Dipeptide salts and their use in cell culture - Patents.com

Abstract

The present invention relates to salts of dipeptides and their use in cell culture.The present invention further relates to a culture medium for culturing cells, preferably plant cells, animal cells or mammalian cells, and the use of the culture medium of the present invention, as well as to a method for producing a cell culture product.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to salts of dipeptides and their use in cell culture. 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 obtained from the methods of using the improved culture media.

[0002] 2. Background of the Invention Chemically defined dipeptides are widely used as precursors of highly soluble and stable amino acids in cell culture media formulation. To increase the solubility of certain limiting amino acids, such as L-tyrosine or L-cystine, highly soluble amino acids, such as glycine, L-alanine, L-proline and L-lysine, can be coupled to form dipeptides, resulting in highly soluble and natural dipeptide precursors of these limiting amino acids. For example, WO 2011 / 133902 discloses cell culture media containing dipeptides, which contain amino acids with low water solubility, in this case tyrosine and cysteine. The authors found that by incorporating tyrosine and cysteine ​​into the dipeptide, the solubility and stability problems of amino acids can 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.

[0003] WO 2012 / 019160 discloses animal cell cultures in which serum-free media is supplemented with a Tyr- and His-containing dipeptide during the production phase. The positive effects of the addition of the Tyr- and His-containing dipeptide on growth and product formation are described.

[0004] L-lysine is particularly suitable as a partner in dipeptide formation to improve the solubility of less soluble amino acids due to its high solubility at neutral pH. This insight has been exploited in the synthesis of highly soluble forms of L-cystine, L-tyrosine, and the branched chain amino acids L-valine, L-leucine, and L-isoleucine. EP 3372671 discloses Lys-containing dipeptides with substantially increased solubility over peptides in which the lysine residues are replaced by L-alanine or L-glycine. These peptides have beneficial effects on cell growth and viability.

[0005] However, in addition to providing highly soluble nutrients in the form of dipeptides, there remains a need to provide these nutrients in a highly biocompatible and stable form. A highly biocompatible form allows these nutrients to be provided in high concentrations to address metabolic or biological processing limitations. Stability must be sufficient to prevent degradation over time in liquid or dry powder media and supplements. Also, sufficient stability is required to transport and store the individual components in dry form, ideally at ambient temperature. Especially in feed media, nutrients are present in higher concentrations. Furthermore, storage media shall contain higher concentrations of soluble nutrients as they are diluted for end use in cell culture. These points have not been fully addressed and there remains a need to improve these parameters.

[0006] 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.

[0007] Surprisingly, it has been found that these parameters can be improved by preparing and using the correct salt forms of dipeptides containing basic amino acids, particularly L-lysine-containing dipeptides.

[0008] Various hydrochloride salts were prepared and compared with basic inner salts of Lys-dipeptides without inorganic counterions, and it was found that the chloride form significantly improved the storage stability of the dry powder.

[0009] We also found that the 2HCl form of L-lysine in peptides was less biocompatible than the 1HCl form at high concentrations in cell culture (this was not due to a pH effect).

[0010] For example, in the case of (Lys-Cys)2, the dihydrochloride (2HCl) form can be provided at a significantly higher concentration than the tetrahydrochloride (4HCl) form. Indeed, high concentrations of the 2HCl form improved cell viability, while the 4HCl form reduced cell viability at the same concentration.

[0011] 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).

[0012] 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.

[0013] 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.

[0014] Preferred embodiments of the present invention are described in further detail in the following detailed description of the invention.

[0015] 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.

[0016] 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.

[0017] The present invention generally relates to a dipeptide salt comprising a dipeptide consisting of two amino acids, the amino acids being natural amino acids, at least one of the amino acids being a basic amino acid and a chloride counterion, wherein the molar ratio of the basic amino acid to the chloride ion is between 0.8 and 1.2.

[0018] In a preferred embodiment of the invention, the dipeptide is Xxx-Yyy or Yyy-Xxx, where Xxx is a basic amino acid and Yyy is another amino acid.

[0019] In a preferred embodiment of the invention, the other amino acid Yyy is selected from cysteine / cystine (Cys) or tyrosine (Tyr).

[0020] In another preferred embodiment, the dipeptide is Xxx-Cys or Cys-Xxx, where the dipeptide salt is in the form (Xxx-Cys)22HCl or (Cys-Xxx)22HCl.

[0021] The basic amino acids are preferably selected from lysine (Lys), arginine (Arg) and histidine (His). The basic amino acids may be at the N-terminal or C-terminal position.

[0022] In a further preferred embodiment, the dipeptide salt has formula I: [ka] The compound is (Lys-Cys)22HCl.

[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] 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)

[0026] 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.

[0027] 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.

[0028] 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.

[0029] The dipeptide salt may be in the form of a solid (crystalline powder, agglomerate, etc.) or may be provided in an aqueous solution. The concentrated stock solution should have a concentration of greater than 25 mM, preferably greater than 100 mM, and most preferably greater than 200 mM.

