Photostable compositions and methods for stabilizing photosensitive components

Abstract

The present invention relates to a photostable composition comprising a water-soluble tyrosine-containing compound having a molecular weight of less than 1 kDa and at least one light-sensitive component, and a method of stabilizing a light-sensitive component comprising the addition of a tyrosine-containing dipeptide.

Description

[0001] The present invention relates to a photostable composition comprising a water-soluble tyrosine-containing compound having a molecular weight below 1 kDa and at least one photosensitizing ingredient, and to a method for stabilizing photosensitizing ingredients comprising adding a tyrosine-containing dipeptide.

[0002] Many organic molecules are sensitive to degradation caused by electromagnetic radiation in the UV and visible spectrum. Examples include certain vitamins and amino acids, but also macromolecules such as proteins composed of amino acids or oligonucleotides such as DNA or mRNA. This poses a challenge to the use of those molecules, for example in pharmaceutical, nutritional or cosmetic applications where product changes and degradation need to be avoided to ensure product safety and efficacy.

[0003] Electromagnetic radiation either interacts directly with photosensitive molecules, leading to degradation reactions, or photochemically excited molecules trigger various types of oxidation reactions, promoting the formation of unwanted products or even degradation (Baptista et al., Photochemistry and Photobiology, 2021, 97: 1456-1483).

[0004] Many photosensitive oxidation reactions involve the formation of reactive oxygen species (ROS). ROS react with biomolecules, including DNA, lipids and proteins, as well as 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 photooxidized due to the combined action of light and oxygen, which leads to oxidation and hydrolysis (Rumecal & McNeill, Environ. Sci Technol. 2011, 45: 5230-5237).

[0005] The photosensitivity of amino acids in protein drug product formulations is a problem that has not been fully resolved. Limited light stability is also a challenge when formulating cell culture media. Amino acids are the main components of cell culture media / feeds used in biopharmaceutical production processes. During production, storage, transportation, rehydration and / or the cell culture process itself (reaction vessel exposed to light), cell culture media / feeds may be accidentally exposed to harmful light. The photodegradation process decomposes sensitive culture medium components, such as amino acids, especially Trp, Tyr, His, Met and Cys, resulting in the accumulation of degradation products. This reactivity leads to color changes in cell culture media / feeds after instability and long-term storage. In this term, Trp mainly promotes the process of unwanted cell culture media / feed browning and the production of toxic degradation products of cell lines (Schnellbaecher et al., Int. J. Mol. Sci. 2021, 22: 6221).

[0006] Cell culture medium / feed is a mixture of various ingredients, including photosensitizers such as riboflavin (vitamin B2). Photosensitizers facilitate photooxidative reactions of sensitive cell culture medium / feed compounds, the most sensitive of which are the amino acids Trp and Tyr (Wu & McCormick, Biochim. Biophys. Acta 1971, 236: 479-486).

[0007] Molecules in solution are particularly sensitive and need to be protected from light by storing them in the dark or using appropriate packaging. Since this is not always possible, light stabilizers and free radical scavengers have been developed for various applications (plastics, etc.) where the product is exposed to light (Yousif & Haddad, Springer Plus 2013, 2:398).

[0008] However, when it comes to cosmetic, nutritional or pharmaceutical formulations that come into direct contact with the body or with isolated cells and tissues, it is important that the photostabilizers are highly biocompatible, non-toxic, and ideally easily metabolized (Kawabata et al., Pharmaceuticals 2020, 13:135).

[0009] Amino acids can fulfill this function to some extent, but not completely.

[0010] Therefore, there is still a need for improved light stabilizers to stabilize light-sensitive ingredients in nutrition, cosmetics, cell culture media / feeds and pharmaceutical product formulations. It is therefore an object of the present invention to provide a method for stabilizing light-sensitive ingredients, especially light-sensitive amino acids, in aqueous solutions.

