CELL CULTURE MEDIA FOR ENHANCED PROTEIN PRODUCTION

Abstract

A cell culture medium is provided which constrains cell growth and enhances antibody production. The high glucose medium of the invention is preferably saturated at 40° C. with essential amino acids.

Description

FIELD OF THE INVENTION

[0001] This invention relates to cell culture media, which improves protein production, constrains cell growth and extends cell longevity in vitro culture, and to methods for the production and use of such media.

BACKGROUND OF THE INVENTION

[0002] The increasing demand for monoclonal antibodies (MABs) useful in research, diagnosis, therapy and purification purposes has created a need to optimize production techniques. The prior art includes improved bioreactor designs and bioreactor operation to increase cell densities or the longevity of the culture by nutrient feedings.

[0003] Bioreactors have been operated in fed-batch, immobilized, perfusion and continuous modes. Alternate strategies, such as the use of temperature, media formulation, including the addition of mouse peritoneal factors, growth inhibitors, autocrine factors or cyclic mononucleotides and hyperstimulation by osmolarity stress, have been used to enhance protein production. These approaches have shown only marginal success.

[0004] Commonly used basal cell culture media are RPMI 1640, DMEM (Dulbecco's modified Eagle's medium), Ham's F12 and DMEM/F12 (DF). Murakami (1989) (1)1 describes a modified medium, eRDF, prepared from RDF(RPMI:DMEM: F12=2:1:1) by 1 A bibliography precedes the claims. enrichment with amino acids, glucose and vitamins. Murakami showed that doubling total amino acids or glucose alone did not increase cell density but concurrent elevation of amino acids and glucose maximized the cellular growth by threefold. Hyper-stimulation of monoclonal antibody production by high osmolarity stress in a eRDF medium is described in Chua et al. (1994) (2) and (1994)(3). However, the maximum IgG concentration achieved was about 300 ug/ml and 270 ug/ml for HG11 and TBC3 cells, respectively, at medium osmolarities about 350 to 400 mOsm. Further increase in osmolarity with NaCl caused a deterioration in antibody production.

[0005] Oh, et al. (1995) (4) reports that hybridomas increased metabolic activities and amino acids uptake via the Na+ dependent symports to compensate for the osmotically elevated external environment.

[0006] Oh, et al. (1996) (5) describes the application of flow cytometry in examining the relationships between total cellular monoclonal antibody content, cell size, and cell cycle distribution of hybridomas subjected to environmental stress.

SUMMARY OF THE INVENTION

[0007] The invention provides cell nutrient media which enhance protein production and prolong in vitro cell viability. The method cell culture utilizing such media are an important aspect of the invention.

[0008] The media and the methods of the invention are applicable to the culture of cells of any type in bioreactors of all kinds.

DETAILED DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1—growth of hybridomas 2HG11 and TBC3 in BTC-28101 and control DMEM/F12 media.

[0010] FIGS. 2a-2d reflect the result of hollow fiber bioreactor experiments in which BTC-28101 was utilized.

[0011] FIG. 2a represents levels of antibody produced.

[0012] FIG. 2b sets forth medium pH data.

[0013] FIG. 2c reports glucose utilization.

[0014] FIG. 2d reports cell viability.

[0015] FIG. 3a—growth of hybridoma 2HG11 in serum-free BTC-28101 and commercial media Hb and PFHM available from Gibco.

[0016] FIG. 3b—IgG concentration in serum-free BTC-28101 and commercial media Hb and PFHM available from Gibco.

[0017] FIG. 4—growth of hybridomas 2HG11 and TBC3 in BTC-28102 and control DMEM/F12 media.

[0018] FIG. 5—growth of CHO cells in BTC-28103 and control IMDM media. IMDM refers to Iscove's Modified Dulbecco's Media.

[0019] FIG. 6 presents a correlation of percent dry weight of amino acid in the media components with MAB production in ug/ml. The figure illustrates an unexpected increase in MAB concentration when the percent amino acid content in the media exceeds about 20%. In the Figure D/F refers to Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12, 1:1 Mixture.

DEFINITIONS

[0020] Cell Culture Medium—Any medium in which cells of any type may be cultured.

