Composition containing dipeptides and trace elements

Abstract

The present invention relates to compositions comprising dipeptides and trace metal ions. More specifically, the present invention relates to improved culture media for use in biotechnological production processes, methods of using such improved media, and products obtained from the methods of using the improved culture media.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a composition comprising at least one dipeptide consisting of two amino acids, said amino acids being natural amino acids, at least one of said amino acids being cysteine ​​(Cys), and at least one trace metal ion.

[0002] Furthermore, the present invention relates to biotechnological production processes. More specifically, the present invention relates to improved culture media for use in biotechnological production processes, methods of using such improved media, and products resulting from the methods of using the improved culture media.

[0003] 2. Background of the Invention Chemically defined dipeptides are often substituted for difficult-to-formulate amino acids in cell culture media used in the production of biopharmaceuticals. The formation of dipeptides increases the stability of glutamine and the solubility of tyrosine and cystine. Well-known examples are alanyl-glutamine, glycyl-glutamine, glycyl-tyrosine, alanyl-tyrosine and N,N'-di-alanyl-cystine. They are efficiently used as nutrients and their uptake and metabolism have been studied in detail (Sanchez-Kopper et al. AMB Expr (2016) 6:48; Verhagen et al. Eng Life Sci. (2020);1-11).

[0004] WO 2011 / 133902 discloses a cell culture medium containing a dipeptide, which contains amino acids with low water solubility, in this case tyrosine and cysteine. Cysteine ​​is not only poorly soluble in water, but is also unstable due to the presence of a reactive thiol group. Furthermore, application of cysteine ​​at higher concentrations, such as 10 mM, may reduce cell viability. The authors found that by incorporating tyrosine and cysteine ​​into the dipeptide, the solubility and stability problems of amino acids may be improved. The higher stability and solubility allow the formulation of chemically defined, highly concentrated media, which are required to carry out enhanced and highly productive industrial cell culture processes.

[0005] Trace elements are micronutrients that all animal and human cells require to support metabolism. Examples of essential trace elements are iron, zinc and copper (Frieden, Journal of Chemical Education (1985), Vol 62, No 11, 917-923). Trace elements can be toxic if provided in excess, and providing enough, but not too much, of these micronutrients can be difficult.

[0006] This is a well-known challenge for the formulation of cell culture media used to cultivate animal or human cells in vitro, e.g. in the context of biopharmaceutical, cell culture-based production of monoclonal antibodies; viral vaccines or cell therapy. Balancing sufficient supply against toxic effects can be difficult, especially when chemically defined, serum-free and even protein-free media are required. Further complications are presented by the fact that trace metals are often present as impurities in other media components such as amino acids.

[0007] The concentrations of trace elements defined in traditional basal medium formulations vary over a relatively wide concentration range between different formulations. For example, zinc and copper concentrations are in the low nanomolar to low micromolar range, with the largest portion traditionally introduced via serum, which contains 10-30 μM of these trace elements (Vargas Arigony et al., BioMed Research International (2013) Article ID 597282).

[0008] When serum-free and chemically defined media are required in modern biopharmaceutical cell culture for improved safety, consistency and performance, these trace elements need to be added in a chemically defined form, which means inorganic salts.

[0009] Published data on the trace metal content of chemically defined biological production media are limited. However, the addition of sufficient amounts of copper and zinc was shown to be important to support growth, product formation, and to directly affect primary metabolism, e.g., lactate consumption. It was also shown to have a positive effect on product quality. Concentration effects were systematically investigated in many publications. For copper, concentrations between 0.05 and 100 μM have been described (Yuk et al., Biotechnol. Prog. (2015), 31; 1: 226-238; Chaderjian et al., Biotechnol. Prog. (2005), 21:550-553). For zinc, concentrations between 3 and 150 μM have been described (Roca et al., Cytotechnoloy (2019) 71:915-924; Graham et al., Applied Microbiology and Biotechnology (2020), 104: 1097-1108). In all cases, a beneficial effect of the addition could be demonstrated, although at higher concentrations, performance or toxicity was reduced.

[0010] There is evidence in the literature that the toxic effects of trace metals are caused by redox reactions with other medium components, leading to the formation of reactive oxygen species (ROS) that become toxic at high concentrations (Keenan et al., In Vitro Cellular & Developmental Biology - Animal (2018) 54:555-558; Graham et al., Biotechnology and Bioengineering. 2019; 116:3446-3456).

[0011] The amino acid L-cysteine ​​participates in redox reactions with metal ions, contributing to ROS formation. In these reactions, L-cysteine ​​is oxidized to L-cystine to form disulfides. This suggests that the formation of ROS in the presence of L-cysteine ​​is 2+ This is described in the Ion literature.

