Photostabilized composition and method for stabilizing a photosensitive component

Abstract

The present invention relates to a photo-stabilized composition containing a water-soluble tyrosine-containing compound having a molecular weight of less than 1 kDa and at least one photosensitive component, and a method for stabilizing a photosensitive component including the addition of a tyrosine-containing dipeptide.

Description

Technical Field

[0001] The present invention relates to a photo-stabilized composition comprising a water-soluble tyrosine-containing compound having a molecular weight of less than 1 kDa and at least one photosensitive component, and a method for stabilizing a photosensitive component including the addition of a tyrosine-containing dipeptide.

Background Art

[0002] Many organic molecules are sensitive to degradation induced by electromagnetic radiation in the ultraviolet and visible spectra. Examples include certain vitamins and amino acids, but also macromolecules such as proteins consisting of amino acids and oligonucleotides such as DNA and mRNA. This poses a problem for the use of these molecules in pharmaceutical, nutritional, or cosmetic applications where it is necessary to avoid product changes and degradation in order to ensure product safety and efficacy, for example.

[0003] Electromagnetic radiation can interact directly with photosensitive molecules to cause decomposition reactions, or photo-chemically excited molecules can initiate various types of oxidation reactions that also contribute to the formation or decomposition of unwanted products (Baptista et al., Photochemistry and Photobiology, 2021, 97:1456 - 1483).

[0004] Many photosensitized oxidation reactions involve the formation of reactive oxygen species (ROS). ROS react with biomolecules including DNA, lipids, proteins, and individual amino acids. The photosensitivity of amino acids, especially tryptophan (Trp), tyrosine (Tyr), histidine (His), methionine (Met), and cystine (Cys) has been studied in detail. These molecules are photo-oxidized by the combined action of light and oxygen, causing oxidation and hydrolysis (Rumecal & McNeill, Environ. Sci Technol. 2011, 45:5230 - 5237).

[0005] The photosensitivity of amino acids in protein pharmaceuticals formulations is an unsolved problem. When formulating cell culture media, low photo-stability is also an issue. Amino acids are the main components of cell culture media or feed used in the manufacturing process of biopharmaceuticals. During production, storage, transportation, rehydration, and / or the cell culture process itself (reaction vessels exposed to light), the cell culture media or feed may be inadvertently exposed to harmful light. The photodegradation process decomposes sensitive media components such as amino acids, especially Trp, Tyr, His, Met, and Cys, and the decomposition products accumulate. This reactivity makes the cell culture media or feed unstable and causes color changes upon long-term storage. In this case, the undesirable browning of the cell culture media or feed and the generation of toxic decomposition products in cell lines are mainly promoted by Trp (Schnellbaecher et al., Int. J. Mol. Sci. 2021, 22:6221).

[0006] Cell culture media or feed is a mixture of various components including photosensitizers such as riboflavin (vitamin B2). Photosensitizers promote the photosensitized oxidation reaction of sensitive compounds in cell culture media or feed, and the most sensitive compounds are Trp and Tyr, which are amino acids (Wu & McCormick, Biochim. Biophys. Acta 1971, 236:479-486).

[0007] Since molecules in solution are particularly sensitive, it is necessary to protect them from light, such as storing them in the dark or using appropriate packaging. Since this is not always possible, photo-stabilizers and radical scavengers have been developed to address various applications where the product is exposed to light (such as plastics) (Yousif & Haddad, SpringerPlus 2013, 2:398).

[0008] However, for cosmetics, nutritional agents, or pharmaceutical preparations that come into direct contact with the body or isolated cells and tissues, it is important that the photo stabilizer has high biocompatibility and low toxicity and is ideally easily metabolized (Kawabata et al., Pharmaceuticals 2020, 13:135).

[0009] Amino acids can perform their functions to some extent, but not completely.

