Cell culture methods
A method of cell culture includes (i) culturing cells in a cell culture medium, and (ii) maintaining at least one metabolite below an inhibitory concentration in the cell culture medium for the at least one metabolite, wherein the at least one metabolite is aconitic acid (AA), leucinic acid (HICA), cytidine monophosphate (CMP), methylsuccinic acid (MSA), trigonelline (TRI), N-acetylputrescinium (NAP), or a combination thereof, and wherein the enzyme comprises ADH5, BCAT1, CAT, GOT1, HADHB, HOGA1, SLC35A1, or a combination thereof.
1 . A method of cell culture comprising
(i) culturing mammalian cells in a cell culture medium, and
(ii) maintaining at least one metabolite below an inhibitory concentration in the cell culture medium for the at least one metabolite by regulating enzyme expression to reduce synthesis of the at least one metabolite,
wherein:
the at least one metabolite is at least one of aconitic acid (AA), leucinic acid (HICA), cytidine monophosphate (CMP), methylsuccinic acid (MSA), trigonelline (TRI), N-acetylputrescinium (NAP), or a combination thereof;
the enzyme to reduce the synthesis of HICA comprises at least one of alcohol dehydrogenase 5 (ADH5), branched-chain amino acid transaminase 1 (BCAT1), solute carrier family 35 member A1 (SLC35A1), glutamic oxaloacetic transaminase 1 (GOT1), hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB), or a combination thereof;
the enzyme to reduce the synthesis of CMP comprises at least one of ADH5, BCAT1, catalase (CAT), GOT1, 4-hydroxy-2-oxoglutarate aldolase 1 (HOGA1), SLC35A1, HADHB or a combination thereof;
the enzyme to reduce the synthesis of MSA comprises at least one of BCAT1, GOT1, HADHB, HOGA1, SLC35A1 or a combination thereof;
the enzyme to reduce the synthesis of TRI comprises at least one of BCAT1, GOT1, HADHB, CAT, HOGA1, SLC35A1, or a combination thereof;
the enzyme to reduce the synthesis of NAP comprises at least one of BCAT1, SLC35A1, HOGA1, GOT1, CAT, HADHB or a combination thereof; and
the enzyme to reduce the synthesis of trans-aconitic acid comprises at least one of BCAT1, ADH5, GOT1, HOGA1, SLC35A1, HADHB, or a combination thereof,
wherein the enzyme comprises ADH5 and BCAT1.
2 . The method of claim 1 , wherein the method further comprises measuring the concentration of the at least one metabolite, and when the measured concentration of the at least one metabolite is above a predefined value, regulating enzyme expression to reduce the synthesis of the at least one metabolite.
3 . The method of claim 2 , wherein regulating enzyme expression to reduce the synthesis of the at least one metabolite comprises adding a recombinant deoxyribonucleic acid (DNA) molecule to overexpress the enzyme, wherein the enzyme is a downstream enzyme.
4 . The method of claim 1 , wherein the mammalian cells are CHO cells or HEK 293 cells.
5 . The method of claim 1 , wherein the enzyme comprises ADH5, BCAT1, GOT1, HADHB, HOGA1, SLC35A1, and CAT.
6 . The method of claim 1 , wherein the concentration of the at least one metabolite is measured using at least one of nuclear magnetic resonance (NMR) spectroscopy, Raman spectroscopy, High/Ultra Performance Liquid Chromatography (H/UPLC), Liquid Chromatography Mass Spectrometer (LC-MS), Gas Chromatography Mass Spectrometer (GC-MS) technology, or a combination thereof.
7 . The method of claim 1 , wherein maintaining the at least one metabolite below an inhibitory concentration in the cell culture medium for the at least one metabolite comprises controlling at least one of temperature, dissolved oxygen level, pH, or a combination thereof.
8 . The method of claim 1 , wherein at least one of:
the mammalian cells are CHO cells, HEK 293 cells, HT-1080 cells, engineered T cells, or engineered natural killer cells;
the cell culture is a batch culture, a fed batch culture, or a perfusion culture; or
a combination thereof.
9 . The method of claim 1 , wherein the mammalian cells express a recombinant protein, a gene product, or a cell product.
10 . The method of claim 9 , wherein the recombinant protein is a monoclonal antibody.