[0012] Because both trace metals and a sufficient source of cysteine ​​are required in the cell culture medium to maximize cell culture productivity, there remains a need to improve the formulation of cell culture media to ensure sufficient trace metals are provided without toxicity. There is also a need to provide sufficient cysteine ​​equivalents while providing sufficient trace metals to the cells. Therefore, the object of the present invention was to provide a cell culture medium that provides a source of cysteine, ensures the supply of trade metals, and has reduced toxicity of both trace metals and cysteine.

[0013] Summary of the Invention The above mentioned shortcomings are addressed by the present invention, which is defined by the terms of the accompanying independent claims. Preferred embodiments of the invention are defined by the dependent claims.

[0014] Surprisingly, it was found that the addition of the dipeptide to cell culture media reduced the toxicity of metal ions. Furthermore, it was found that L-cysteine-induced metal toxicity could be reduced by the addition of the dipeptide. Finally, it was found that L-cysteine-induced metal toxicity could be completely suppressed by replacing L-cysteine ​​with cystine dipeptide, which actually increased cell viability when compared to the respective controls.

[0015] The composition according to the present invention comprises at least one dipeptide consisting of two amino acids (the amino acids are natural amino acids, and at least one of the amino acids is cysteine ​​(Cys)) and at least one trace metal ion, wherein the molar ratio of the dipeptide to the trace metal ion is 10,000 to 20.

[0016] The compositions according to the invention may also be a cosmetic product, a dietary supplement, a nutrient solution for clinical nutrition, or a constituent part of a cell or tissue culture medium (basal, fed-batch or perfusion medium).

[0017] The present invention further relates to the use of the culture medium of the present invention for culturing cells, preferably plant cells, animal cells or mammalian cells.

[0018] Another aspect of the invention relates to a method for producing a cell culture product, comprising the steps of: (i) providing cells capable of producing said cell culture product; (ii) contacting said cells with a culture medium according to the invention; and (iii) obtaining said cell culture product from said culture medium or from said cells.

[0019] Preferred embodiments of the present invention are described in further detail in the following detailed description of the invention.

[0020] Detailed Description of the Invention In the context of the present invention, the expression "natural amino acids" shall be understood to include both the L- and D-forms of the 20 amino acids listed above. However, the L-forms are preferred. In one embodiment, the term "amino acids" also includes analogs or derivatives of these amino acids.

[0021] A "free amino acid" according to the present invention, e.g. "free" cysteine, is understood to be an amino acid having its amino group and its (α-) carboxylic acid functionality in free form, i.e. not covalently linked to other molecules, e.g. not forming a peptide bond. Free amino acids may be present as salts or in the form of hydrates. When referring to amino acids as part of or in a dipeptide, this shall be understood to refer to that part of the respective dipeptide structure which is derived from the respective amino acid according to known mechanisms of biochemistry and peptide biosynthesis.

[0022] The present invention generally relates to a composition comprising at least one dipeptide consisting of two amino acids, the amino acids being natural amino acids, at least one of the amino acids being cysteine ​​(Cys), and at least one trace metal ion, the molar ratio of the dipeptide to the trace metal ion being 10,000-20.

[0023] "Peptide" means a peptide that is formed by an α-peptide bond (R 1 -CO-NH-R 2 It is to be understood that the term "amino acid" is a molecule comprising at least two amino acids covalently bonded to each other by an amino acid chain.

[0024] "Dipeptide" means an α-peptide bond (R 1 -CO-NH-R 2 It is to be understood that the term "amino acid" is a molecule comprising two amino acids covalently bonded to each other by an amino acid chain.

[0025] The expression "Xxx," when used herein in reference to an amino acid, shall be understood to refer to any of the naturally occurring amino acids as defined below.

[0026] In the context of the present invention, an "amino acid" shall be understood to be a molecule comprising an amino functional group (-NH2) and a carboxylic acid functional group (-COOH) together with a side chain specific for each amino acid. In the context of the present invention, both α-amino acids and β-amino acids are included. The preferred amino acids of the present invention are the α-amino acids, in particular the 20 "natural amino" acids, which include cystine, as follows: Alanine (Ala / A) Arginine (Arg / R) Asparagine (Asn / N) Aspartic acid (Asp / D) Cysteine ​​(Cys / C) Cystine (Cyss / C2) Glutamic acid (Glu / E) Glutamine (Gln / Q) Glycine (Gly / G) Histidine (His / H) Isoleucine (Ile / I) Leucine (Leu / L) Lysine (Lys / K) Methionine (Met / M) Phenylalanine (Phe / F) Proline (Pro / P) Serine (Ser / S) Threonine (Thr / T) Tryptophan (Trp / W) Tyrosine (Tyr / Y) Valin (Val / V)

[0027] In the context of the present invention, the expression "natural amino acids" shall be understood to include both the L- and D-forms of the twenty amino acids listed above. However, the L-forms are preferred. In one embodiment, the term "amino acids" also includes analogs or derivatives of these amino acids.