Prior Art Documents

Non-Patent Documents

[0010]

Non-Patent Document 1

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[0011] Summary of the Invention Therefore, there is still a need for improved photo stabilizers for stabilizing photosensitive components in nutrition, cosmetics, cell culture media / feed, and pharmaceutical preparations. Accordingly, an object of the present invention was to provide a method for stabilizing photosensitive components, particularly photosensitive amino acids, in an aqueous solution.

[0012] Surprisingly, when tyrosine derivatives are added to an aqueous solution containing tryptophan or riboflavin, and to cell culture media or feeds, it has been found that they reduce or prevent their browning, and as a result, contribute to photo-stabilization. In particular, the addition of Tyr-dipeptides resulted in a decrease in color development.

[0013] Accordingly, the present invention relates to a photo-stabilizing composition comprising a water-soluble tyrosine-containing compound having a molecular weight of less than 1 kDa and at least one photosensitive component, wherein the water-soluble tyrosine-containing compound is present at a concentration of at least 0.5 mM and the composition is a culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0014]

Figure 1

Figure 2

[0015] In a preferred embodiment, the culture medium is a cell culture medium selected from a basal medium, a feeding medium or a perfusion medium.

[0016] The present invention further relates to a method for stabilizing a photosensitive component, comprising adding a tyrosine-containing dipeptide selected from Xxx-Tyr or Tyr-Xxx, wherein Xxx is a natural amino acid and the dipeptide is present at a concentration of at least 0.5 mM.

[0017] Another aspect of the present invention relates to the use of tyrosine-containing dipeptides for stabilizing photosensitive components in cell culture, cosmetics, food applications, parenteral nutrition or pharmaceutical formulations.

[0018] Preferred embodiments of the present invention are described in more detail in the following detailed description of the present invention.

[0019] "Peptide" should be understood as a molecule containing at least two amino acids covalently bonded to each other by an alpha-peptide bond (R 1 -CO-NH-R 2 ).

[0020] "Dipeptide" should be understood as a molecule containing two amino acids covalently bonded to each other by a peptide bond (R 1 -CO-NH-R 2 ) and may exist in the form of salts or hydrates.

[0021] In the context of the present invention, "amino acid" is to be understood as a molecule containing an amino functional group (-NH 2 ) and a carboxylic acid functional group (-COOH), together with a side chain specific to each amino acid. Both alpha-amino acids and beta-amino acids are included in the context of the present invention. Preferred amino acids of the present invention are alpha-amino acids, in particular the 20 "natural amino" acids including cystine below. 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) Valine (Val / V)

[0022] In the context of the present invention, the expression "natural amino acid" should be understood to include both the L-form and the D-form of the 20 amino acids listed above. However, the L-form is preferred. In one embodiment, the term "amino acid" also includes analogs or derivatives of those amino acids.

[0023] The "free amino acid" (e.g., "free cysteine") according to the present invention is understood to be an amino acid having its amino and its (alpha-) carboxylic acid functional groups in free form, i.e., not covalently bonded to other molecules, e.g., not forming a peptide bond. The free amino acid can also exist as a salt or in a hydrated form. When referring to an amino acid that is part of or within a dipeptide, this should be understood to refer to that part of the respective dipeptide structure that is derived from the respective amino acid, according to known mechanisms of biochemistry and peptide biosynthesis.

[0024] The "growth factor" according to the present invention should be understood as any naturally occurring substance that can stimulate cell growth, proliferation, and cell differentiation. Preferred growth factors are in the form of proteins or steroid hormones. According to one embodiment of the present invention, the expression "growth factor" refers to fibroblast growth factor (FGF) including acidic FGF and basic FGF, insulin, insulin-like growth factor (IGF), epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), and transforming growth factor (TGF) including TGF alpha and TGF beta, cytokines such as interleukin 1, 2, 6, granulocyte stimulating factor, and leukocyte inhibitory factor (LIF).

