Composition for culturing animal cells

Abstract

Provided are a composition for culturing animal cells and a technology related thereto. The composition for culturing animal cells contains an amino acid source and an α–Ketoglutaric acid and has a property (A) and / or (B). (A): The α–Ketoglutaric acid is a salt of an α–Ketoglutaric acid. (B): The amino acid source is a free-form L-cysteine anhydrate.

Description

Composition for animal cell culture

[0001] The present invention relates to compositions for animal cell culture and related techniques.

[0002] Culture media containing amino acids such as cysteine ​​(Cys) are widely used for animal cell culture. Cys undergoes spontaneous oxidation in the culture medium and is converted to cystine ((Cys)2). Because (Cys)2 has low solubility, it easily precipitates in liquid culture media. For this reason, Cys is a factor in the storage instability of liquid culture media.

[0003] Cys can be condensed with α-keto acids such as pyruvic acid (Pyr) and α-ketoglutaric acid to form thiazolidine derivatives (Non-Patent Document 1). It has been reported that the use of α-keto acids stabilizes the culture medium, and that thiazolidine derivatives are effective for culturing animal cells (Non-Patent Document 1).

[0004] Kuschelewski J et al., Antioxidant effect of thiazolidine molecules in cell culture media improves stability and performance. Biotechnol Prog. 2017 May;33(3):759-770.

[0005] An objective of the present invention is to provide a composition for animal cell culture and techniques related thereto.

[0006] The present inventors have discovered that problems such as caking and a decrease in the cysteine ​​content occur in a mixed powder of a specific form of an amino acid source such as cysteine ​​and a specific form of α-ketoglutaric acid, and that these problems can be solved by adjusting the forms of the amino acid source and α-ketoglutaric acid, and have completed the present invention.

[0007] That is, the present invention can be exemplified as follows. [1] A composition for animal cell culture, comprising an amino acid source and α-ketoglutaric acid, and having the following property (A) and / or (B): (A) the α-ketoglutaric acid is a salt of α-ketoglutaric acid; (B) the amino acid source is free L-cysteine ​​anhydrate. [2] The composition having at least the property (A). [3] The composition in the form of a powder. [4] The composition in the form of a medium or a medium additive. [5] The composition in the form of a medium that is a basal medium, a fed-batch medium, or a perfusion medium. [6] The composition in the form of an alkali metal salt or an alkaline earth metal salt of α-ketoglutaric acid. [7] The composition in the form of a sodium salt or a potassium salt of α-ketoglutaric acid. [8] The composition as described above, wherein the salt of α-ketoglutaric acid is monosodium α-ketoglutarate or disodium α-ketoglutarate, [9] The composition as described above, wherein the amino acid source is cysteine, glycine, asparagine, glutamic acid, lysine, phenylalanine, methionine, ornithine, tyrosine, or a glycine-containing dipeptide.

[10] The composition, having one or more properties selected from the group consisting of the following properties (1) to (10): (1) the cysteine ​​is free L-cysteine ​​or L-cysteine ​​hydrochloride; (2) the glycine is free glycine or free glycyl-L-tyrosine; (3) the asparagine is free L-asparagine; (4) the glutamic acid is monosodium L-glutamate; (5) the lysine is L-lysine hydrochloride; (6) the phenylalanine is free L-phenylalanine; (7) the methionine is free L-methionine; (8) the ornithine is L-ornithine hydrochloride; (9) the tyrosine is L-tyrosine disodium; (10) the glycine-containing dipeptide is free glycyl-L-tyrosine.

[11] The composition satisfies one or more properties selected from the group consisting of the following properties (1a) to (10a): (1a) the cysteine ​​is free L-cysteine ​​anhydrate or L-cysteine ​​hydrochloride monohydrate; (2a) the glycine is free glycine or free glycyl-L-tyrosine; (3a) the asparagine is free L-asparagine monohydrate; (4a) the glutamic acid is L-glutamic acid monosodium monohydrate; (5a) the lysine is L-lysine hydrochloride; (6a) the phenylalanine is free L-phenylalanine; (7a) the methionine is free L-methionine; (8a) the ornithine is L-ornithine hydrochloride; (9a) the tyrosine is L-tyrosine disodium dihydrate; (10a) The glycine-containing dipeptide is glycyl-L-tyrosine dihydrate in its free form.