[0028] A "free amino acid" (e.g., "free cysteine") according to the present invention is understood to be an amino acid having its amino group and its (α-) carboxylic acid functionality in free form, i.e. not covalently bound to other molecules, e.g. not forming a peptide bond. Free amino acids may be present as salts or in the form of hydrates. When referring to an amino acid as part of or in a dipeptide, this shall be understood to refer to that part of the respective dipeptide structure which is derived from the respective amino acid according to known mechanisms of biochemistry and peptide biosynthesis.

[0029] The expression "N-acylated" shall be understood to mean, with respect to a chemical compound, such as an amino acid, that the N-acylated compound is modified by the addition of an acyl group to the nitrogen functional group of said compound. Preferably, the acyl group is added to the α-amino group of the amino acid.

[0030] The molar ratio of the dipeptide to the trace metal ion is preferably 5,000-20, more preferably 1,000-20.

[0031] In a preferred embodiment, the trace metals are selected from iron, lithium, zinc, copper, chromium, nickel, cobalt, vanadium, molybdenum, and manganese, and the trace metal ions are preferably copper ions. 2+ It is particularly preferred to use the .sup.3-.sup.4 ion.

[0032] In another preferred embodiment, the dipeptide concentration is 0.1 to 200 mM, preferably 0.2 to 20 mM, and most preferably 0.5 to 10 mM, and the trace metal ion concentration is 0.1 to 400 μM, preferably 0.2 to 100 μM, and most preferably 0.5 to 20 μM.

[0033] In another preferred configuration, the dipeptide is present in the culture medium at a concentration of at least 1 mM, preferably at least 10 mM, more preferably at least 50 mM, more preferably at least 100 mM. At such high concentrations, the composition according to the invention offers the advantage that the cysteine-containing dipeptide stabilizes cysteine ​​against oxidative precipitation.

[0034] When the dipeptide is Xxx-Cys Cys-Xxx, where Xxx is a natural amino acid, the dipeptide is preferably Ala-Cys, Cys-Ala, Lys-Cys or Cys-Lys.

[0035] It is particularly preferred that when the dipeptide is Xxx-Cys or Cys-Xxx and is in an oxidized and dimerized form, whereby the dimerized dipeptide is linked via a disulfide bond.

[0036] In another preferred configuration, the composition further comprises free cysteine.

[0037] The composition can be prepared by mixing a specified molar ratio of the dipeptide with each trace metal in an appropriate ratio to prepare a powdered product or liquid stock solution that can be added to cell culture medium. Alternatively, the dipeptide can be added to a cell culture medium that already contains each trace metal. Optionally, an appropriate amount of cysteine ​​can be added to the composition or directly added to the cell culture medium to prepare a composition that further contains cysteine.

[0038] When the dipeptide is a Cys-dipeptide, the preferred molar ratio of dipeptide-bound cysteine ​​(via a disulfide bond) to free cysteine ​​is 10 or less, preferably 4 or less, more preferably 2 or less, more preferably 1 or less, more preferably 0.5 or less, and most preferably 0.2 or less. The mixture may be in the form of a solid (crystalline powder, agglomerate, etc.) or an aqueous solution. In the case of an aqueous solution, cysteine ​​is added at a concentration of at least 1 mM, preferably at least 10 mM, more preferably at least 50 mM, and most preferably at least 100 mM, and the dipeptide is added at the appropriate molar ratio as described above.

[0039] The composition may be prepared by mixing at least one dipeptide, one amino acid of which is cysteine ​​(Cys), with a cysteine ​​source selected from free cysteine ​​and optionally cystine (Cys-Cys).The present invention thus relates to a culture medium comprising the composition.

[0040] In the context of the present invention, Cys-peptides that form disulfide bonds via oxidized cysteine ​​residues shall be described as (Xxx-Cys)2. The peptides may exist as salts or in the form of hydrates. Such disulfide bond-mediated dimers of Cys-dipeptides, e.g. (Xxx-Cys)2, are still considered to be dipeptides in the sense of the present invention.

[0041] Preferably, the composition has a pH value of at least 5, or preferably at least 6, at 25°C.

