integrin α IIbβ 3, beta-amyloid, reticulon 4 (RTN4)/Neurite Outgrowth Inhibitor (NOGO-A), nerve growth factor (NGF), LINGO-1, myelin-associated glycoprotein, or integrin α4β7.
75. The method of claim 70, wherein said monoclonal antibody is trastuzumab, pertuzumab, infliximab, adalimumab, bevacizumab, ranibizumab, natalizumab, rituximab, alemtuzumab, daclizumab, efalizumab, golimumab, certolizumab, cetuximab, panitumumab, palivizumab, abciximab, basiliximab, ibritumomab, omalizumab, eculizumab, abciximab, alirocumab, basiliximab, belimumab, blinatumomab, brentuximab, canakinumab, capromab, daratumumab, denosumab, dinutuximab, eculizumab, elotuzumab, evolocumab, idarucizumab, ipilimumab, mepolizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, palivizumab, pembrolizumab, ramucirumab, raxibacumab, ecukinumab, siltuximab, tocilizumab, ustekinumab, alacizumab, denosumab, blosozumab, romosozumab, stamulumab, alirocumab, ascrinvacumab, enoticumab, evinacumab, evolocumab, icrucumab, inclacumab, nesvacumab, orticumab, ramucirumab, rinucumab, vesencumab, bococizumab, caplacizumab, demcizumab, etaracizumab, idarucizumab, ralpancizumab, tadocizumab, aducanumab, atinumab, fasinumab, fulranumab, gantenerumab, opicinumab, bapineuzumab, crenezumab, ozanezumab, ponezumab, refanezumab, solanezumab, tanezumab, and vedolizumab.
76- 77. (canceled)
78. A kit comprising:
(a) (i) a mammalian cell culture basal medium and/or (ii) a mammalian cell culture feed medium; and
(b) one or more sources of lithium ions.
79. The kit of claim 78, further comprising (c) ethanol and/or (d) one or more fatty acids.
80. The kit of claim 79, wherein said one or more fatty acids is selected from Butyric (C4), Valeric (C5), Caproic (C6), Enanthic (C7), Caprylic (C8), Pelargonic (C9), Capric (C10), Undecylic (C11), Lauric (C12), Tridecylic (C13), Myristic (C14), Pentadecanoic (C15), Palmitic (C16), Margaric (C17), Stearic (C18), Nonadecylic (C19), Arachidic (C20), Heneicosylic (C21), Behenic (C22), Tricosylic (C23), Lignoceric (C24), Pentacosylic (C25), Cerotic (C26), Heptacosylic (C27), Montanic (C28), Nonacosylic (C29), Melissic (C30), Hentriacontylic (C31), Lacceroic (C32), Psyllic (C33), Geddic (C34), Ceroplastic (C35), Hexatriacontylic (C36), Heptatriacontanoic (C37), or Octatriacontanoic (C38) acids.
81. The kit of claim 79, wherein said one or more fatty acids is selected from the group consisting of wherein the fatty acid is one or more of thymol, cholesteryl acetate, methyl octanoate, 1-octanoyl-rac-glycerol, oleic acid, linoleic acid, linolenic acid, cholesterol, palmitic acid, stearic acid, and myristic acid.
82. The kit of claim 78, further comprising (e) written instructions for culturing mammalian cells.
83. The kit of claim 78, wherein said one or more sources of lithium ions is selected from the group of one or more of lithium acetate, lithium chloride, lithium carbonate, lithium oxybutyrate, lithium orotate, lithium bromide, lithium citrate, lithium fluoride, lithium iodide, lithium nitrate, and lithium sulfate.
84. A recombinant polypeptide produced by culturing an engineered mammalian cell in the medium of claim 1 under suitable conditions for the production of said recombinant polypeptide.
85- 89. (canceled)