[0011] Surprisingly, it was found that the addition of tyrosine derivatives to aqueous solutions containing tryptophan or riboflavin and to cell culture media / feeds reduces or even prevents their browning and thus contributes to photostability. In particular, the addition of Tyr dipeptides results in a reduction in color formation.

[0012] Therefore, the present invention relates to a photostable composition comprising a water-soluble tyrosine-containing compound having a molecular weight below 1 kDa and at least one photosensitizing component, wherein the water-soluble tyrosine-containing compound is present in a concentration of at least 0.5 mM, and wherein the composition is a culture medium.

[0013] In a preferred embodiment, the culture medium is a cell culture medium selected from a basal medium, a feed medium or a perfusion medium.

[0014] Furthermore, the present invention 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, wherein the dipeptide is present in a concentration of at least 0.5 mM.

[0015] Another aspect of the present invention relates to the use of a tyrosine-containing dipeptide for stabilizing photosensitive ingredients in cell culture, cosmetics, food applications, parenteral nutrition or pharmaceutical product formulations.

[0016] Preferred embodiments of the present invention are described in further detail in the following detailed description of the invention.

[0017] A "peptide" is understood to be a molecule comprising at least two amino acids covalently linked to each other via an alpha peptide bond (R1-CO-NH-R2).

[0018] A "dipeptide" is understood to be a molecule comprising two amino acids covalently linked to one another via a peptide bond (R1-CO-NH-R2), which may also exist in the form of a salt or a hydrate.

[0019] In the context of the present invention, "amino acid" is understood to be a molecule comprising an amino functional group (-NH2) and a carboxylic acid functional group (-COOH) as well as side chains specific to the respective amino acids. In the context of the present invention, α and β amino acids are included. Preferred amino acids of the present invention are α amino acids, in particular the following 20 "natural amino acids", including cystine:

[0020] Alanine (Ala / A)

[0021] Arginine (Arg / R)

[0022] Asparagine (Asn / N)

[0023] Aspartic acid (Asp / D)

[0024] Cysteine ​​(Cys / C)

[0025] Cystine (Cyss / C2)

[0026] Glutamate (Glu / E)

[0027] Glutamine (Gin / Q.)

[0028] Glycine (Gly / G)

[0029] Histidine (His / H)

[0030] Isoleucine (Ile / I)

[0031] Leucine (Leu / L)

[0032] Lysine (Lys / K)

[0033] Methionine (Met / M)

[0034] Phenylalanine (Phe / F)

[0035] Proline (Pro / P)

[0036] Serine (Ser / S)

[0037] Threonine (Thr / T)

[0038] Tryptophan (Trp / W)

[0039] Tyrosine (Tyr / Y)

[0040] Valine (Val / V)

[0041] In the context of the present invention, the expression "natural amino acid" is understood to include 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.

[0042] According to the invention, a "free amino acid" (e.g. "free cysteine") is understood to be an amino acid which has its amino group and its (α-)carboxyl function in free form, i.e. not covalently bound to other molecules, e.g. not forming peptide bonds. A free amino acid may also be present in the form of a salt or in the form of a hydrate. When referring to an amino acid which is part of a dipeptide or an amino acid in a dipeptide, this is to be understood as referring to that part of the respective dipeptide structure which is derived from the respective amino acid according to the known biochemical and peptide biosynthetic mechanisms.

[0043] According to the present invention, "growth factor" 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" should be interpreted as referring to a growth factor selected from the list consisting of: fibroblast growth factor (FGF) (including acidic FGF and basic FGF), insulin, insulin-like growth factor (IGF), epithelial growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), and transforming growth factor (TGF) (including TGFα and TGFβ), cytokines (such as interleukin 1, 2, 6), granulocyte stimulating factor and leukocyte inhibitory factor (LIF).