[0025] The "culture medium" according to the present invention is a liquid or solid medium containing nutrients, and it is understood that the culture medium, except for the medium for parenteral nutrition, is suitable for providing nutrients to the cells in the culture and supporting the life and / or formation of products. The cultured cells according to the present invention can be bacterial cells, yeast cells, fungal cells, animal cells such as mammalian cells or insect cells, and / or plant cells, such as algae. Typically, the culture medium provides essential and non-essential amino acids, vitamins, at least one energy source, lipids, and trace elements, all of which are required by the cells to maintain life, growth, and / or product formation. The culture medium may also contain components that enhance growth and / or survival beyond a minimum rate, including hormones and growth factors. The culture medium preferably has a pH and salt concentration that support the lifespan, growth, and / or product formation of the cells. The culture medium according to the present invention preferably contains all the nutrients necessary to maintain the lifespan and proliferation of the cell culture. A preferred culture medium is a defined medium.

[0026] The "chemically defined medium" according to the present invention is a medium that does not contain cell extracts, cell hydrolysates, or protein hydrolysates. A chemically defined medium does not contain components of unknown composition. As generally understood by those skilled in the art, a chemically defined medium usually does not contain animal-derived components. All components of a chemically defined medium have known chemical structures. Culture media other than the defined culture medium may be referred to as "complex" culture media.

[0027] "Cell culture medium" should be understood as a culture medium suitable for maintaining the life, growth and / or product formation of animal cells and / or plant cells.

[0028] "Basal medium" or "basal culture medium" should be understood as a solution or substance containing nutrients from which cell culture is initiated.

[0029] "Feeding medium" should be understood as a solution or substance supplied to cells after the start of the culture process. In certain embodiments, the feeding medium contains one or more components not present in the basal medium. The feeding medium can also lack one or more components present in the basal medium. Preferably, the concentration of nutrients in the feeding medium exceeds the concentration in the basal medium to avoid loss of productivity due to dilution.

[0030] "Perfusion medium" should be understood as a solution or substance containing nutrients that are continuously added after the start of cell culture in which harvest is continuously removed.

[0031] The compositions and methods according to the present invention improve the photo-stability of photosensitive components. One of the negative aspects of limited photo-stability is, for example, the brown color development promoted by tryptophan. In this context, photo-stability can be characterized, for example, by color change under exposure to sunlight during the day.

[0032] In a preferred configuration, the water-soluble tyrosine-containing compound is present at a concentration of at least 1 mM, or at least 2 mM, or at least 5 mM, or at least 10 mM, or at least 20 mM.

[0033] In another preferred embodiment, the water-soluble tyrosine-containing compound is selected from phosphorylated tyrosine or a dipeptide, preferably Ala-Tyr or Gly-Tyr.

[0034] In another preferred embodiment, the photosensitive component is selected from aromatic compounds, preferably selected from riboflavin and aromatic amino acids or derivatives thereof, preferably selected from tryptophan and tyrosine.

[0035] In a preferred configuration of the present invention, the cell culture medium further contains free tyrosine.

[0036] The photosensitive component according to the present invention is selected from aromatic compounds, preferably selected from riboflavin and aromatic amino acids or derivatives thereof, preferably selected from tryptophan and tyrosine. These components are known to contribute to color development under exposure.

[0037] In a preferred configuration, the derivative of the aromatic amino acid is a soluble derivative of tryptophan or tyrosine, preferably selected from phosphorylated tyrosine.

[0038] In a specific configuration of the present invention, the photosensitive component is present in an amount of at least 0.5 mM, preferably at least 1 mM, more preferably at least 2 mM.

[0039] Another aspect of the present invention relates to a method for stabilizing a photosensitive component.

[0040] In a preferred embodiment of the method according to the present invention, the photosensitive component is present in an aqueous solution.

[0041] In another preferred embodiment, the dipeptide is selected from Ala-Tyr or Gly-Tyr.

[0042] In another preferred embodiment, the photosensitive component is selected from aromatic compounds, preferably selected from riboflavin and aromatic amino acids or derivatives thereof, preferably selected from tryptophan and tyrosine, and more preferably selected from tryptophan.