[0021] Bioreactor—Any device in which cells may be cultured. Includes stationary flasks, spinner flasks and hollow fiber bioreactors.

[0022] Basal Medium—A cell culture medium that contains all of the ingredients essential to cell metabolism, e.g., amino acids, lipids, carbohydrates, vitamins and mineral salts RPMI, DMEM, Ham's 12, and RDF are examples of basal media.

[0023] Essential Amino Acids—Arg, Cys, Gln, His, Pro, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr and Val.

[0024] Non-Essential Amino Acids—Ala, Asn, Asp, Gln, Gly, Ser.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The invention provides a method for improving protein production in cultures of protein producing cells. In particular, the invention comprises culturing hybridomas antibody producing cells in a high osmolarity aqueous medium comprising a high concentration of amino acids, in particular the essential amino acids, and an energy source such as glucose or sucrose. The medium is substantially saturated at around 40° C. with an amino acid or acids essential to the metabolism of the culture cells. The medium of the invention may contain 5.50 to 20 grams per liter of total or gross amino acids in solution or suspension and 5.50 to 20 grams per liter of a carbohydrate energy source, preferably glucose, in solution. The gross amino acids comprise at least 20%, preferably, from about 25% to about 50% of the total dry weight of the medium components. Cells may appropriately be adapted to the high osmolarity media of this invention by passaging.

[0026] The osmolarity of the medium is from 320 to 450. Sodium chloride is the preferred osmolyte.

[0027] The media and the methods of the invention are useful in all forms of bioreactors. The benefits of the invention are realized in static, batch, shaker flask, and spinner flask and hollow fiber bioreactor culture procedures.

[0028] Cells of all kinds may be cultured in any of the methods of the invention. Culture of recombinant protein expressing mammalian cells, e.g., CHO cells, is an important aspect of the invention. Many types of mammalian cells, which contain recombinant protein containing expression vectors are known. See, e.g., Acklin, C., et al. (Recombinant human brain-derived neurotrophic factor (&Ggr;HuBDNF). Disulfide structure and characterization of BDNF expressed in CHO cells) Int. J. Pept. Protein Res. (1993) 41:548-52; Fukushima, K., et al., (N-linked sugar chain structure of recombinant human lymphotoxin produced by CHO cells; the functional role of carbohydrate as to its lectin-like character and clearance velocity) ABB (1993) 304:144-53; Hayakawa, T., et al. (In vivo biological activities of recombinant human erythropoietin analogs produced by CHO cells, BHK cells and C127 cells) Biologicals (1992) 20:253-7; Israel, D. I., et al. (Expression and characterization of bone morphogenetic protein-2 in Chinese hamster ovary cells) GF (1992) 7:139-50; Langley, K. E., et al. (Purification and characterization of soluble forms of human and rat stem cell factor recombinantly expressed by Escherichia coli and by Chinese hamster ovary cells) ABB (1992) 295:21-8; Lu, H. S., et al. (Post-translational processing of membrane-associated recombinant human stem cell factor expressed in Chinese hamster ovary cells) 298:150-8; Malik, N., et al., (Amplification and expression of heterologous oncostatin M in Chinese hamster ovary cells) DNA Cell Biol. (1992) 11:453-9; Nagao, M., et al. (Production and ligand-binding characteristics of the soluble form of murine erythropoietin receptor) Biochem. Biophys. Res. Commun. (1992) 188:888-97; Rice, K. G., et. al. (Quantitative mapping of the n-linked sialyloligosaccharides of recombinant erythropoietin; combination of direct high-performance anion-exchange chromatography and 2-aminopyridine derivatization) Anal. Biochem. (1992) 206:278-87; Schmelzer, C. H., et al. (Purification and partial characterization of recombinant human differentiation-stimulating factor) Protein Expr. Purif. (1990) 1:54-62; Schmelzer, C. H., et al. (Biochemical characterization of human nerve growth factor) J. Neurochem. (1992) 59:1675-83; Sima, N., et al. (Tumor cytotoxic factor/hepatocyte growth factor from human fibroblasts; cloning of its cDNA, purification and characterization of recombinant protein) Biochem. Biophys. Res. Commun. (1992) 180:1151-8; Sun, X. J., et al. (Expression and function of IRS-1 in insulin signal transmission) J. Biol Chem (1992) 267:22662-72; Suzuki, A., et al. (Biochemical properties of amphibian bone morphogenetic protein-4 expressed in CHO cells) BJ (1993) 291:413-7; Tressel, T. J., et al. (Purification and characterization of human recombinant insulin-like growth factor binding protein 3 expressed in Chinese hamster ovary cells) Biochem. Biophys. Res. Commun. (1991) 178:625-33. See also Lucas, B. K., et al. (High-level production of recombinant proteins in CHO cells using a dicistronic DHFR intron expression vector) (1996) Nucleic Acids Res. 24: 1774-9.