[0042] In an advantageous configuration of the invention, the molar ratio of peptide-bound cysteines to free cysteines is between 0.1 and 10, preferably between 0.2 and 4 or less, most preferably between 0.5 and 2. In a preferred embodiment, the dipeptide is either in the reduced state (=free thiols) or in the oxidized state (=disulfide bonds), preferably in the oxidized state.

[0043] In another embodiment, the composition comprises a mixed disulfide of a dipeptide and a cysteine ​​source.

[0044] In a preferred embodiment of the invention, the dipeptide further comprises one or more naturally occurring amino acids having a solubility of at least more than 10 g / l in the pH range between pH 6 and pH 9, preferably selected from glycine (Gly), alanine (Ala), serine (Ser), proline (Pro), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys) or arginine (Arg).

[0045] In a preferred embodiment, the dipeptide is not N-acylated. Although N-acylation is known to improve the thermal stability of certain dipeptides, it has been found that N-acylated dipeptides can also lead to reduced viable cell density and viability.

[0046] The present invention also relates to a cosmetic product, a dietary supplement or a nutritional solution for clinical nutrition comprising a composition according to the invention.

[0047] Cosmetics can be shampoos, conditioners, lotions, creams, or other formulations used to treat the skin or hair. Dietary supplements can be in liquid form, such as syrups or shots, or in solid form, such as capsules, softgels, gummies, etc. The compositions can also be part of a nutritional solution for clinical enteral or parenteral nutrition, for example, an amino acid solution such as Aminoven (Fresenius Kabi).

[0048] Additionally, the present invention also refers to cell or tissue culture media.

[0049] Another subject of the present invention relates to a cell or tissue culture medium comprising a composition according to the invention, further comprising at least one carbohydrate, at least one free amino acid, at least one inorganic salt, a buffer and / or at least one vitamin. In a particularly preferred embodiment, the culture medium comprises all of at least one carbohydrate, at least one free amino acid, at least one inorganic salt, a buffer and at least one vitamin.

[0050] In one embodiment of the present invention, the culture medium does not contain growth factors. According to this embodiment, the dipeptide of the present invention can be used instead of growth factors to promote the growth and / or proliferation of cells in culture. In another embodiment of the present invention, the culture medium does not contain lipids.

[0051] According to another embodiment of the invention, the culture medium is in liquid form, gel, powder, granulate, pellet form, or tablet form.

[0052] In a preferred embodiment, the culture medium of the present invention is a defined medium or a serum-free medium. For example, the composition of the present invention can be supplemented with CHOMACS CD medium from Miltenyi Biotech (Bergisch Gladbach, Germany), PowerCHO-2 CD medium available from LONZA (Basel, Switzerland), Acti-CHO P medium from PAA (PAA Laboratories, Patching, Austria), Ex-Cell CD CHO medium available from SAFC, SFM4CHO medium, and CDM4CHO medium from ThermoFisher (Waltham, USA). The dipeptide of the present invention can also be supplemented with DMEM medium (Life Technologies Corp., Carlsbad, USA). However, the present invention is not limited to supplementing the above-mentioned media.

[0053] In another preferred embodiment, the culture medium is a liquid medium in a concentrated form (volume / volume) of 2x, 3x, 3.33x, 4x, 5x, or 10x the concentration of the above medium used. This allows the preparation of a "ready to use" culture medium by simply diluting the concentrated medium with the respective volume of sterile water. Such concentrated forms of the medium of the present invention can also be used by adding them to the culture, for example in a fed-batch or perfusion process.

[0054] The cell culture medium of the present invention (basal medium, feed medium or perfusion medium of cell or tissue culture) can preferably contain all nutrients required for continuous growth and product formation. The formulations for preparing culture medium, especially cell culture medium, are well known to those skilled in the art (see, for example, Cell Culture Technology for Pharmaceutical and Cell-Based Therapies, Oeztuerk and Wei-Shou Hu eds., Taylor and Francis Group 2006). Various culture media are commercially available from various sources.

[0055] The culture medium of the present invention may preferably contain a carbohydrate source. The main carbohydrate used in cell culture media is glucose, which is routinely supplemented at 5 to 25 mM. In addition, any hexose, such as galactose, fructose, or mannose, or a combination, may be used.

[0056] Culture media may also typically include at least essential amino acids (i.e., His, Ile, Leu, Lys, Met, Phe, Thr, Try, Val), as well as non-essential amino acids. Non-essential amino acids are typically included in cell culture media when the cell line cannot synthesize the amino acid or when the cell line cannot produce sufficient amounts of the amino acid to support maximal growth. In addition, mammalian cells can also use glutamine as a primary energy source. Glutamine is often included in higher concentrations than other amino acids (2-8 mM). However, as noted above, glutamine can spontaneously decompose to form ammonia, and certain cell lines produce ammonia more quickly, which is toxic.

