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US 6,048,728 AGranted
Cell culture medium for enhanced cell growth, culture longevity, and product expression
AssigneeChiron Corporation
InventorsDuane Inlow, Brian Maiorella, William Howarth
Life of this patent
Estimating the term…
Abstract
Protein-free cell culture media supplements are described consisting of synergistic combinations of medium components, which when added to cell culture media, either serum supplemented or serum-free, enhance cell growth, culture longevity and product expression.
Claims39
A method of growing cells, comprising the step of contacting said cells with a cell culture medium comprising the following components:
- (a) a first reagent selected from the group consisting of glutamine, glutamate, and asparagine, said first reagent being present at a concentration of at least 8 mM;
- (b) phospholipid precursors comprising at least choline and ethanolamine;
- (c) tryptophan;
- and
- (d) an amino acid other than tryptophan and other than said first reagent of (a);
- and wherein said components are at concentrations effective to increase culture longevity and product expression by maintaining said cells in a pseudo-stationary growth phase for about 100 hours or longer.
3 more elements
A method of growing cells, comprising the step of contacting said cells with a cell culture medium comprising:
- ______________________________________ Component Concentration (mg/L) ______________________________________ Ca(NO.sub.3).sub.2.4H.sub.2 O 40-50 KCl 400-560 MgSO.sub.4 [73.5] 73.26-242.8 NaCl [5,300] 4300-6,000 NaH.sub.2 PO.sub.4.H.sub.2 O 625-[732] 830 Na.sub.2 HPO.sub.4 [320.4] 190.66-400.5 Glucose [2,700] 1000-[4,800] 5250 Glutathione 0.4-0.5 HEPES 2383.2-2979 Sodium pyruvate 44-1155 NaHCO.sub.3 2350-2850 p-Aminobenzoate 0.464-1[.0] Biotin 0.1-0.2 Calcium pantothenate [0.170] 1.7-4.0 Folic acid 1.6[0]-[4.0] 5 Nicotinamide 2[.0]-5[.0] Pyridoxine hydrochloride 0.48-[1.0] 4 Riboflavin 0.256-0.6 Thiamine hydrochloride 2.016-5[.0] Vitamin B12 0.0025-0.24 Choline chloride 22.8-[23.5] 75 Inositol [24.9] 24.88-[41.0] 50 Ethanolamine 0.8-[10.0] 20 [Glycerol] [0-200] Glutamine 1160-[1180] 5844 PLURONIC .RTM. polyol F68 1000 [Transferrin] [4.0-5.0] [Na.sub.2 SeO.sub.3 ] [.004-.005] [FeCl.sub.3 ] [0-1.6] [(NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 O] [0-0.1] CoCl.sub.2.6H.sub.2 O .005-[0.1] 0.4 [CuCl.sub.2.2H.sub.2 O] [0-0.1] MnCl.sub.2.4H.sub.2 O .002-[0.1] 0.4 [ZnCl.sub.2 ] [0-0.1] [FeSO.sub.4.7H.sub.2 O] [0-0.11] [Monothioglycerol] [0-10] [Citrate] [0-258] Arginine 194.1-794 [Aspartate] Asparagine 125-[280] 1400 [Cystine] [49-195] Glutamic acid 50-308 Glycine 38-[56] 250 Histidine 45.2-[120] 300 Isoleucine 145.2-[282] 1000 Leucine 95.2-[282] 1200 Lysine hydrochloride 153.7-[317] 1000 Methionine 52-[217.6] 400 Phenylalanine 70-[232] 400 Proline 50-108 Serine 52.4-[136] 400 Threonine 72.2-[194] 500 Tryptophan 19-[140] 250 Tyrosine 66.4-[158] 500 Valine 97-[191] 600 ______________________________________
A method of growing cells, comprising the step of contacting said cells with a cell culture medium comprising the following components:
- (a) a first reagent selected from the group consisting of glutamine, glutamate, and asparagine, said first reagent being present at a concentration of at least 8 mM;
- (b) phospholipid precursors comprising at least choline and ethanolamine;
- (c) tryptophan;
- and
- (d) an amino acid other than tryptophan and other than said first reagent of (a);
- and wherein said components are at concentrations effective to increase product expression of said cells to about 470 mg per liter or more of final desired protein product yield at the end of cell culture.