[0044] According to the present invention, "medium" should be understood as a liquid or solid medium containing nutrients, which is suitable for nourishing and supporting the life and / or product formation of cells in the culture, excluding the medium for parenteral nutrition. According to the present invention, the cells cultured can be bacterial cells, yeast cells, fungal cells, animal cells (such as mammalian cells or insect cells), and / or plant cells (such as algae). Usually, 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 for the cell to maintain life, growth and / or product formation. The culture medium may also contain components that enhance growth and / or survival to exceed the minimum rate (minimal rate), including hormones and growth factors. The culture medium preferably has pH and salt concentrations that support the life, growth and / or product formation of the cells. According to the present invention, the culture medium preferably includes all nutrients necessary for the life and proliferation of the cell culture. A preferred culture medium is a determined culture medium.

[0045] According to the present invention, a "chemically defined medium" 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 is generally understood by those skilled in the art, a chemically defined medium generally does not contain components of animal origin. All components of a chemically defined medium have known chemical structures. Mediums other than defined mediums may be referred to as "complex" mediums.

[0046] "Cell culture medium" is understood to be a culture medium suitable for maintaining the life, proliferation and / or product formation of animal cells and / or plant cells.

[0047] "Basal medium" or "basal culture medium" is understood as a solution or substance containing nutrient substances in which a cell culture is initiated.

[0048] "Feed medium" is understood to be a solution or substance with which the cells are fed after the culture process has begun. In certain embodiments, the feed medium contains one or more components that are not present in the basal medium. The feed medium may also lack one or more components present in the basal medium. Preferably, the concentration of nutrients in the feed medium exceeds that in the basal medium to avoid loss of productivity through dilution.

[0049] "Perfusion medium" is understood to be a solution or substance containing nutrients which is added continuously after the start of the cell culture and from which the harvest is removed continuously.

[0050] The compositions and methods according to the invention enhance the photostability of light-sensitive ingredients. A negative aspect of limited photostability is brown formation, such as that promoted by tryptophan. In this context, photostability can be characterized, for example, by a color change upon exposure to sunlight.

[0051] In preferred arrangements, 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.

[0052] In another preferred embodiment, the water-soluble tyrosine-containing compound is selected from phosphotyrosine or a dipeptide, preferably Ala-Tyr or Gly-Tyr.

[0053] In another preferred embodiment, the photosensitive component is selected from aromatic compounds, preferably from riboflavin and aromatic amino acids or their derivatives, preferably from tryptophan and tyrosine.

[0054] In a preferred configuration of the present invention, the cell culture medium further comprises free tyrosine.

[0055] The light-sensitive component according to the invention is selected from aromatic compounds, preferably from riboflavin and aromatic amino acids or their derivatives, preferably from tryptophan and tyrosine. Those components are known to contribute to the color formation under light exposure.

[0056] In a preferred arrangement, the derivative of an aromatic amino acid is a soluble derivative of tryptophan or tyrosine, preferably selected from phosphotyrosine.

[0057] In a particular configuration of the invention, the photosensitizing component is present in an amount of at least 0.5 mM, preferably at least 1 mM, more preferably at least 2 mM.

[0058] Another aspect of the present invention relates to a method for stabilizing a photosensitive composition.

[0059] In a preferred embodiment of the method according to the invention, the photosensitizing component is present in an aqueous solution.

[0060] In another preferred embodiment, the dipeptide is selected from Ala-Tyr or Gly-Tyr.

[0061] In another preferred embodiment, the photosensitive component is selected from aromatic compounds, preferably from riboflavin and aromatic amino acids or derivatives thereof, preferably from tryptophan and tyrosine, more preferably from tryptophan.

[0062] In another preferred configuration of the method according to the invention, the molar ratio of dipeptide to tryptophan is about 10:1 to 1:10, preferably about 5:1 to 1:5.

[0063] 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.

[0064] In another preferred embodiment, a cysteine ​​derivative is added, preferably selected from a cysteine-containing dipeptide or a cysteine ​​salt, preferably the sodium S-sulfate salt of cysteine.

[0065] 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.

[0066] According to the present invention, the cultivation of cells can be carried out in batch culture, fed-batch culture or continuous culture.

[0067] 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.