EXAMPLE 1

Medium BTC 28101

[0029] The dry powder form of Medium BTC-28101 was prepared as two separate components (A) and (B) as listed in Table I. The ingredients were milled to fine dry powder prior to use. To prepare the medium, Component (A) was dissolved in 90% by volume of pyrogen-free water. The mixture was warmed to around 40° C. and stirred for one hour to fully dissolve the powder, and then cooled down to room temperature. Component (B) was added and stirred another hour to dissolve. pH was adjusted to 7.0 by addition of NaOH. Water was added to make up to the desired volume. The osmolarity of the medium was in the range of 330-335 mOsm/Kg. 1 TABLE I Composition of Medium BTC-28101 in mg/L Component (A) Amino Acids Alanine 13.4 Arginine.HCl 1,162.9 Asparagine.H2O 189.2 Aspartic acid 80.0 Cystine.2HCl 105.4 Cysteine.HCl.H2O 105.4 Glutamic acid 79.4 Glutamine 1,997.2 Glycine 85.6 Histidine.HCl.H2O 150.9 Hydroxyproline 63.0 Isoleucine 314.8 Leucine 330.6 Lysine.HCl 394.6 Methionine 98.4 Phenylalanine 148.6 Proline 110.6 Serine 170.2 Threonine 221.6 Tryptophan 36.8 Tyrosine 174.0 Valine 218.0 Component (B) Mineral Salts CaCl2 (anh) 82.1 CuSO4.5H2O 0.00075 FeSO4.7H2O 0.220 KCl 372.8 MgSO4 (anh.) 52.4 NaCl 6,136.2 Na2HPO4 (anh.) 484.1 ZnSO4.7H2O 0.230 Vitamins Biotin 0.102 D-Ca pantothenate 1.240 Folic acid 8.800 Putrescine.2HCl 0.040 Niacinamide 1.510 Para-aminobenzoic 0.510 acid Pyridoxine.HCl 0.520 Pyridoxal.HCl 1.000 Riboflavin 0.210 Thiamine.HCl 1.585 Vitamin B12 0.342 Carbohydrates and derivatives D-glucose 6,846.0 Na Pyruvate 110.0 Nucleic acid derivatives Thymidine 5.7 Hypoxanthine 1.0 Lipids and derivatives Choline bitartrate 55.7 i-inositiol 104.5 Linoleic acid 0.020 Lipoic acid 0.050 Thiol compound Glutathione (reduced) 0.490 Buffers HEPES 3,570.0 NaHCO3 1,130.0 PH indicator Phenol red 6.0 The composition of the medium BTC-28101 is: Glucose (mg/l) 6.846 Amino acids (mg/l) 6.251 Amino acids (% d.w.*) 24.8 *dry weight of media ingredients

EXAMPLE 2

Effect of Medium BTC-28101 on IgG Production in Hybridomas in Shake Flask Culture

[0030] This example compares cell growth and monoclonal antibody production in two hybridoma cell lines 2H11 (antihuman chronic gonadotropin) and TBC3 (antihuman IgG) in the serum supplemented BTC-28101 medium of Example 1 versus DMEM/F12 (Dulbecco's modified Eagle's medium; Ham's F12.1-1).