[0043] In another preferred configuration of the method according to the present invention, the molar ratio of dipeptide to tryptophan is from about 10:1 to 1:10, preferably from about 5:1 to 1:5.

[0044] In another preferred embodiment, the dipeptide is added to the aqueous solution at a concentration of at least 1 mM, or at least 2 mM, or at least 5 mM, or at least 10 mM, or at least 20 mM.

[0045] In another preferred embodiment, a cysteine derivative, preferably a cysteine-containing dipeptide or cysteine salt, preferably the sodium S-sulfate salt of cysteine, is added.

[0046] In another preferred embodiment, the aqueous solution is a cell culture medium, and the cultured cells are cultured in the presence of a tyrosine-containing dipeptide.

[0047] The culturing of cells according to the present invention can be carried out by batch culture, fed-batch culture or continuous culture.

[0048] In a preferred embodiment, the culture medium further comprises at least one carbohydrate, at least one free amino acid, at least one inorganic salt, a buffer and / or at least one vitamin.

[0049] 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.

[0050] In a preferred embodiment, the culture medium of the present invention is a defined medium or a serum-free medium. For example, the composition for intervention can be supplemented with Freestyle™ F17 medium, Freestyle™ 293 medium, all Expi293™ medium (Waltham, USA) from Gibco™ ThermoFisher, TheraPEAK™ SfAAV™ medium (Basel, Switzerland) from LONZA, HEK ViP NB from Sartorius Xell (Bielefeld, Germany), HyClone™ SFM4HEK293 (Marlborough, USA) from Cytiva. The dipeptide of the present invention may be supplemented to DMEM medium (Life Technologies Corp., Carlsbad, USA). However, the present invention is not limited to the supplementation of the above media.

[0051] The cell culture medium of the present invention (cell or tissue culture basal medium, feeding medium or perfusion medium) preferably may contain all nutrients necessary for sustained growth and product formation. Recipes for preparing culture media, particularly cell culture media, 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.

[0052] 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 typically supplemented at 5-25 mM. Additionally, any hexose such as galactose, fructose, or mannose, or combinations thereof, can be used.

[0053] The culture medium may typically also contain at least essential amino acids (i.e., His, Ile, Leu, Lys, Met, Phe, Thr, Try, Val) as well as non-essential amino acids. When a cell line is unable to synthesize amino acids or cannot produce sufficient amounts of amino acids to support maximum growth, non-essential amino acids are typically included in the cell culture medium. Additionally, mammalian cells can also use glutamine as a major energy source. Glutamine is often included at a higher concentration than other amino acids (2-8 mM). However, as noted above, glutamine can spontaneously degrade to form ammonia, and certain cell lines produce ammonia more rapidly, which is toxic.

[0054] The culture medium of the present invention may preferably contain salts. Salts are added to cell culture media to maintain isotonic conditions and prevent osmotic imbalances. The weight osmolality of the culture medium of the present invention is about 300 mOsm / kg, although many cell lines can tolerate variations of about 10 percent or more from this value. The weight osmolality of some insect cell cultures tends to be higher than 300 mOsm / kg, which may be 0.5 percent, 1 percent, 2-5 percent, 5-10 percent, 10-15 percent, 15-20 percent, 20-25 percent, 25-30 percent higher than 300 mOsm / kg. The most commonly used salts in cell culture media include Na + , K + , Mg 2+ , Ca 2+ , Cl - , SO 4 2- , PO 4 3- , and HCO 3 -(For example, CaCl 2 , KCl, NaCl, NaHCO 3 , Na 2 HPO 4 ) is included.

[0055] 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 are CaCl 2 , Fe(NO 3 ) 3 , MgCl 2 , MgSO 4 , MnCl 2 , NaCl, NaHCO 3 , Na 2 HPO 4 and other salts, as well as selenium, vanadium and zinc and other trace elements in the form of ions. These inorganic salts and trace elements can be commercially obtained, for example, from Sigma (Saint Louis, Missouri).