[0057] The culture medium of the present invention may preferably contain salts. Salts are added to cell culture media to maintain isotonicity and prevent osmotic imbalance. The osmotic pressure of the culture medium of the present invention is about 300 mOsm / kg, but many cell lines can tolerate variations of about 10% or more of this value. The osmotic pressure of some insect cell cultures tends to be higher than 300 mOsm / kg, which may be 0.5%, 1%, 2-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30% higher than 300 mOsm / kg. The most commonly used salt in cell culture media is Na + , K + , Mg 2+ , Ca 2+ , Cl - , SO4 2- , PO4 3- and HCO3 - (e.g., CaCl2, KCl, NaCl, NaHCO3, Na2HPO4).

[0058] Other inorganic elements (including additional trace elements) may be present in the culture medium. These include Mn, Cu, Zn, Mo, Va, Se, Fe, Ca, Mg, Si, and Ni. Many of these elements are involved in enzyme activity. They may be provided in the form of salts such as CaCl2, Fe(NO3)3, MgCl2, MgSO4, MnCl2, NaCl, NaHCO3, Na2HPO4, and ions of trace elements such as selenium, vanadium, and zinc. These inorganic salts and trace elements can be obtained commercially, for example, from Sigma (St. Louis, MO).

[0059] The culture medium of the present invention preferably contains vitamins. Vitamins are typically used by cells as cofactors. The vitamin requirements of each cell line are highly variable, but extra vitamins are generally required when the cell culture medium contains little or no serum or when the cells are growing at high density. Exemplary vitamins preferably present in the culture medium of the present invention include biotin, choline chloride, folic acid, i-inositol, nicotinamide, D-Ca ++ -Pantothenic acid, pyridoxal, riboflavin, thiamine, pyridoxine, niacinamide, A, B6, B 12 , C, D3, E, K, and p-aminobenzoic acid (PABA).

[0060] The culture medium of the present invention may also contain serum. Serum is the supernatant of clotted blood. Serum components include adhesion factors, micronutrients (e.g., trace elements), growth factors (e.g., hormones, proteases), and protective elements (e.g., antitoxins, antioxidants, antiproteases). Serum can be obtained from a variety of animal sources, including human, bovine or horse serum. When included in the cell culture medium according to the present invention, serum is typically added at a concentration of 5-10% (by volume). A preferred cell culture medium is serum-free.

[0061] To promote cell growth in the absence or in serum or serum-reduced medium, one or more of the following polypeptides may be added to the cell culture medium of the present invention: for example, fibroblast growth factors (FGFs), such as acidic FGF and basic FGF, insulin, insulin-like growth factors (IGFs), epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), and transforming growth factors (TGFs), such as TGFα and TGFβ, cytokines, such as interleukin 1, 2, 6, granulocyte stimulating factor, leukocyte inhibitory factor (LIF), and the like.

[0062] In other embodiments, the cell culture medium does not contain polypeptides (i.e., peptides having more than 20 amino acids).

[0063] Also, one or more lipids, such as linoleic acid, linolenic acid, arachidonic acid, palmitoleic acid, oleic acid, polyenoic acid, and / or fatty acids of 12, 14, 16, 18, 20 or 24 carbon atoms (each carbon atom being branched or unbranched), phospholipids, lecithin (phosphatidylcholine), and cholesterol, can be added to the cell culture medium of the present invention. One or more of these lipids can be included in serum-free medium as supplements. Phosphatidic acid and lysophosphatidic acid stimulate the growth of certain anchorage-dependent cells, such as MDCK, mouse epithelial cells, and other renal cell lines, while phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol stimulate the growth of human fibroblasts in serum-free medium. Ethanolamine and cholesterol have also been shown to promote the growth of certain cell lines. In certain embodiments, the cell culture medium is lipid-free.

[0064] One or more carrier proteins, such as bovine serum albumin (BSA) or transferrin, may also be added to the cell culture medium. Carrier proteins may aid in the transport of certain nutrients or trace elements. BSA is typically used as a carrier for lipids such as linoleic acid and oleic acid, which are insoluble in aqueous solutions. In addition, BSA may also function as a carrier for certain metals, such as Fe, Cu, and Ni. In protein-free formulations, non-animal-derived alternatives to BSA, such as cyclodextrin, may be used as lipid carriers.

[0065] One or more adhesion proteins, such as fibronectin, laminin, and pronectin, can also be added to the cell culture medium to promote attachment of anchorage-dependent cells to the substrate.