[0068] In one embodiment of the present invention, the culture medium does not contain growth factors. According to this embodiment, the dipeptides 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 any lipids.

[0069] In a preferred embodiment, the culture medium of the present invention is a defined culture medium or a serum-free culture medium. For example, the composition of the intervention can be supplemented with all Gibco TM Freestyle™ F17 medium, Freestyle™ 293 medium, Expi293™ medium from ThermoFisher (Waltham, USA), TheraPEAK™ SfAAV™ medium from LONZA (Basel, Switzerland), HEK ViP NB from Sartorius Xell (Bielefeld, Germany), HyClone from Cytiva (Marlborough, USA) TM SFM4HEK293. The dipeptide of the present invention can also be supplemented to DMEM medium (Life Technologies Corp., Carlsbad, USA). However, the present invention is not limited to supplementing the above culture medium.

[0070] The cell culture medium (cell or tissue basal medium, feed medium or perfusion medium) of the present invention may preferably contain all nutrients required for sustained growth and product formation. The formulations for preparing culture media, in particular cell culture media, are well known to those skilled in the art (see, for example, Cell Culture Technology for Pharmaceutical and Cell-Based Therapies, and Wei-Shou Hu eds., Taylor and Francis Group 2006). Various culture media are commercially available from various sources.

[0071] The culture medium of the present invention may preferably include a carbohydrate source. The main carbohydrate used in the cell culture medium is glucose, which is usually supplemented with 5 to 25 mM. In addition, any hexose such as galactose, fructose or mannose or a combination thereof may be used.

[0072] Culture medium can also usually include at least essential amino acids (i.e. His, Ile, Leu, Lys, Met, Phe, Thr, Try, Val) and non-essential amino acids. If the cell line cannot synthesize amino acids, or if the cell line cannot produce enough amino acids to support maximum growth, non-essential amino acids are usually included in the cell culture medium. In addition, mammalian cells can also use glutamine as the main energy source. Often include glutamine (2-8mM) with a concentration higher than other amino acids. However, as mentioned above, glutamine can spontaneously decompose to form ammonia, and some cell lines produce ammonia faster, which is toxic.

[0073] The culture medium of the present invention may preferably contain salts. Salts are added to the cell culture medium to maintain isotonic conditions and prevent osmotic imbalance. The osmolality of the culture medium of the present invention is about 300 mOsm / kg, although many cell lines can tolerate variations of about 10% or more of this value. The osmolality of some insect cell cultures tends to be higher than 300 mOsm / kg, and this 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 salts in cell culture media include Na + , K + Mg 2+ , Ca 2+ , Cl - 、SO4 2- PO4 3- and HCO3 - (e.g., CaCl2, KCl, NaCl, NaHCO3, Na2HPO4).

[0074] Other inorganic elements may be present in the culture medium. They include Mn, Cu, Zn, Mo, Va, Se, Fe, Ca, Mg, Si and Ni. Many of these elements participate in enzyme activity. They can be provided in the form of ions of salts (such as CaCl2, Fe(NO3)3, MgCl2, MgSO4, MnCl2, NaCl, NaHCO3, Na2HPO4) and trace elements (such as selenium, vanadium and zinc). These inorganic salts and trace elements can be obtained commercially, for example, from Sigma (Saint Louis, Missouri).

[0075] The culture medium of the present invention preferably comprises vitamins. Vitamins are usually used as cofactors by cells. The vitamin requirements of each cell line vary greatly, although if the cell culture medium contains little or no serum or if the cells are grown at high density, additional vitamins are usually 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, nicotinamide (niacinamide), vitamin A, B6, B12, C, D3, E, K and para-aminobenzoic acid (PABA).

[0076] The culture medium of the present invention may also contain serum. Serum is the supernatant of coagulated blood. Serum components include attachment factors, trace nutrients (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 a cell culture medium according to the present invention, serum is typically added at a concentration of 5-10% (volume). Preferred cell culture media are serum-free.