11 . The method of claim 1 , wherein cell growth and/or productivity are increased as compared to a control culture, wherein the control culture is identical to the culture of claim 1 except it is not cultured using step (ii).
12 . The method of claim 11 , wherein the cell growth is determined by maximum viable cell density and is increased by at least 5% as compared to the control culture.
13 . The method of claim 1 , wherein at least one of:
the at least one metabolite includes AA and concentration of AA in the cell culture medium is lower than 440 μM,
the at least one metabolite includes HICA and concentration of HICA in the cell culture medium is lower than 23.5 μM,
the at least one metabolite includes CMP and concentration of CMP in the cell culture medium is lower than 10 μM,
the at least one metabolite includes MSA and concentration of MSA in the cell culture medium is lower than 3.75 μM,
the at least one metabolite includes TRI and concentration of TRI in the cell culture medium is lower than 0.35 μM,
the at least one metabolite includes NAP and concentration of NAP in the cell culture medium is lower than 0.3 μM, or
a combination thereof.
14 . The method of claim 13 , wherein the concentration of the at least one metabolite is measured using at least one of nuclear magnetic resonance (NMR) spectroscopy, Raman spectroscopy, High/Ultra Performance Liquid Chromatography (H/UPLC), Liquid Chromatography Mass Spectrometer (LC-MS), Gas Chromatography Mass Spectrometer (GC-MS) technology, or a combination thereof.
15 . The method of claim 1 , wherein the enzyme further comprises at least one of GOT1, HADHB, HOGA1, SLC35A1, CAT, or a combination thereof.
16 . A method of cell culture comprising
(i) culturing mammalian cells in a cell culture medium, and
(ii) maintaining at least one metabolite below an inhibitory concentration in the cell culture medium for the at least one metabolite by regulating enzyme expression to reduce synthesis of the at least one metabolite,
wherein the at least one metabolite is at least one of aconitic acid (AA), leucinic acid (HICA), cytidine monophosphate (CMP), methylsuccinic acid (MSA), trigonelline (TRI), N-acetylputrescinium (NAP), or a combination thereof, and
wherein the enzyme comprises alcohol dehydrogenase 5 (ADH5), branched-chain amino acid transaminase 1 (BCAT1), catalase (CAT), Glutamic oxaloacetic transaminase 1 (GOT1), Hydroxyacyl-CoA Dehydrogenase Trifunctional Multienzyme Complex Subunit Beta (HADHB), 4-hydroxy-2-oxoglutarate aldolase 1 (HOGA1), and solute carrier family 35 member A1 (SLC35A1).
17 . The method of claim 16 , further comprising measuring the concentration of the at least one metabolite, and when the measured concentration of the at least one metabolite is above a predefined value, regulating enzyme expression to reduce the synthesis of the at least one metabolite.
18 . The method of claim 17 , wherein regulating enzyme expression to reduce the synthesis of the at least one metabolite comprises adding a recombinant deoxyribonucleic acid (DNA) molecule to overexpress the enzyme, wherein the enzyme is a downstream enzyme.
19 . The method of claim 16 , wherein at least one of:
maintaining the at least one metabolite below an inhibitory concentration in the cell culture medium for the at least one metabolite comprises controlling at least one of temperature, dissolved oxygen level, pH, or a combination thereof;
the mammalian cells are CHO cells, HEK 293 cells, HT-1080 cells, engineered T cells, or engineered natural killer cells;
the cell culture is a batch culture, a fed batch culture, or a perfusion culture;
the cells express a recombinant protein, a gene product, or a cell product;
cell growth and/or productivity are increased as compared to a control culture, wherein the control culture is identical to the culture of claim 1 except it is not cultured using step (ii); or
a combination thereof.
20 . The method of claim 16 , wherein at least one of:
the at least one metabolite includes AA and concentration of AA in the cell culture medium is lower than 440 μM,
the at least one metabolite includes HICA and concentration of HICA in the cell culture medium is lower than 23.5 μM,
the at least one metabolite includes CMP and concentration of CMP in the cell culture medium is lower than 10 μM,
the at least one metabolite includes MSA and concentration of MSA in the cell culture medium is lower than 3.75 μM,
the at least one metabolite includes TRI and concentration of TRI in the cell culture medium is lower than 0.35 μM,
the at least one metabolite includes NAP and concentration of NAP in the cell culture medium is lower than 0.3 μM, or
a combination thereof.