[0066] The cell culture medium may optionally contain one or more buffers. Suitable buffers include, but are not limited to, N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid] (HEPES), MOPS, MES, phosphate, bicarbonate and other buffers suitable for use in cell culture applications. Suitable buffers provide buffering capacity without substantial cytotoxicity to cultured cells. Selection of suitable buffers is within the scope of those skilled in the art of cell culture.

[0067] Polyanionic or polycationic compounds can be added to the culture medium to prevent cell aggregation and promote growth of cells in suspension.

[0068] In a preferred embodiment, the culture medium is in liquid form. However, the culture medium may also be a solid medium, such as a gelatinous medium, such as an agar medium, a carrageen medium or a gelatin-containing medium (powder, agglomerated powder, instantiated powder, etc.). Preferably, the culture medium is in a sterile form.

[0069] The culture medium of the present invention may be in concentrated form. It may be, for example, in a concentrated form of 2 to 100 times (relative to the concentration that supports cell growth and product formation), preferably 2 times, 3 times, 3.33 times, 4 times, 5 times, 10 times, 20 times, 50 times or 100 times. Such concentrated culture medium is useful for preparing a culture medium for use by diluting the concentrated culture medium with an aqueous solvent such as water. Such concentrated culture medium may be used in batch culture, but is also advantageously used in fed-batch or continuous culture, in which the concentrated nutrient composition is added to the ongoing culture of cells, for example to replenish nutrients consumed by the cells during the culture.

[0070] In another embodiment of the invention, the culture medium is in a dry form, such as in the form of a dry powder, or in the form of granules, or in the form of pellets, or in the form of tablets.

[0071] The present invention also relates to the use of the culture medium of the present invention for culturing cells. Another aspect of the present invention relates to the use of the culture medium of the present invention for producing a cell culture product.

[0072] A preferred embodiment of the present invention relates to the use of the culture medium according to the present invention for culturing animal or plant cells, most preferably mammalian cells. In a specific embodiment, the cells to be cultured are CHO cells, COS cells, VERO cells, BHK cells, HEK cells, HELA cells, AE-1 cells, NS0 cells, insect cells, fibroblasts, muscle cells, neuronal cells, stem cells, skin cells, endothelial cells and hybridoma cells. The preferred cells of the present invention are CHO cells and hybridoma cells. The most preferred cells of the present invention are CHO cells. The particularly preferred CHO cells of the present invention are CHO DG44 cells and CHO DP12 cells.

[0073] Also included within the scope of the present invention is a method of culturing cells, said method comprising contacting said cells with a cell culture medium according to the present invention. In one embodiment of the present invention, the method of culturing cells comprises contacting the cells with a basal culture medium under conditions supporting the culture of the cells, and supplementing the basal cell culture medium with an enriched medium according to the present invention. In a preferred embodiment, the basal culture medium is supplemented with an enriched feed or medium for more than one day.

[0074] Another aspect of the present invention relates to a method for producing a culture medium according to the present invention, said culture medium comprising a composition according to the present invention. The method for producing a culture medium according to the present invention comprises at least one step of adding a composition according to the present invention to the culture medium. Similarly, one aspect of the present invention relates to the use of a composition according to the present invention for producing a cell culture medium.

[0075] Another aspect of the invention relates to a method of amending a culture medium, said amending said culture medium comprising adding a composition of the invention to said culture medium.

[0076] Another aspect of the invention relates to a method of producing a liquid culture medium, said method comprising the steps of providing a solid medium according to the invention, for example in the form of a dry powder, or in the form of granules, or in the form of pellets, or in the form of a tablet, and dissolving said solid culture medium in an aqueous medium, such as water.

[0077] Another aspect of the invention relates to the use of a composition according to the invention in a culture medium for culturing cells.Another aspect of the invention relates to the use of a composition according to the invention for cell culture.

[0078] The present invention also relates to a method for producing a cell culture product, comprising the steps of (i) providing cells capable of producing said cell culture product, (ii) contacting said cells with a culture medium of the present invention, and (iii) obtaining said cell culture product from said culture medium or from said cells. Similarly, the present invention relates to the use of a composition according to the present invention for producing a cell culture product.

[0079] In preferred methods, the cell culture product is a therapeutic protein, a diagnostic protein, a polysaccharide such as heparin, an antibody, a monoclonal antibody, a growth factor, an interleukin, a virus, a virus-like particle or an enzyme.