[0031] The experiment was set up in shaker flasks with 100 ml media supplemented with 10% FBS (Fetal Bovine Serum). Inoculum cells were adapted and maintained by daily passaging at 2×105/ml with the respective fresh medium for at least a week, and the viability of each inoculum culture was above 90% before use. Batch culture was started by inoculating at 2×105/ml into the respective medium. Samples were taken daily to follow the cell growth by trypan blue staining and hemocytometer counting. Monoclonal antibody concentration in the culture supernatant was determined by ELISA analysis. The effect on cell growth is shown in FIG. 1. Maximum concentration of Ig at the end of the cultures are summarized in Table II: 2 TABLE II Maximum Ig Concentration in the Cultures with BTC-28101 and Control DMEM/F12 Media Max Ig Concentration (ug/ml) Cell Line DMEM/F12 BTC-28101 2HG11 50 270 TBC3 84 450

EXAMPLE 3

[0032] Hybridoma cell line TH12 (anti-theophylline) was cultured in either the BTC-28101 media of Example 1 or a DMEM formulation. Cells were inoculated into 100 ml of BTC-28101 or control medium DMEM at 2×105/ml in 250 ml spinner flasks, both media were supplemented with 10% FBS. Similar procedure as stated in Example 2 was followed for preparing the inoculum cultures, and for monitoring the batch. TH12 produced higher concentrations of antibody in BTC-28101 than in the formulation of DMEM. As Table III shows, cell numbers and cell viability were also higher in 28101. 3 TABLE III TH12 Batch Culture:Cell Counts and Viabilities DATE MEDIUM CELL COUNT VIABILITY D0 BTC-28101   2 × 105/ml 100% 1/30/95 DMEM   2 × 105/ml  90% D1 BTC-28101 5.6 × 105/ml 100% 1/31/95 DMEM 2.6 × 105/ml 100% D2 BTC-28101 2.4 × 106/ml  98% 2/1/95 DMEM 0.8 × 106/ml  91% D3 BTC-28101   3 × 106/ml  98% 2/2/95 DMEM 2.2 × 106/ml  97% D4 BTC-28101 3.4 × 106/ml  93% 2/3/95 DMEM 1.4 × 106/ml  77% D5 BTC-28101 3.8 × 106/ml  69% 2/4/95 DMEM 0.5 × 106/ml  32% D6 BTC-28101 8.4 × 105/ml  25% 2/5/95 DMEM 3.6 × 105/ml  20% D7 BTC-28101 5% day 7 2/6/95 DMEM only Viabilities in both media were <5%. Total media volumes collected for analyses.

[0033] Table IV demonstrates enhanced Ig production and specific antibody titer when BTC-28101 is used. 4 TABLE IV TH12 Batch Culture: Ig Concentrations and Specific Antibody Titers. DATE MEDIUM Ig mg/Ml1 TITER2 D1 BTC-28101 539 ug/ml 1,600 1/31/95 DMEM 447 ug/ml   400 D2 BTC-28101 468 ug/ml 12,800 2/1/95 DMEM 397 ug/ml   3,200 D3 BTC-28101 681 ug/ml 12,800 2/2/95 DMEM 440 ug/ml   6,400 D4 BTC-28101 752 ug/ml   6,400 2/3/95 DMEM 518 ug/ml   3,200 D5 BTC-28101 823 ug/ml 25,600 2/4/95 DMEM 553 ug/ml   6,400 D6 BTC-28101 1,500 ug/ml 25,600 2/5/95 DMEM   489 ug/ml   6,400 D7 BTC-28101 1,190 ug/ml 25,600 2/6/95 DMEM   560 ug/ml   3,200 1Ig concentrations determined by precipitating each sample with saturated ammonium sulfate # and reading optical densities at 280 nm. 2Specific antibody titers determined in an indirect ELISA with theophylline-BSA on the # solid phase.

EXAMPLE 4

[0034] Hybridoma cell line DI16 (anti-Dirofilaria immitis) was cultured in either BTC-28101 or DMEM. DI16 produced higher concentrations of antibody in BTC-28101 than in the in-house formulation of DMEM. Table V shows that cell numbers and cell viability were also higher in BTC-28101. 5 TABLE V DI16 Batch Culture:Cell Counts and Viabilities DATE MEDIUM CELL COUNT VIABILITY D0 BTC-28101   2 × 105/ml 98% 3/31/95 DMEM   2 × 105/ml 98% D3 BTC-28101 7.2 × 105/ml 83% 4/03/95 DMEM 8.4 × 105/ml 99% D4 BTC-28101 5.4 × 105/ml 57% 4/04/95 DMEM 1.5 × 105/ml 26% D5 BTC-28101   6 × 105/ml 58% 4/05/95 DMEM 0.5 × 105/ml  8% D6 BTC-28101 3.9 × 105/ml 30% 4/06/95 DMEM —  0% D7 BTC-28101 3.5 × 105/ml 21% 4/07/95 DMEM —  0% D8 BTC-28101 1.8 × 105/ml 10% 4/08/95 DMEM —  0%