[0056] 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 vary greatly, but generally, when the cell culture medium contains little or no serum, or when cells are grown at high density, extra vitamins are required. 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, B12, C, D3, E, K and p-aminobenzoic acid (PABA).

[0057] The culture medium of the present invention may also contain serum. Serum is the supernatant of clotted blood. Serum components include attachment factors, micronutrients (e.g., trace elements), growth factors (e.g., hormones, proteases), and protective elements (e.g., antitoxins, antioxidants, antiproteases). Serum is available from a variety of animal sources, including human, bovine, or equine 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). Preferred cell culture media are serum-free.

[0058] One or more lipids such as linoleic acid, linolenic acid, arachidonic acid, palmitoleic acid, oleic acid, polyunsaturated fatty acids, and / or fatty acids having 12, 14, 16, 18, 20, 22, or 24 carbon atoms (each carbon atom being branched or unbranched), phospholipids, lecithin (phosphatidylcholine), and cholesterol can also be added to the cell culture medium of the present invention. One or more of these lipids can be included as adjunct substances in serum-free media. Phosphatidic acid and lysophosphatidic acid stimulate the growth of certain substrate-dependent cells such as MDCK, mouse epithelium, and other kidney cell lines, and phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol stimulate the growth of human fibroblasts in serum-free media. Ethanolamine and cholesterol have also been shown to promote the growth of certain cell lines. In certain embodiments, the cell culture medium does not contain lipids.

[0059] One or more carrier proteins such as bovine serum albumin (BSA) or transferrin can also be added to the cell culture medium. Carrier proteins can serve to transport specific nutrients or trace elements. BSA is typically used as a carrier for lipids such as linoleic acid and oleic acid that are insoluble in aqueous solutions. Additionally, BSA can 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 can be used as lipid carriers.

[0060] To help promote the attachment of anchorage-dependent cells to the substrate, one or more attachment proteins such as fibronectin, laminin, and propeptin can also be added to the cell culture medium.

[0061] The cell culture medium can 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, phosphates, bicarbonates, and other buffers suitable for use in cell culture applications. A suitable buffer is one that provides buffering capacity without substantial cytotoxicity to the cultured cells. The selection of a suitable buffer is within the purview of those skilled in the art of cell culture.

[0062] A polyanionic or polycationic compound can be added to the culture medium to prevent cell aggregation and promote the growth of cells in suspension.

[0063] In a preferred embodiment, the culture medium is in liquid form. However, the culture medium can also be a solid medium such as a gel-like medium, for example, an agar, carrageenan, or gelatin-containing medium (powder, aggregated powder, instantized powder, etc.). Preferably, the culture medium is in a sterile form.

[0064] The culture medium of the present invention can be in a concentrated form. It can be, for example, in a 2-fold to 100-fold concentrated form (relative to the concentration that supports cell growth and product formation), preferably in a 2-fold, 3-fold, 3.33-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold concentrated form. Such a concentrated culture medium serves to prepare a culture medium for use by diluting the concentrated culture medium with an aqueous solvent such as water. Such a concentrated culture medium can be used in batch culture, but is also advantageously used in fed-batch or continuous culture where a concentrated nutrient composition is added to the ongoing culture of cells, for example, to replenish nutrients consumed by the cells during culture.

[0065] In other embodiments of the present 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.

[0066] Another aspect of the present invention relates to the use of tyrosine-containing dipeptides for stabilizing photosensitive components in cell culture, cosmetics, food applications, parenteral nutrition or pharmaceutical formulations.

[0067] In a preferred configuration, the use is as a cell culture medium, preferably a basal medium, a feeding medium or a perfusion medium, for stabilizing photosensitive components in cell culture.

[0068] In a preferred embodiment, the cells are selected from the group consisting of CHO cells, COS cells, VERO cells, BHK cells, HEK cells, HELA cells, AE-1 cells, NS0 cells, insect cells, algal cells, fibroblasts, muscle cells, nerve cells, stem cells, skin cells, endothelial cells, immune cells such as NK or T cells, and hybridoma cells.