[0080] The cultivation of the cells according to the invention can be carried out as batch, fed-batch or continuous culture. [Brief description of the drawings]

[0081] [Figure 1-A] FIG. 1 shows the cell viability of MSCs treated with a combination of Cu(II) and L-cysteine ​​at the indicated concentrations. [Figure 1-B] FIG. 1 shows cell viability of CHO cells treated with a combination of Cu(II) and L-cysteine ​​at the indicated concentrations. [Figure 2-A] FIG. 1 shows the cell viability of MSCs treated with a combination of Cu(II) and (Ala-Cys)2 at the indicated concentrations. [Figure 2-B] FIG. 1 shows cell viability of CHO cells treated with a combination of Cu(II) and (Ala-Cys)2 at the indicated concentrations. [Figure 3-A] FIG. 1 shows the cell viability of MSCs treated with a combination of Cu(II) and (Lys-Cys)2 at the indicated concentrations. [Figure 3-B] FIG. 1 shows cell viability of CHO cells treated with a combination of Cu(II) and (Lys-Cys)2 at the indicated concentrations. [Figure 4] FIG. 13: Control experiment showing the effect of addition of single components at various concentrations on cell viability.

[0082] Working Example material: [Table 1]

[0083] [Table 2]

[0084] method: In vitro cytotoxicity assay Human mesenchymal stem cells (MSCs) were cultured in MesenCult™-ACF Plus Culture Kit (Stemcell Technologies). Media was prepared according to the manufacturer's instructions with the addition of 30 mg / mL gentamicin and 2 mM l-glutamine. Agarabi CHO cells were cultured in ActiPro medium supplemented with 6 mM l-glutamine. "Complete growth medium" refers to the medium supplemented with the supplements used to culture the corresponding cell type.

[0085] Stock solutions of Cu(II), l-cysteine, N,N'-di-l-alanyl-l-cystine ((Ala-Cys)2) and N,N'-di-l-lysyl-l-cystine ((Lys-Cys)2) were dissolved in the corresponding complete growth medium immediately before their use. In assays performed on MSCs combining Cu(II) with l-cysteine, (Ala-Cys)2 or (Lys-Cys)2, the dissolved single compounds were mixed at 2x treatment concentration. On MSCs, l-cysteine ​​and dipeptides were tested at 0.5 mM, 1 mM and 5 mM, and Cu(II) was tested at 0.2 μM, 1 μM and 5 μM. For treatment of Agarabi CHO cells, serial dilutions of l-cysteine, (Ala-Cys)2 and (Lys-Cys)2 stock solutions in complete growth medium were performed with 2-fold treatment concentration at each dilution step. In Agarabi CHO cells, l-cysteine ​​and the dipeptide were tested at 2.5 mM, 5 mM and 10 mM, and Cu(II) was tested at 2.5 μM, 10 μM and 40 μM.

[0086] The cytotoxicity of test compounds and their combinations was assayed in MSCs and Agarabi CHO cells cultured at 5,000 cells / well in white 96-well cell culture plates. MSCs (50 μL / well) were cultured for 24 hours in a CO2 incubator (37°C, 5% CO2, 95% humidity) to ensure cell attachment. Following this, the prepared 2x concentrated test compounds and mixtures (50 μL / well) were added. The plates were incubated for 24 hours (37°C, 5% CO2, 95% humidity). Single compounds, as well as MSCs cultured in complete growth medium without additional compounds, were used as controls. Each condition was tested in triplicate. Agarabi CHO cells were seeded (50 μL / well) with complete growth medium and complete growth medium containing 2x concentrated Cu(II) test concentrations. This was immediately followed by the addition of 50 μL of the prepared serial dilutions of l-cysteine, (Ala-Cys)2 and (Lys-Cys)2. Agarabi CHO cells were incubated for 96 hours (37°C, 5% CO2, 95% humidity). All single components on Agarabi CHO cells ("single component controls"), Agarabi CHO cells in complete growth medium without the addition of test compounds ("untreated cells"), and complete growth medium with or without the combination of Cu(II) with l-cysteine, (Ala-Cys)2 or (Lys-Cys)2 ("medium blank") were used as controls. For controls, these compounds were used at the highest concentrations tested. Each condition was tested in quadruplicate.

[0087] After incubation, viability of treated cells was determined based on adenosine triphosphate (ATP) quantification using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions.

[0088] To calculate cell viability, the resulting luminescence values ​​were averaged and normalized to the signal emitted by untreated cells.