[12] The composition wherein the amino acid source is cysteine.

[13] The composition wherein the cysteine ​​is free L-cysteine ​​anhydrate or L-cysteine ​​hydrochloride monohydrate.

[14] The composition wherein the cysteine ​​is free L-cysteine ​​anhydrate.

[15] The composition wherein the content of α-ketoglutaric acid is 1 to 20 times the content of the amino acid source in terms of molar ratio.

[16] A method for producing a target substance, comprising: culturing animal cells capable of producing the target substance using the composition; and recovering the target substance.

[17] The method wherein the target substance is a protein or a virus.

[18] A method for culturing animal cells, comprising culturing animal cells using the composition.

[19] Use of a salt of α-ketoglutaric acid for preventing caking of a composition containing an amino acid source,

[20] the amino acid source being cysteine, glycine, asparagine, glutamic acid, lysine, phenylalanine, methionine, ornithine, tyrosine, or a glycine-containing dipeptide.

[21] The use, which satisfies one or more conditions selected from the following conditions (1) to (10): (1) the cysteine ​​is free L-cysteine ​​or L-cysteine ​​hydrochloride; (2) the glycine is free glycine or free glycyl-L-tyrosine; (3) the asparagine is free L-asparagine; (4) the glutamic acid is monosodium L-glutamate; (5) the lysine is L-lysine hydrochloride; (6) the phenylalanine is free L-phenylalanine; (7) the methionine is free L-methionine; (8) the ornithine is L-ornithine hydrochloride; (9) the tyrosine is disodium L-tyrosine; (10) the glycine-containing dipeptide is free glycyl-L-tyrosine.

[22] The use, which satisfies one or more conditions selected from the following conditions (1a) to (10a): (1a) the cysteine ​​is free L-cysteine ​​anhydrate or L-cysteine ​​hydrochloride monohydrate; (2a) the glycine is free glycine or free glycyl-L-tyrosine; (3a) the asparagine is free L-asparagine monohydrate; (4a) the glutamic acid is L-glutamic acid monosodium monohydrate; (5a) the lysine is L-lysine hydrochloride; (6a) the phenylalanine is free L-phenylalanine; (7a) the methionine is free L-methionine; (8a) the ornithine is L-ornithine hydrochloride; (9a) the tyrosine is L-tyrosine disodium dihydrate; (10a) The glycine-containing dipeptide is glycyl-L-tyrosine dihydrate in a free form.

[23] The use as described above, wherein the amino acid source is cysteine.

[24] The use of a salt of α-ketoglutaric acid for preventing a decrease in the cysteine ​​content in a cysteine-containing composition.

[25] The use as described above, wherein the salt of α-ketoglutaric acid is an alkali metal salt or alkaline earth metal salt of α-ketoglutaric acid.

[26] The aforementioned use, wherein the salt of α-ketoglutaric acid is a sodium salt or a potassium salt of α-ketoglutaric acid.

[27] The aforementioned use, wherein the salt of α-ketoglutaric acid is monosodium α-ketoglutarate or disodium α-ketoglutarate.

[28] The aforementioned use, wherein the cysteine ​​is free L-cysteine ​​anhydrate or L-cysteine ​​hydrochloride monohydrate.

[29] The aforementioned use, wherein the cysteine ​​is free L-cysteine ​​anhydrate.

[30] The aforementioned use, wherein the content of the salt of α-ketoglutaric acid in the composition is 1 to 20 times the molar ratio of the content of the amino acid source or the cysteine ​​in the composition.

[31] A method for producing a composition for animal cell culture, wherein the composition is the composition according to any one of [1] to

[15] , and the method comprises mixing the amino acid source and the α-ketoglutaric acid.