[0030] The dipeptide salts may also be used with other commonly used dipeptides that do not contain basic amino acids, such as Ala-Gln, Gly-Gln, Ala-Tyr, Gly-Tyr or Ala-Cys or (Ala-Cys)2.

[0031] In the context of the present invention, Cys-peptides that form disulfide bonds through oxidized cysteine ​​residues shall be described by (Xxx-Cys)2 or (Cys-Xxx)2. Peptides may also exist in the form of hydrates. Such dimers of Cys-dipeptides via disulfide bonds, such as (Xxx-Cys)2, are still considered dipeptides in the sense of the present invention.

[0032] Preferably, the composition has a pH value of at least 5, or preferably at least 6, at 25°C.

[0033] In a preferred embodiment, the dipeptide salt is either in the reduced state (=free thiol) or in the oxidized state (=disulfide bond), preferably in the oxidized state.

[0034] 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.

[0035] The present invention also relates to a cosmetic product, a dietary supplement, a nutritional solution for clinical nutrition, or a biopharmaceutical preparation comprising a composition according to the invention.

[0036] The cosmetic product may be a shampoo, conditioner, lotion, cream, or other formulation used to treat the skin or hair. The dietary supplement may be in liquid form, such as a syrup or shot, or in solid form, such as a capsule, softgel, or gummies. The composition may also be part of a nutritional solution for clinical enteral or parenteral nutrition, e.g., an amino acid solution such as Aminoven (Fresenius Kabi). The composition may also be part of a biopharmaceutical formulation, preferably selected from an antibody or vaccine formulation.

[0037] Additionally, the present invention also relates to a cell or tissue culture medium.

[0038] 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.

[0039] In one embodiment of the present invention, the culture medium does not contain growth factors. According to this embodiment, the dipeptide salt of the present invention can be used in place 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.

[0040] According to another embodiment of the invention, the culture medium is in liquid form, gel, powder, granulate, pellet form, or tablet form.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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 5-25 mM. In addition, any hexose, such as galactose, fructose, or mannose, or a combination, may be used.

[0045] 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.

[0046] 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).

[0047] Other inorganic 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).

[0048] 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).

[0049] 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.

[0050] To promote cell growth in serum-free 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.

[0051] In other embodiments, the cell culture medium does not contain polypeptides (i.e., peptides having more than 20 amino acids).

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] Polyanionic or polycationic compounds can be added to the culture medium to prevent cell aggregation and promote growth of cells in suspension.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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, insect cells, fibroblasts, muscle cells, nerve 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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]

[0070] [Figure 1] FIG. 1 shows the effect of various salt forms of (Lys-Cys)2 on the viability of CHO-K1 cells compared to a control with added medium without (Lys-Cys)2. [Diagram 2] FIG. 1 shows the effect of different salt forms of (Lys-Cys)2 on MSC cell viability compared to a control with added medium without (Lys-Cys)2.

[0071] Working Example material: [Table 1] [Table 2]

[0072] method: Preparation of (Lys-Cys)2 salt Peptide synthesis was performed with commonly used protecting groups in solution. The raw product was purified by chromatographic methods, and finally, various salt forms were prepared by ion-exchange chromatography. The chloride-free internal salt (basic form), the dihydrochloride salt (two of the four lysine amino groups were protonated with two chloride ions as counterions), and the tetrachloride form (all four lysine amino groups were protonated with four chloride ions as counterions) were obtained by subsequent lyophilization.

[0073] Therefore, the stock solution of (Lys-Cys)2 was desalted by strong acid IEX. Elution with ammonia allows the generation of the free base of (Lys-Cys)2. Further purification was performed using an adsorption resin. Once the free base was purified, it was dried by lyophilization. Alternatively, the (Lys-Cys)2 free base was desalted by elution with dilute HCl (pH=5) to obtain (Lys-Cys) 2. It can be converted to the 2HCl salt. (Lys-Cys) 2. The dihydrochloride can be isolated by crystallization in EtOH followed by drying.

[0074] To confirm and characterize the 2HCl salt, a titration curve of (Lys-Cys)2 free base with 0.1N HCl was performed. The pH jump confirmed the stoichiometry of 1 mole of (Lys-Cys)2 free base to 2 moles of HCl. A back titration of (Lys-Cys)2.2HCl with 0.1N NaOH also confirmed the stoichiometry.

[0075] (Lys-Cys) by salifying the (Lys-Cys)2 free base with excess HCl using more than 4 equivalents of dilute HCl (pH = 2.0). 2. The 4HCl salt was produced and isolated by crystallization in butanol followed by drying.

[0076] In vitro cytotoxicity assay in mesenchymal stem cells and Chinese hamster ovary cells (subclone K1): The assays were performed with human bone marrow stromal cells, mesenchymal stem cells (MSCs) or Chinese hamster ovary cells (subclone K1), respectively. In the first step, cells were seeded in clear 96-well cell culture plates and incubated for 24 hours in a CO2-incubator (37°C, 5% CO2, 95% humidity) at a cell density of 10,000 cells / well and a final volume of 100 μl / well. After a resting period of 24 hours, the supernatant was discarded and the prepared dipeptide test compounds were added to the cells in a final volume of 100 μl / well. Controls were placed in medium without dipeptide.