Example

[0069] Materials:

Table 1

[0070]

Table 2

[0071] Method: Preparation of test samples An aqueous solution of 2.5 mM L-tryptophan (Trp) and 0.013 mM riboflavin was prepared in phosphate-buffered saline (D-PBS) and minimum essential medium (MEM). Using these solutions, the dipeptides L-alanyl-L-tyrosine dihydrate (Ala-Tyr) and glycyl-L-tyrosine dihydrate (Gly-Tyr) and the amino acids L-tyrosine (Tyr), L-alanine (Ala), and glycine (Gly) were dissolved at several concentrations to make solvent mixtures. In Example 1, the dipeptides were tested at concentrations of 0.5 mM, 2.5 mM, and 10 mM, and Tyr was tested at a concentration of 0.5 mM. PBS and MEM, and 2.5 mM Trp dissolved in PBS and MEM were used as controls. In Example 2, the dipeptides and Tyr were tested at concentrations of 2 mM, Ala and Gly were tested at a concentration of 32 mM, and the solvent (PBS or MEM) was used as a control ("solvent background").

[0072] Detection of brown color The samples were exposed to electromagnetic radiation (sunlight) at room temperature for 7 days (Example 2) and 14 days (Example 1). Relative quantification of the brown color of the samples in Example 1 was obtained by measuring the absorbance at 330 nm. The intensity of the brown color of the samples in Example 2 was visually determined using a brown index. The numbers from 1 to 5 correspond to the intensity of the brown color of the solution (0, no brown; 1, weak brown; 2, medium brown; 3, normal brown; 4, dark brown; 5, strong brown).

[0073] Example 1: Reduction in the brownness of tryptophan solutions containing Tyr derivatives When a non-colored aqueous solution of PBS or MEM containing 2.5 mM Trp is exposed to electromagnetic radiation at room temperature, a strong color change to brown occurs. The addition of 0.5 mM Tyr or Tyr-dipeptides (Ala-Tyr or Gly-Tyr) has been shown to reduce this coloring, especially with both Tyr-dipeptides having a greater effect. If the concentration of the dipeptide is up to 10 mM, it exceeds the solubility of Tyr and further reduces or completely prevents the color change. The results are shown in Figures 1 and 2.

[0074] Figure 1 shows a comparison of the effects of PBS alone and Trp dissolved in PBS on the browning of various aqueous solutions containing Trp and Tyr derivatives dissolved in phosphate-buffered saline (PBS). The intensity of brown color was measured as the difference in absorbance at 330 nm.

[0075] Figure 2 shows a comparison of the effects of MEM alone and Trp dissolved in MEM on the browning of various aqueous solutions containing Trp and Tyr derivatives dissolved in minimum essential medium (MEM). The intensity of brown color was measured as the difference in absorbance at 330 nm.

[0076] Example 2: Reduction in the degree of browning of riboflavin solutions containing Tyr derivatives An aqueous solution of PBS or MEM containing 0.013 mM riboflavin showed a weak fluorescent yellow-green color. When exposed to electromagnetic radiation at room temperature for 7 days, the MEM solution underwent browning, while the yellow-green color in the PBS solution disappeared. The intensity of brown was classified as "normal brown" based on the applied browning index.

[0077] Similarly, control solutions of MEM further supplemented with 0.013 mM riboflavin and amino acids such as Ala and Gly at concentrations up to 32 mM also changed color to "normal brown", while the same control solutions in PBS remained colorless. Other control solutions of PBS and MEM without riboflavin were colorless on day 0. When irradiated with electromagnetic radiation at room temperature for 7 days, the PBS solution remained colorless, while the MEM solution changed to a "weak brown" color. The same was observed for control solutions without riboflavin and further supplemented with amino acids such as Ala and Gly at concentrations up to 32 mM.