[0008] 1 is a diagram (photograph) showing the degree of caking of a mixed powder of L-cysteine ​​and α-ketoglutaric acid when stored at 25°C. 2 is a diagram (photograph) showing the degree of caking of a mixed powder of L-cysteine ​​and α-ketoglutaric acid when stored at 40°C. 3 is a diagram (photograph) showing the degree of caking of a mixed powder of various amino acid sources and α-ketoglutaric acid immediately after preparation. 4 is a diagram (photograph) showing the degree of caking of a mixed powder of various amino acid sources and α-ketoglutaric acid when stored at 4°C. 5 is a diagram (photograph) showing the degree of caking of a mixed powder of various amino acid sources and α-ketoglutaric acid when stored at 25°C. 6 is a diagram (photograph) showing the degree of caking of a mixed powder of various amino acid sources and α-ketoglutaric acid when stored at 40°C. 7 is a diagram showing the change over time in the L-cysteine ​​content of a mixed powder of L-cysteine ​​hydrochloride monohydrate and free α-ketoglutaric acid when stored at 25°C or 40°C. 1 is a diagram showing the change over time in the L-cysteine ​​content when a mixed powder of free L-cysteine ​​anhydrate and free α-ketoglutaric acid is stored at 25° C. or 40° C. 2 is a diagram showing the change over time in the L-cysteine ​​content when a mixed powder of L-cysteine ​​hydrochloride monohydrate and monosodium α-ketoglutarate is stored at 25° C. or 40° C. 3 is a diagram showing the change over time in the L-cysteine ​​content when a mixed powder of free L-cysteine ​​anhydrate and monosodium α-ketoglutarate is stored at 25° C. or 40° C. 4 is a diagram showing the change over time in the L-cysteine ​​content when a mixed powder of free L-cysteine ​​anhydrate and monosodium α-ketoglutarate is stored at 25° C. or 40° C.

[0009] <1> Composition of the Present Invention The composition of the present invention is a composition containing an amino acid source and α-ketoglutaric acid.

[0010] The amino acid source and α-ketoglutaric acid are collectively referred to as the "active ingredients."

[0011] The composition of the present invention can be used, for example, for culturing animal cells. That is, the composition of the present invention may be a composition for animal cell culture. Specifically, the composition of the present invention can be used for culturing animal cells, for example, in the embodiment described in the method of the present invention described below.

[0012] The composition of the present invention may be, for example, a culture medium. Examples of culture media include basal medium, feed medium, and perfusion medium. "Basal medium" may refer to the medium used at the start of culture. Basal medium is also called "initial medium." "Fed-batch medium" may refer to the medium supplied to a culture system after the start of culture in fed-batch culture. "Perfusion medium" may refer to the medium supplied to a culture system after the start of culture in continuous culture (this is not limited to perfusion culture).

[0013] The composition of the present invention may be, for example, a medium additive. The term "medium additive" may refer to a composition that is added to a medium for use. Media to which the medium additive is added include basal medium, feed medium, and perfusion medium.

[0014] The composition of the present invention has the following properties (A) and / or (B): (A) the α-ketoglutaric acid is a salt of α-ketoglutaric acid; (B) the amino acid source is free L-cysteine ​​anhydrate.

[0015] The composition of the present invention may have only one of the properties (A) and (B), or may have both the properties (A) and (B). The composition of the present invention may particularly have at least the property (A). The composition of the present invention may more particularly have both the properties (A) and (B).

[0016] The composition of the present invention has properties (A) and / or (B), which can improve the stability of the composition of the present invention, i.e., can provide an effect of improving the stability of the composition of the present invention. This effect is also referred to as a "stability-improving effect." Specifically, the composition of the present invention has properties (A) and / or (B), which can improve the stability of the composition of the present invention compared to a composition not having properties (A) and / or (B).

[0017] An example of improving the stability of a composition is preventing the composition from caking. Furthermore, when the composition of the present invention contains cysteine ​​(specifically, when cysteine ​​is selected as the amino acid source of the active ingredient), an example of improving the stability of the composition is preventing a decrease in the cysteine ​​content of the composition. "Preventing the composition from caking" means reducing the degree of caking of the composition, and also includes the case where the composition does not caking at all. "Preventing a decrease in the cysteine ​​content of the composition" means reducing the degree of decrease in the cysteine ​​content of the composition, and also includes the case where the cysteine ​​content of the composition does not decrease at all. The effect of preventing the composition from caking is also referred to as an "anti-caking effect." The effect of preventing a decrease in the cysteine ​​content of a composition is also referred to as a "cysteine ​​decrease prevention effect." That is, the anti-caking effect and / or the cysteine ​​decrease prevention effect may be obtained by the composition of the present invention having properties (A) and / or (B). That is, the composition of the present invention may be prevented from caking and / or from reducing the cysteine ​​content in the composition due to the composition having properties (A) and / or (B). Specifically, the composition of the present invention may be prevented from caking and / or from reducing the cysteine ​​content in the composition due to the composition having properties (A) and / or (B), compared to a case in which the composition of the present invention does not have properties (A) and / or (B).