3 more elements
A method of growing cells, comprising the step of contacting said cells with a cell culture medium comprising the following components at the concentration set forth:
- ______________________________________ Component Concentration (mg/L) ______________________________________ Choline chloride >22.8 Ethanolamine >0.8 Glutamine >1160 Tryptophan >19 ______________________________________
- and at least one amino acid selected from the group consisting of the following amino acids at the concentrations set forth:
- ______________________________________ Amino Acid Concentration (mg/L) ______________________________________ Arginine .gtoreq.194.1 Asparagine .gtoreq.125 Aspartate .gtoreq.0 Cystine or cysteine .gtoreq.0 Glutamic acid .gtoreq.50 Glycine .gtoreq.38 Histidine .gtoreq.45.2 Isoleucine .gtoreq.145.2 Leucine .gtoreq.95.2 Lysine hydrochloride .gtoreq.153.7 Methionine .gtoreq.52 Phenylalanine .gtoreq.70 Proline .gtoreq.50 Serine .gtoreq.52.4 Threonine .gtoreq.72.2 Tyrosine .gtoreq.66.4 Valine .gtoreq.97 ______________________________________
A kit for prolonging the pseudo-stationary growth phase of cells grown in culture, comprising:
- (a) a first reagent selected from the group consisting of glutamine, glutamate, and asparagine, said first reagent providing a concentration of at least 5 mM;
- (b) phospholipid precursors comprising at least choline and ethanolamine;
- (c) tryptophan;
- and
- (d) an essential amino acid for growth of said cells other than tryptophan and other than said first reagent of (a);
- and
- wherein said components are at concentrations effective to increase culture longevity and product expression by maintaining said cells in a pseudo-stationary growth phase for about 100 hours or longer.
4 more elements
A method of growing cells, comprising the step of contacting said cells with a cell culture medium comprising:
- ______________________________________ Component Concentration (mg/L) ______________________________________ Ca(NO.sub.3).sub.2.4H.sub.2 O 35-55 KCl 350-600 MgSO.sub.4 70-250 NaCl 3700-6100 NaH.sub.2 PO.sub.4.H.sub.2 O 600-900 Na.sub.2 HPO.sub.4 180-450 Glucose 900-5300 Glutathione 0.3-0.6 HEPES 2300-2990 Sodium pyruvate 40-1120 NaHCO.sub.3 2340-2900 p-Aminobenzoate 0.40-1.50 Biotin 0.05-0.3 Calcium pantothenate 1.50-4.50 Folic acid 1.0-5.5 Nicotinamide 1.5-6.0 Pyridoxine hydrochloride 0.4-4.5 Riboflavin 0.20-0.70 Thiamine hydrochloride 2.4-5.5 Vitamin B12 0.0020-0.30 Choline chloride 10-80 Inositol 23-55 Ethanolamine 0.7-25 Glutamine 1160-6700 Pluronic .RTM. polyol F68 800-1200 (NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 O 0.003-0.5 CoCl.sub.2.6H.sub.2 O 0.005-0.5 CuCl.sub.2.2H.sub.2 O 0.01-0.5 MnCl.sub.2.4H.sub.2 O 0.01-0.5 ZnCl.sub.2 0.003-0.5 Monothioglycerol 0.7-12 Citrate 1-300 Arginine 190-1420 Glutamic acid 45-350 Glycine 30-300 Histidine 50-350 Isoleucine 1040-1050 Leucine 150-1250 Lysine hydrochloride 145-1050 Methionine 50-450 Phenylalanine 65-450 Proline 45-150 Serine 50-450 Threonine 70-600 Tryptophan 18-320 Tyrosine 60-550 Valine 90-650 ______________________________________
Dependencies are inferred from the claim text.