[0035] Table VI reports comparative Ig titers and concentration. 6 TABLE VI D116 Batch Culture: Ig Titers and Concentrations DATE MEDIUM Ig TITER1 Ig mg/ml2 D3 BTC-28101 1:1,024 1.09 4/03/95 DMEM 1:256 0.770 D4 BTC-28101 1:2,048 0.882 4/04/95 DMEM 1:512 0.926 D5 BTC-28101 1:2,048 0.940 4/05/95 DMEM 1:512 0.654 D6 BTC-28101 1:2,048 1.28 4/06/95 DMEM 1:512 0.746 D7 BTC-28101 1:2,048 1.11 4/07/95 DMEM Culture Culture terminated terminated D8 BTC-28101 1:2,048 1.39 4/08/95 DMEM Culture Culture terminated terminated 1Ig titers determined by titrating samples in a mouse Ig capture ELISA. 2Ig concentrations determined by precipitating each sample with saturated ammonium sulfate # and reading optical densities at 280 nm; mg/ml = O.D. 280 × dilution factor ÷ 1.41 # extinction coefficient.

EXAMPLE 5

[0036] Hybridoma cell line NP11 (anti-N-acetylprocainamide) was cultured in either BTC-28101 or DMEM. This cell line was slightly slower than the other cell lines to respond to BTC-28101 with enhanced levels of antibody production; variations for different cell lines are not surprising. It is significant that the BTC-28101 culture produced substantial levels of antibody when cultures in DMEM were no longer viable. The ability to keep cultures producing for longer periods of time is a significant advantage of BTC-28101. See Table VII. 7 TABLE VII NP11 Batch Culture:Cell Counts and Viabilities DATE MEDIUM CELL COUNT VIABILITY D0 BTC-28101   2 × 105/ml 100% 4/07/95 DMEM   2 × 105/ml 100% D1 BTC-28101  1.4 × 105/ml 100% 4/08/95 DMEM  2.3 × 105/ml 100% D3 BTC-28101   8 × 105/ml  98% 4/10/95 DMEM 1.04 × 106/ml  84% D4 BTC-28101  8.4 × 105/ml  81% 4/11/95 DMEM  6.7 × 105/ml  55% D5 BTC-28101  1.2 × 106/ml  72% 4/12/95 DMEM  2.9 × 105/ml  21% D6 BTC-28101  8.4 × 105/ml  52% 4/13/95 DMEM   1 × 105/ml  0% D7 BTC-28101  7.6 × 105/ml  41% 4/14/95 DMEM —  0% D10 BTC-28101  1.3 × 105/ml  5% 4/17/95 DMEM —  0%

EXAMPLE 6

Effect of BTC-28101 on IgG Production in Hollow Fiber Culture

[0037] Performance of the hybridoma in a “mini” hollow fiber bioreactor (UniSyn Technologies, Inc.'s “Mini Mouse” bioreactor) supplied with FBS supplemented BTC-28101 was compared with the control FBS supplemented DMEM. The results are shown in Table VIII. Comparable levels of antibody were produced by this hybridoma in the control DMEM and in BTC-28101. However, the control culture was terminated after day 13 when viability was <10%. In contrary, the cells in BTC-28101 remained highly viable, and the culture was terminated only because of shortage of medium supply. 8 TABLE VIII Comparison of Ig Titer of Hollow Fiber Culture in BTC-28101 and Control DMEM Media Time (day) BTC-28101 DMEM  2 1:6,400 1:51,200  5 1:102,400 1:204,800  7 1:204,800 1:204,800  9 1:204,800 1:102,400 12 1:204,800 1:102,400 14 1:204,800 — 16 1:102,400 — 19 1:51,200 —