[0018] For example, the stability of the composition of the present invention may be improved by having only one of the properties (A) and (B) compared to a composition of the present invention that does not have either the property (A) or the property (B). The caking of the composition may be particularly prevented by having only one of the properties (A) and the property (B) compared to a composition of the present invention that does not have either the property (A) or the property (B).

[0019] For example, when the composition of the present invention has both properties (A) and (B), the stability of the composition of the present invention may be improved compared to when the composition of the present invention has neither property (A) nor property (B). When the composition of the present invention has both properties (A) and (B), the composition may be particularly prevented from caking and / or the cysteine ​​content may be prevented from decreasing compared to when the composition of the present invention has neither property (A) nor property (B). When the composition of the present invention has both properties (A) and (B), the composition may be more particularly prevented from caking and the cysteine ​​content may be prevented from decreasing compared to when the composition of the present invention has neither property (A) nor property (B).

[0020] For example, when the composition of the present invention has both properties (A) and (B), the stability of the composition of the present invention may be improved compared to when the composition of the present invention has only one of properties (A) and (B). When the composition of the present invention has both properties (A) and (B), the composition may be particularly prevented from caking and / or from losing its cysteine ​​content compared to when the composition of the present invention has only one of properties (A) and (B). When the composition of the present invention has both properties (A) and (B), the composition may be more particularly prevented from caking and from losing its cysteine ​​content compared to when the composition of the present invention has only one of properties (A) and (B).

[0021] An example of the composition of the present invention not having property (A) is when α-ketoglutaric acid is free α-ketoglutaric acid. An example of the composition of the present invention not having property (B) is when the amino acid source is a cysteine ​​other than free L-cysteine, specifically, when the amino acid source is L-cysteine ​​hydrochloride monohydrate or when the amino acid source is L-cysteine ​​hydrochloride anhydrate.

[0022] The stability-improving effect can be confirmed by comparing the relevant parameters of the composition of the present invention after storage under specified conditions between a composition of the present invention having property (A) and / or (B) and a composition of the present invention not having property (A) and / or (B).

[0023] That is, the anti-caking effect can be confirmed by comparing the degree of caking of the composition of the present invention after storage under specified conditions between compositions having and not having properties (A) and / or (B). The degree of caking can be confirmed, for example, by visual inspection with the naked eye. The degree of caking can also be confirmed, for example, using the particle size (e.g., average particle size D50) of the composition as an indicator. That is, the smaller the particle size (e.g., average particle size D50) of the composition, the lower the degree of caking. "Average particle size D50" may refer to the particle size corresponding to a passing mass percentage of 50% on a particle size accumulation curve graph. The average particle size D50 can be measured, for example, using a low-tap sieve shaker.

[0024] The cysteine ​​reduction prevention effect can be confirmed by comparing the degree of reduction in the cysteine ​​content in the composition of the present invention after storage under specified conditions between compositions of the present invention having and not having properties (A) and / or (B). The degree of reduction in cysteine ​​content can be confirmed using the ratio of the cysteine ​​content in the composition of the present invention before and after storage as an indicator. That is, the greater the ratio of the cysteine ​​content in the composition of the present invention after storage to the cysteine ​​content in the composition of the present invention before storage, the lower the degree of reduction in cysteine ​​content. The cysteine ​​content can be measured, for example, by the method described in WO2021 / 060517A.