[0089] Example 1: Replacement of cysteine ​​with a dipeptide eliminates the adverse effects of trace metals and further contributes to improved survival Addition of L-cysteine ​​and Cu(II) to the cell culture medium at several concentrations significantly decreased cell viability in a concentration-dependent manner (Figure 1A and Figure 1B). Replacement of L-cysteine ​​with equimolar amounts of the dipeptides (Ala-Cys)2 and (Lys-Cys)2 did not show any toxic effect. On the contrary, combining the dipeptides with Cu(II) had a beneficial effect, further improving viability (Figure 2A and Figure 2B, Figure 3A and Figure 3B). This opened the possibility of increasing the Cu(II) concentration in the cell culture medium using dipeptides instead of L-cysteine, as well as the concentration of the L-cysteine ​​source. Addition of the individual compounds at the respective concentrations was examined as a control. Toxicity at the highest concentration tested was only observed with L-cysteine ​​(Figure 4).

[0090] Figures 1-A and 1-B show the effect of adding Cu(II) and L-cysteine ​​at different concentrations and ratios on the viability of MSCs and CHO cells, respectively. A significant increase in toxicity was observed with increasing concentrations of Cu and L-cysteine.

[0091] Figures 2-A and 2-B show the effect of adding Cu(II) and (Ala-Cys)2 at different concentrations and ratios on the viability of MSCs and CHO cells, respectively. In contrast to Figures 1-A and 1-B, no toxic effects were observed. For selected combinations of (Ala-Cys)2 and Cu(II), a significant positive effect on viability was observed.

[0092] Figures 3-A and 3-B show the effect of the addition of Cu(II) and (Ala-Cys)2 at different concentrations and ratios on the viability of MSCs and CHO cells, respectively. In contrast to Figures 1-A and 1-B, no toxic effects were observed. For selected combinations of (Lys-Cys)2 and Cu(II), a significant positive effect on viability was observed.

[0093] Figure 4 shows control experiments showing the effect of addition of single components (L-cysteine, (Ala-Cys)2, (Lys-Cys)2, Cu(II)) at various concentrations on the viability of MSCs. Only L-cysteine ​​had a negative effect on viability. Cu(II) up to 5 μM (MSCs) or 40 μM (CHO cells) had no negative effect on viability.

[0094] In summary, we surprisingly found that Cu(II)-catalyzed L-cysteine ​​toxicity could be alleviated by the addition of peptides and when L-cysteine ​​was replaced by Cys-peptides, thus allowing cells to be safely supplied with higher concentrations of trace metals and even improving their viability.

Claims

1. at least one dipeptide consisting of two amino acids, said amino acids being natural amino acids, at least one of said amino acids being cysteine ​​(Cys); - at least one trace metal ion; Including, A composition, wherein the molar ratio of the dipeptide to the trace metal ion is 10,000 to 20.

2. 2. The composition of claim 1, wherein the molar ratio of said dipeptide to said trace metal ions is 5000 to 20.

3. 2. The composition of claim 1, wherein the trace metals are selected from iron, lithium, zinc, copper, chromium, nickel, cobalt, vanadium, molybdenum, and manganese.

4. 2. The composition according to claim 1, wherein the concentration of the dipeptide is 0.1 to 200 mM and the concentration of the trace metal ion is 0.1 to 400 μM.

5. 2. The composition of claim 1, wherein the dipeptide is Xxx-Cys or Cys-Xxx, where Xxx is a naturally occurring amino acid.

6. 6. The composition of claim 5, wherein the dipeptide is Xxx-Cys or Cys-Xxx and is in an oxidized and dimerized form.

7. The composition of claim 1 further comprising free cysteine.

8. 2. The composition of claim 1, wherein the molar ratio of said dipeptide-bound cysteine ​​to said free cysteine ​​is about 0.1 to 10.

9. A cosmetic product, a dietary supplement, or a nutritional solution for clinical nutrition, comprising a composition according to any one of claims 1 to 8.

10. 9. A cell culture medium comprising the composition of any one of claims 1 to 8, wherein the culture medium further comprises at least one carbohydrate, and / or at least one additional free amino acid, and / or at least one inorganic salt, and / or a buffering agent and / or at least one vitamin.

11. 11. The culture medium of claim 10, wherein the culture medium is in liquid form, gel, powder, granulate, pellet form, or tablet form.

12. The culture medium according to claim 10, wherein the culture medium is in a form that is 2 to 100 times concentrated relative to the concentration of the culture medium used.

13. Use of the culture medium according to claim 10 for cell culture.

14. 14. The use according to claim 13, wherein the cells are selected from the list consisting of CHO cells, COS cells, VERO cells, BHK cells, HEK cells, HELA cells, AE-1 cells, NS0 cells, insect cells, fibroblasts, muscle cells, nerve cells, stem cells, skin cells, endothelial cells, immune cells, and hybridoma cells.

15. 1. A method for producing a cell culture product, comprising: - providing cells capable of producing said cell culture product; - contacting the cells with a culture medium according to claim 10; - obtaining said cell culture product from said culture medium or said cells; A method comprising:

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