EXAMPLE 7

Hollow Fiber Bioreactors—28101 Medium

[0038] This hollow fiber bioreactor experiment involved a particular cell line which normally produces a few hundred ug/ml of MAB in conventional method. FIG. 2a indicates that this cell line performed substantially better in BTC-28101. Data for pH of the medium (FIG. 2b), glucose utilization (FIG. 2c), and cell viability (FIG. 2d) are presented. Cells growing in BTC-28101 in hollow fiber bioreactors do not appear to utilize glucose from the medium at the rate normally seen with conventional media. Monitoring of glucose utilization is a standard means of monitoring the progress of cells in hollow fiber bioreactors—the higher the level of glucose utilization, the better the cells are growing.

EXAMPLE 8

Spinner Flask Experiments with BTC 28101 Medium

[0039] Anti-theophylline hybridoma cells were inoculated into 250 ml of Difco's preparation of BTC 28101 or DMEM at 2×105 cells/ml in 500 ml spinner flasks. Both media were supplemented with 10% FBS, 2% L-glutamine, and 1% pen-strep. Five ml samples were collected from each flask on the days indicated. Cell viability was determined each day samples were collected, and antibody concentrations were determined for all samples by radial immunodiffusion (RID) after all had been collected. Until that time, the samples (with cell material removed by centrifugation) were stored at −20° C. Antibody concentrations, as determined by RID, and cell viabilities are described in Table IX: 9 TABLE IX &mgr;g/ml Ab Cell Viability Day DMEM Difco DMEM Difco 0  <125* <125 95% 95% 1 <125 <125   94% 95% 3 <125 <125   95% 95% 4 <125 <125   58% 86% 7 <125 176 0 27% 8 562 0 21% 9 473 0 23% 14  1,035   0 19% Day 0 = 12/30/96 *The lowest concentration RID standard used was 125 ug/ml.

[0040] It is significant that the cell line utilized produced 1 mg/ml under conditions described. Specifically, the spinner flasks did not provide ideal culture conditions. Once the experiment was set up, the medium was never replaced or replenished. Consequently, metabolites and dead cells continued to accumulate.

EXAMPLE 9

[0041] Anti-theophylline hybridoma cells were inoculated into 100 ml of Difco's preparation of BTC 281010 or in-house medium (DMEM) at 2×105 cells/ml in 250 ml spinner flasks. All other parameters were as described for Example 8. Antibody concentrations, as determined by RID, and cell viabilities are described in Table X: 10 TABLE X &mgr;g/ml Ab Cell Viability Day DMEM Difco DMEM Difco 1  <125* <125   95% 99% 3 <125 156 87% 99% 4 <125 209 49% 74% 5 <125 436 16% 65% 6 <125 417  9% 39% 7 <125 417 0 14% 9 400 0 10% Day 0 = 1/18/97 *The lowest concentration RID standard used was 125 ug/ml.

[0042] Note that the culture volume in Example 9 was one-half that in Example 8. Consequently, nutrients may have depleted more quickly and metabolites or other materials accumulated in inhibitory concentrations more rapidly.

EXAMPLE 10

BTC-28101 on IgG Production in Serum-Free Culture

[0043] This example compares cell growth and monoclonal antibody production in a hybridoma cell line (2HG11) in serum-free BTC-28101 and other commercially available serum-free media available from Gibco.

[0044] Hybridoma 2HG11 has been adapted to serum-free conditions in the respective media. All media were supplemented with insulin, transferrin, ethanolamine and selenite. Cells were inoculated into 100 ml of BTC-28101 or control media at 2×105/ml in 250 ml shaker flasks. The results on growth and IgG production are shown in FIGS. 3a and 3b.