[0078] Addition of any type of aqueous solution of aromatic amino acids such as Tyr (2 mM) in combination with exposure to electromagnetic radiation for 7 days at room temperature was found to result in the "strong brown" coloring of all solutions. Interestingly, compared to this "strong brown", it was shown that when 2 mM of Tyr was replaced equimolarly with 2 mM of Tyr-dipeptides such as Ala-Tyr and Gly-Tyr, the brown color clearly decreased towards the intensity classified as "normal brown".

[0079]

Table 3

Claims

**Claim 1** A photo-stabilizing composition comprising a water-soluble tyrosine-containing compound having a molecular weight of less than 1 kDa and at least one photosensitive component, wherein the water-soluble tyrosine-containing compound is present at a concentration of at least 0.5 mM, and the composition is a culture medium. **Claim 2** The composition according to claim 1, wherein the culture medium is a cell culture medium selected from a basal medium, a feeding medium, or a perfusion medium. **Claim 3** The composition according to claim 1 or 2, wherein the water-soluble tyrosine-containing compound is present at a concentration of at least 1 mM, or at least 2 mM, or at least 5 mM, or at least 10 mM, or at least 20 mM. **Claim 4** The composition according to any one of claims 1 to 3, wherein the water-soluble tyrosine-containing compound is selected from phosphorylated tyrosine or a dipeptide, preferably Ala-Tyr or Gly-Tyr. **Claim 5** The composition according to any one of claims 1 to 4, wherein the photosensitive component is selected from aromatic compounds, preferably selected from riboflavin and aromatic amino acids or derivatives thereof, preferably selected from tryptophan and tyrosine. **Claim 6** The composition according to any one of claims 1 to 5, wherein the photosensitive component is present in an amount of at least 0.5 mM, preferably at least 1 mM, more preferably at least 2 mM. **Claim 7** A method for stabilizing a photosensitive component, comprising adding a tyrosine-containing dipeptide selected from Xxx-Tyr or Tyr-Xxx, wherein Xxx is a natural amino acid, and the dipeptide is present at a concentration of at least 0.5 mM. **Claim 8** The method according to claim 7, wherein the photosensitive component is present in an aqueous solution. **Claim 9** The method according to claim 7 or 8, wherein the dipeptide is selected from Ala-Tyr or Gly-Tyr. **Claim 10** The method according to any one of claims 7 to 9, wherein the photosensitive component is selected from aromatic compounds, preferably selected from riboflavin and aromatic amino acids or derivatives thereof, preferably selected from tryptophan and tyrosine, more preferably selected from tryptophan. **Claim 11** The method according to claim 10, wherein the molar ratio of the dipeptide to tryptophan is from about 10:1 to 1:10, preferably from about 5:1 to 1:

5. **Claim 12** The method according to any one of claims 7 to 11, wherein the dipeptide is added to the aqueous solution at a concentration of at least 1 mM, or at least 2 mM, or at least 5 mM, or at least 10 mM, or at least 20 mM.

13. The method according to any one of claims 7 to 12, wherein a cysteine derivative selected preferably from a cysteine-containing dipeptide or a cysteine salt, preferably the sodium S-sulfate salt of cysteine, is added.

14. The method according to any one of claims 7 to 13, wherein the aqueous solution is a cell culture medium and the cultured cells are cultured in the presence of the tyrosine-containing dipeptide.

15. Use of a tyrosine-containing dipeptide for stabilizing a photosensitive component in cell culture, cosmetics, food applications, parenteral nutrition or pharmaceutical formulations.

16. Use according to claim 15, as a cell culture medium, preferably a basal medium, a feeding medium or a perfusion medium, for stabilizing a photosensitive component in cell culture.

17. The use according to claim 16, wherein the cells are selected from the group consisting of CHO cells, COS cells, VERO cells, BHK cells, HEK cells, HELA cells, AE-1 cells, NS0 cells, insect cells, algal cells, fibroblasts, muscle cells, nerve cells, stem cells, skin cells, endothelial cells, immune cells such as NK or T cells, and hybridoma cells.

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