[0077] All samples were dissolved directly in the relevant cell culture medium. The pH value of the samples was pre-adjusted to pH 7 to eliminate any effect on cell viability due to potentially lower pH values.

[0078] Various salt forms of (Lys-Cys)2 were tested at two different concentrations, and each sample was tested in triplicate.

[0079] After 24 h of incubation in a CO2-incubator (37°C, 5% CO2, 95% humidity), the assay reagent was prepared according to the manufacturer's manual and 20 μl of the reagent was added to the wells to give a final volume of 120 μl / well.

[0080] Reduction of MTS to a formazan product by metabolically active cells resulted in a formazan absorbance signal that could be measured at 490 nm wavelength in a multiplate reader, which was directly proportional to the number of viable cells in culture.

[0081] Example 1: Effect of different salt forms of Cys-peptides on cell viability CHO-K1 and MSC cells were cultured for 24 hours before adding various (Lys-Cys)2 salts. Cells were cultured in the presence of the dihydrochloride and tetrahydrochloride forms of (Lys-Cys)2, and each peptide was applied at two different concentrations, 1 mM and 10 mM. After 24 hours of culture with the dipeptides, cell viability was assessed using the CellTiter 96® aqueous non-radioactive cell proliferation assay (MTS). The results of these viability assays for CHO-K1 and MSC cells are shown in Figures 1 and 2.

[0082] Figure 1 shows the effect of various salt forms of (Lys-Cys)2 on the viability of CHO-K1 cells compared to a control with added medium without (Lys-Cys)2. Error bars represent standard deviation.

[0083] Figure 2 shows the effect of different salt forms of (Lys-Cys)2 on the viability of MSC cells compared to a control with added medium without (Lys-Cys)2. Error bars represent standard deviation.

[0084] Within these viability assessments, peptides containing basic amino acids where the basic amino acids are fully protonated and have two chloride counterions were found to be less biocompatible in cell culture at high concentrations (and this was not caused by pH effects) than peptides containing basic amino acids where the basic amino acids are only partially protonated and have one chloride counterion. For example, in the case of (Lys-Cys)2, the dihydrochloride (2HCl) form could be provided at significantly higher concentrations than the tetrahydrochloride (4HCl) form. Indeed, the 2HCl form at high concentrations improved cell viability, while the 4HCl form reduced cell viability at the same concentrations.

[0085] Example 2: Improved storage stability of the chloride salt versus the base form Storage stability studies at 25°C and 60°C were performed on various salt forms of (Lys-Cys)2. Stability was measured by HPLC method. Both the dihydrochloride and tetrahydrochloride salts were found to be significantly more stable than the chloride-free base form.

Claims

1. A dipeptide salt comprising a dipeptide consisting of two amino acids, wherein the amino acids are natural amino acids, and at least one amino acid is a basic amino acid and a chloride counterion, wherein the molar ratio of the basic amino acid to the chloride ion is 0.8 to 1.

2.

2. 2. The dipeptide salt of claim 1, wherein the dipeptide is Xxx-Yyy or Yyy-Xxx, where Xxx is a basic amino acid and Yyy is another amino acid.

3. 3. The dipeptide salt of claim 2, wherein the further amino acid Yyy is selected from cysteine / cystine (Cys) or tyrosine (Tyr).

4. The dipeptide is Xxx-Cys or Cys-Xxx, and the dipeptide salt is (Xxx-Cys) ２ 2HCl or (Cys-Xxx) ２ 2. The dipeptide salt of claim 1 in the form of dihydrochloride.

5. 2. The dipeptide salt of claim 1, wherein the basic amino acid is selected from lysine (Lys), arginine (Arg) and histidine (His).

6. The dipeptide salt is of Formula I: 【Chemical 1】 (Lys-Cys) ２ 2. The dipeptide salt of claim 1, wherein the dipeptide salt is 2HCI.

7. 7. A cosmetic product, a dietary supplement, a nutritional solution for clinical nutrition or a biopharmaceutical preparation comprising a dipeptide salt according to any one of claims 1 to 6.

8. 7. A cell culture medium comprising a dipeptide salt according to claim 1, further comprising at least one carbohydrate, and / or at least one additional free amino acid, and / or at least one inorganic salt, and / or a buffer and / or at least one vitamin.

9. 9. The culture medium of claim 8, wherein the culture medium is in liquid form, gel, powder, granule, pellet form, or tablet form.

10. 9. The culture medium according to claim 8, wherein the culture medium is in a form that is 2 to 100 times concentrated relative to the concentration of the culture medium used.

11. Use of the culture medium according to claim 8 for culturing cells.

12. 12. The use according to claim 11, 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, insect cells, fibroblasts, muscle cells, neuronal cells, stem cells, skin cells, endothelial cells, immune cells such as NK cells or T cells, and hybridoma cells.

13. 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 8; - obtaining said cell culture product from said culture medium or said cells; A method comprising:

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