EXAMPLE 11

Preparation and Use of BTC-28102 to Culture Hybridomas

[0045] Nutrient contents of the Medium BTC-28101 was further enhanced to formulate Medium-28102. To prepare this medium, Component (C) was prepared according to the composition in Table XI and milled to dry fine powder. The powder was sterilized by gamma-irradiation and added to 100 ml of BTC-28101, constituting the Medium BTC-28102. Osmolarity of the medium was around 400 mOsm/Kg. 11 TABLE XI Composition of Supplement to Medium BTC-28101 to Make Up BTC-28102 Component (C) in mg Alanine 2.0 Arginine.HCl 174.4 Asparagine.H2O 28.4 Aspartic acid 12.0 Cystine.2HCl 31.6 Glutamic acid 11.9 Glutamine 299.6 Glycine 12.8 Histidine.HCl.H2O 22.6 Hydroxyproline 9.5 Isoleucine 47.2 Leucine 49.6 Lysine.HCl 59.2 Methionine 14.8 Phenylalanine 22.3 Proline 16.6 Serine 25.5 Threonine 33.2 Tryptophan 5.5 Tyrosine 26.1 Valine 32.7 Glucose 342.3

[0046] Cystine is utilized in lieu of cysteine which is toxic to cells at high concentration. 12 The composition of medium BTC-28102 is: Glucose 10.269 g/l Amino Acids 15.628 g/l Amino Acids ( % d.w. of 41.1 media ingredients)

[0047] Inoculum cells were adapted to Medium BTC-28101 following the protocol stated in Example 2 and inoculated at 2×105/ml when starting the shaker batch, along with the control cells in 100 ml of DMEM/F12 medium. The effects on cell growth are shown in FIG. 4. Maximum concentration of Ig in the culture are summarized in Table XII. 13 TABLE XII Maximum Ig Concentration in the cultures with BTC-28102 and Control DMEM/F12 Media Max Ig Concentration (&mgr;g/ml) Cell Line DMEM/F12 BTC-28102 2HG11 50  490 TBC3 84 1200

EXAMPLE 12

Preparation and Use of BTC-28103 to Culture CHO Cells

[0048] This invention illustrates use of the invention to culture mammalian cells that express natural or recombinant protein. BTC-28103 was prepared as in BTC-28101 but the buffer contents of HEPES and NaHCO were increased to 8330 mg/l and 2650 mg/l, respectively. As a result, osmolarity of the medium was increased to 360 mOsm/Kg. CHO (Chinese hamster ovary) cells were adapted to grow in suspension and cultured in 100 ml of BTC-28103 and the control IMDM in shaker flasks, both supplied with 10% FBS, thymidine and hypoxanthine. Growth of the cultures was followed daily by hemocytometer counting and presented in FIG. 5.

[0049] The media of the invention is useful to culture protein expressing cell lines in the various forms of available bioreactors. In particular, media of this invention may be used as the intracapillary medium in hollow fiber bioreactor culture of recombinant protein expressing CHO cells.

EXAMPLE 13

[0050] Table XIII indicates the composition of the commercially available media RPMI, D/F and eRDF and of the 28101 (Example 1) and 28102 (Example 11) media of the invention. 14 TABLE XIII RPMI D/F eRDF 28101 28102 Glucose 2.00 3.15  3.42 6.846 10.269 mg/l Amino acids 1.04 1.11  3.1 6.251 15.628 mg/l Amino acids 5.6 6.6 16 24.8 41.1 (d.w. of components)

[0051] The correlation of % amino acid content in medium with MAB production is presented in FIG. 6.

[0052] A novel cell culture media which improves protein production by cells of all types including mammalian cells which express recombinant protein vectors has been disclosed. The invention will substantially enhance the cost effectiveness of cell culture procedures generally including the production of monoclonal antibodies.

BIBLIOGRAPHY

[0053] (1) Murakami, H. (1989) Serum-free media used for cultivation of hybridomas. In: A. Mizrahi(Ed.), Advances in Biotechnological Processes, Vol. 11, Monoclonal Antibodies: Production and Application. Alan R. Liss, New York, pp. 107-141.

[0054] (2) Chua, F., et al. (1994) Journal of Immunological Methods 167:109-119.

[0055] (3) Chua, F., et al. (1994) Journal of Biotechnology 37:265-275.

[0056] (4) Oh, S. K. W., et al. (1995) Biotechnology and Bioengineering 48:525-535.

[0057] (5) Oh, S. K. W., et al. (1996) “Flow Cytometric Studies of Osmotically Stressed and Sodium Butyrate-Treated Hybridoma Cells” in Flow Cytometry Applications in Cell Culture, Marcel Dekker, Inc., (Eds. M. Al-Rubeai and A. N. Emery) New York, Basel, Hong Kong, pp. 101-119.