[0077] One or more lipids can also be added to the cell culture medium of the present invention, such as linoleic acid, linolenic acid, arachidonic acid, palmitoleic acid, oleic acid, polyenoic acid and / or 12,14,16,18,20,22 or 24 carbon atoms (each carbon atom is branched or non-branched) fatty acid, phospholipids, lecithin (phosphatidylcholine) and cholesterol.One or more of these lipids can be included in the serum-free medium as fill-in. Phosphatidic acid and lysophosphatidic acid stimulate the growth of some anchor-dependent cells (such as MDCK, mouse epithelial cells and other kidney 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 some cell lines.In certain embodiments, the cell culture medium does not contain lipid.

[0078] 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 help transport certain nutrients or trace elements. BSA is generally used as a carrier of lipids such as linoleic acid and oleic acid that are insoluble in aqueous solution. In addition, BSA can also serve as a carrier of certain metals such as Fe, Cu and Ni. In protein-free preparations, BSA substitutes such as cyclodextrins from non-animal sources can be used as lipid carriers.

[0079] One or more attachment proteins such as fibronectin, laminin, and probentin may also be added to the cell culture medium to help promote anchorage-dependent cell attachment to the matrix.

[0080] 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, phosphates, bicarbonates, and other buffers suitable for cell culture applications. Suitable buffers are those that provide buffering capacity without substantial cytotoxicity to the cultured cells. The selection of suitable buffers is within the ordinary skill of the art of cell culture.

[0081] Polyanionic or polycationic compounds can be added to the culture medium to prevent cell aggregation and promote growth of cells in suspension.

[0082] In a preferred embodiment, the culture medium is in liquid form. However, the culture medium may also be a solid culture medium, such as a gel-like culture medium, for example a culture medium (powder, aggregated powder, instant powder, etc.) containing agar, carrageenan or gelatin. Preferably, the culture medium is in sterile form.

[0083] The culture medium of the present invention can be in concentrated form. It can be, for example, in 2 to 100 times concentrated form, preferably in 2 times, 3 times, 3.33 times, 4 times, 5 times, 10 times, 20 times, 50 times or 100 times concentrated form (relative to the concentration that supports the growth of cells and product formation). This concentrated culture medium helps to be used for preparing culture medium by diluting the concentrated culture medium with an aqueous solvent such as water. This concentrated culture medium can be used for batch culture, but is also advantageously used for fed-batch or continuous culture, in which the concentrated nutrient composition is added to the ongoing cell culture, for example to supplement the nutrients consumed by the cells during the culture.

[0084] In other embodiments of the present invention, the culture medium is in dry form, such as dry powder form, or granule form, or pellet form, or tablet form.

[0085] Another aspect of the invention relates to the use of a tyrosine-containing dipeptide for stabilizing photosensitive ingredients in cell culture, cosmetics, food applications, parenteral nutrition or pharmaceutical product formulations.

[0086] In a preferred configuration, the use is in a cell culture medium for stabilizing photosensitive components in cell culture, preferably as basal medium, feed medium or perfusion medium.

[0087] In a preferred embodiment, the cell is selected from CHO cells, COS cells, VERO cells, BHK cells, HEK cells, HELA cells, AE-1 cells, NSO cells, insect cells, algae cells, fibroblasts, muscle cells, neural cells, stem cells, skin cells, endothelial cells, immune cells such as NK or T cells, and hybridoma cells. Example

[0088] Material:

[0089] Table 1: Materials used for radiation testing

[0090] Material supplier L-Tryptophan (Trp) Evonik rexim SAS,Ham(France) L-Tyrosine (Tyr) Sigma-Aldrich,Missouri(USA) Glycine (Gly) Evonik rexim SAS,Ham(France) L-Alanine (Ala) Evonik rexim SAS,Ham(France) (-)-Riboflavin Sigma-Aldrich,Missouri(USA) L-Alanyl-L-Tyrosine Dihydrate (Ala-Tyr) Evonik Operations GmbH,Darmstadt(Germany) Glycyl-L-tyrosine dihydrate (Gly-Tyr) Evonik Operations GmbH,Darmstadt(Germany) 50mL transparent centrifuge tube Greiner Bio-One,Kremsmünster(Austria) 50mL brown centrifuge tube Greiner Bio-One,Kremsmünster(Austria) 2mL brown safety lock tube Eppendorf,Hamburg (Germany) Transparent flat bottom 96-well plate Greiner Bio-One,Kremsmünster(Austria) 0.22μm syringe filter Corning,Kaiserslautern(Germany) 50mL disposable syringe, luer lock solo Braun, Melsungen (Germany) D-PBS without calcium or magnesium Invitrogen, Carlsbad (USA) Minimum Essential Medium (MEM) Invitrogen, Carlsbad (USA) Aluminum foil Carl Roth,Karlsruhe (Germany) Bottle cap sterile filter Thermo scientific,Massachusetts(USA)

[0091] Table 2: Equipment for radiation testing

[0092]

[0093] method:

[0094] Preparation of test samples

[0095] In phosphate buffered saline (D-PBS) and in minimum essential medium (MEM), prepare an aqueous solution of 2.5mM L-tryptophan (Trp) and 0.013mM riboflavin. These solutions are used to dissolve the dipeptide L-alanyl-L-tyrosine dihydrate (Ala-Tyr) and glycyl-L-tyrosine dihydrate (Gly-Tyr) of different concentrations and amino acid L-tyrosine (Tyr), L-alanine (Ala) and glycine (Gly) to prepare a solvent mixture. In Example 1, dipeptide was tested at a concentration of 0.5mM, 2.5mM and 10mM, and Tyr was tested at a concentration of 0.5mM. PBS and MEM and the 2.5mM Trp dissolved in PBS and MEM were used as controls. In Example 2, dipeptide and Tyr were tested at a concentration of 2mM, and Ala and Gly were tested at a concentration of 32mM, and solvent (PBS or MEM) was used as a control (" solvent background ").

[0096] Brown detection

[0097] The samples were exposed to electromagnetic radiation (sunlight) at room temperature for 7 days (Example 2) and 14 days (Example 1). The relative quantification of the brown color of the sample of Example 1 was obtained by measuring the absorbance at 330 nm. The intensity of the brown color of the sample of Example 2 was determined by eye using the Brown Index. The numbers 1 to 5 correspond to the intensity of the brown color of the solution (0, no brown; 1, light brown; 2, medium brown; 3, normal brown; 4, intensive brown color; 5, dark brown).

[0098] Example 1: Reduction of brown color of tryptophan solution containing Tyr derivatives

[0099] At room temperature, exposure of a colorless aqueous solution of PBS or MEM containing 2.5 mM Trp to electromagnetic radiation results in a strong change in color to brown. Addition of 0.5 mM Tyr or Tyr dipeptide (Ala-Tyr or Gly-Tyr) has been shown to reduce this coloration, especially for the two Tyr dipeptides, the effect is greater. Dipeptide concentrations up to 10 mM, above the solubility of Tyr, lead to a further reduction or even complete prevention of the color change. Results are shown in Figure 2. Figure 1 and Figure 2 shown.

[0100] Figure 1 Shown is the effect of different aqueous solutions containing Trp and Tyr derivatives dissolved in phosphate buffered saline (PBS) on brown coloration compared to controls of PBS only and Trp dissolved in PBS. The intensity of the brown color was measured as the difference in absorbance at 330 nm.

[0101] Figure 2 Shown is the effect of different aqueous solutions containing Trp and Tyr derivatives dissolved in minimum essential medium (MEM) on brown coloration compared to controls of MEM only and Trp dissolved in MEM. The intensity of the brown color was measured as the difference in absorbance at 330 nm.