Claims

1. In a cell culture medium comprising an aqueous solution of amino acids and a carbohydrate energy source for cells cultured in said medium,

the improvement wherein said aqueous solution

(i) is substantially saturated with said amino acids at a temperature of 30° to 50° C.;

(ii) has an osmolarity of 320 to 450 mOsm;

(iii) contains 5.50 to 20 grams per liter of amino acids and 5.50 to 20 grams per liter of said carbohydrate energy source

and wherein the dry weight of the amino acids in solution in said medium comprises at least 20% of the total dry weight of all solid components present in said medium.

2. The claim 1 cell culture medium in which said carbohydrate energy source is glucose.

3. The claim 1 cell culture medium in which said osmolarity of from 320 to 450 mOsm is provided at least in part by sodium salt.

4. The claim 1 or claim 2 cell culture medium in which said osmolarity of from 320 to 450 mOsm is provided at least in part by a sodium chloride osmolyte.

5. The claim 1 cell culture medium further comprising a suspension of undissolved amino acids in said aqueous solution substantially saturated with amino acids

wherein said substantial saturation is maintained as cell growth consumes amino acids in solution in said aqueous medium.

6. In an aqueous basal cell culture medium, the improvement which comprises providing in said basal medium

(i) a concentration of one or more amino acids reactive with cell transport system A to provide a total amino acid concentration of 5.50 to 20 grams per liter;

(ii) an osmolarity of from 320 to 420 mOsm

 said osmolarity being provided by a sodium salt osmolyte.

7. The claim 6 cell culture medium in which the osmolyte (ii) is sodium chloride.

8. The claim 6 cell culture medium in which said one or more amino acids reactive with cell transport system A are zwitterionic amino acids.

9. The claim 6 cell culture medium in which said one or more amino acids reactive with cell transport system A are selected from the group consisting of alanine, glycine, histidine, methionine, proline and serine.

10. The claim 6 or claim 7 or claim 8 cell culture medium in which said basal cell culture medium is RPMI, DMEM, Ham's F12, RDF or eRDF.

11. The cell culture medium 28101, 28102 or 28103.

12. The method which comprises culturing a cell in the culture medium of claim 1, claim 2, or claim 3, or claim 11.

13. A method which comprises:

(i) providing the cell culture medium of claim 1 or claim 2 or claim 3 or claim 11;

(ii) culturing a CHO cell containing a recombinant expression vector in the medium provided in step (i); and

(iii) recovering the recombinant protein expressed by said CHO cell.

14. The claim 13 method in which the CHO cell containing a recombinant expression vector is a dicistronic DHFR intron expression vector.

15. A method which comprises culturing a CHO cell in culture medium 28103.

16. The method of claim 13 wherein said cell cultured in said culture medium is a hybridoma.

17. The method which comprises culturing a mammalian cell in the culture medium of claim 1, claim 2 or claim 3 or claim 11.

18. The method which comprises culturing a mammalian cell having an expression vector for a recombinant protein in a culture medium of claim 1, claim 2 or claim 3 or claim 11.

19. The method which comprises culturing a CHO cell or a BHK cell or a CO5 cell or a Namaliva cell having expression vector for a recombinant protein in a culture medium of claim 1, claim 2, claim 3 or claim 11.

20. The method which comprises culturing a cell in the culture medium of claim 6 or claim 7 or claim 8 or claim 9.

21. The method which comprises culturing a mammalian cell in the culture medium of claim 7 or claim 8 or claim 9.

22. The method which comprises culturing a mammalian cell having an expression vector for a recombinant protein in a culture medium of claim 7 or claim 8 or claim 9.

23. The method which comprises culturing a CHO cell or a BHK cell or a CO5 cell or a Namaliva cell having expression vector for a recombinant protein in a culture medium of claim 7 or claim 8 or claim 9.

24. A dry mixture of cell culture medium components comprising amino acids, carbohydrates and vitamins,

wherein said amino acids component comprise at least 20% of the dry weight of said mixture.

25. The claim 24 mixture wherein said amino acids component comprises from 30% to 50% of the dry weight of said mixture.