[0102] Example 2: Reduction of brown color of riboflavin solution containing Tyr derivatives

[0103] Aqueous solutions of PBS or MEM containing 0.013 mM riboflavin showed a fluorescent yellow-green color for one week. Exposure to electromagnetic radiation for seven days at room temperature resulted in a brown coloration of the MEM solution, while the yellow-green color in the PBS solution disappeared. Based on the applied brown index, the intensity of the brown color was classified as "normal brown".

[0104] Similarly, the color of the MEM control solution containing 0.013mM riboflavin and additionally added amino acids such as Ala and Gly at a concentration of up to 32mM also turned into "normal brown", while the same control solution in PBS remained colorless. Other control solutions of PBS and MEM without riboflavin were colorless on day 0. After seven days of electromagnetic radiation at room temperature, the PBS solution remained colorless, while the color of the MEM solution turned into "light brown". The same situation was observed for the control solution without riboflavin and additionally added amino acids such as Ala and Gly at a concentration of up to 32mM.

[0105] It was found that the addition of an aromatic amino acid such as Tyr (2 mM) to any type of aqueous solution used and exposure to electromagnetic radiation at room temperature for seven days resulted in a "dark brown" coloration of all solutions. Interestingly, in comparison to this "dark brown", equimolar replacement of 2 mM Tyr with 2 mM Tyr-dipeptides (such as Ala-Tyr and Gly-Tyr) showed a clear reduction in the intensity of the brown color towards that classified as "normal brown".

[0106] Table 3: Effect of different aqueous solutions containing tyrosine (Tyr) or Tyr derivatives with or without riboflavin on brown coloration. Control solutions included the single amino acids glycine (Gly) and alanine (Ala) and solvent ("solvent background", phosphate buffered saline (PBS) or minimum essential medium (MEM)). Brown index: 0, no brown; 1, light brown; 2, medium brown; 3, normal brown; 4, rich brown; 5, dark brown

[0107]

Claims

1. A light-stable composition comprising Water-soluble tyrosine-containing compounds with molecular weight 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 wherein the composition is a culture medium.

2. The composition according to claim 1, wherein the culture medium is a cell culture medium selected from a basal medium, a feed medium or a perfusion medium.

3. The composition of 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.

4. A composition according to any one of the preceding claims, wherein the water-soluble tyrosine-containing compound is selected from phosphotyrosine or a dipeptide, preferably Ala-Tyr or Gly-Tyr.

5. The composition according to any of the preceding claims, wherein the photosensitive component is selected from aromatic compounds, preferably from riboflavin and aromatic amino acids or derivatives thereof, preferably from tryptophan and tyrosine.

6. The composition according to any of the preceding claims, wherein the photosensitizing component is present in an amount of at least 0.5 mM, preferably at least 1 mM, more preferably at least 2 mM.

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, wherein the dipeptide is present at a concentration of at least 0.5 mM.

8. The method of claim 7, wherein the photosensitizing component is present in an aqueous solution.

9. The method according to claim 7 or 8, wherein the dipeptide is selected from Ala-Tyr or Gly-Tyr.

10. The method according to any one of claims 7 to 9, wherein the photosensitive component is selected from aromatic compounds, preferably from riboflavin and aromatic amino acids or derivatives thereof, preferably from tryptophan and tyrosine, more preferably from tryptophan.

11. The method according to claim 10, wherein the molar ratio of the dipeptide to tryptophan is about 10:1 to 1:10, preferably about 5:1 to 1:

5.

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

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 photosensitive ingredients in cell culture, cosmetics, food applications, parenteral nutrition or pharmaceutical product formulations.

16. The use according to claim 15, in a cell culture medium for stabilizing photosensitive components in cell culture, preferably as a basal medium, a feed medium or a perfusion medium.

17. The method of claim 16, wherein the cell is selected from the group consisting of CHO cells, COS cells, VERO cells, BHK cells, HEK cells, HELA cells, AE-1 cells, NSO cells, insect cells, algae cells, fibroblasts, muscle cells, neural cells, stem cells, skin cells, endothelial cells, immune cells such as NK or T cells, and hybridoma cells.

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