[0025] The predetermined storage conditions include those under which caking and / or a decrease in cysteine ​​content occurs when the composition of the present invention does not have either property (A) or (B). Specific examples of the predetermined storage conditions include storage under light-shielded conditions at 25°C for 3 days, 25°C for 7 days, 25°C for 14 days, 25°C for 21 days, 40°C for 3 days, 40°C for 7 days, 40°C for 14 days, or 40°C for 21 days. Storage may be carried out, for example, in the presence of a moisture absorbent. Examples of moisture absorbents include silica gel, quicklime (calcium oxide), calcium chloride, and zeolite. Silica gel is particularly preferred. Storage may also be carried out, for example, in a packaging material with moisture absorption properties. Examples of packaging materials with moisture absorption properties include aluminum pouches with moisture-absorbing film. That is, more specifically, the predetermined storage conditions include storage in the presence of a moisture absorbent or in a packaging material having a moisture-absorbing function, under light-shielded conditions, for 3 days at 25° C., 7 days at 25° C., 14 days at 25° C., 21 days at 25° C., 3 days at 40° C., 7 days at 40° C., 14 days at 40° C., or 21 days at 40° C. The stability-improving effect (e.g., anti-caking effect and / or anti-cysteine ​​reduction effect) may be confirmed, for example, under one or more conditions selected from the above-exemplified conditions.

[0026] "Amino acid source" is a general term for amino acids and compounds that produce them. The amino acid source is not particularly limited as long as it provides a stability-improving effect. For example, the amino acid source may be one that causes caking when mixed with free α-ketoglutaric acid. Whether or not caking occurs in a mixture of an amino acid source and free α-ketoglutaric acid can be confirmed by checking whether or not caking occurs in the mixture after storage under specified conditions. The specified storage conditions described above for confirming the stability-improving effect can be applied mutatis mutandis to the specified storage conditions.

[0027] Examples of amino acid sources include glycine sources, alanine sources, valine sources, leucine sources, isoleucine sources, cysteine ​​sources, methionine sources, phenylalanine sources, tyrosine sources, tryptophan sources, histidine sources, lysine sources, arginine sources, serine sources, threonine sources, aspartic acid sources, glutamic acid sources, asparagine sources, glutamine sources, proline sources, and ornithine sources. That is, examples of amino acids include glycine, alanine, valine, leucine, isoleucine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, histidine, lysine, arginine, serine, threonine, aspartic acid, glutamic acid, asparagine, glutamine, proline, and ornithine. Examples of amino acid sources include, in particular, cysteine ​​sources, glycine sources, asparagine sources, glutamic acid sources, lysine sources, phenylalanine sources, methionine sources, ornithine sources, and tyrosine sources. Specifically, amino acids include cysteine, glycine, asparagine, glutamic acid, lysine, phenylalanine, methionine, ornithine, and tyrosine. More specifically, amino acid sources include cysteine ​​sources, glycine sources, asparagine sources, glutamic acid sources, lysine sources, phenylalanine sources, and methionine sources. More specifically, amino acids include cysteine, glycine, asparagine, glutamic acid, lysine, phenylalanine, and methionine. More specifically, amino acids include cysteine ​​sources. More specifically, amino acids include cysteine ​​sources. More specifically, amino acids include cysteine. Compounds that yield amino acids include compounds that yield amino acids upon hydrolysis. Compounds that yield amino acids upon hydrolysis include peptides containing amino acids as building blocks. Peptides include dipeptides and tripeptides. Peptides include, in particular, peptides containing glycine as a building block, such as dipeptides containing glycine as a building block. Peptides containing glycine as a building block are also referred to as "glycine-containing peptides." The position of glycine in the glycine-containing peptide is not particularly limited, and the position of glycine in the glycine-containing peptide may be, for example, the N-terminus, the C-terminus, or any other position.The position of glycine in the glycine-containing peptide may particularly be the N-terminus. Glycine-containing dipeptides particularly include dipeptides of glycine and any of the amino acids exemplified above. Glycine-containing dipeptides, more particularly, include glycyltyrosine. Glycyltyrosine is an example of a glycine source and also an example of a tyrosine source. Examples of amino acid sources (which may cause caking when mixed with, for example, free α-ketoglutaric acid) particularly include cysteine, glycine, asparagine, glutamic acid, lysine, phenylalanine, methionine, ornithine, tyrosine, and glycine-containing dipeptides. Examples of amino acid sources (which may cause caking when mixed with, for example, free α-ketoglutaric acid) particularly include cysteine, glycine, asparagine, glutamic acid, lysine, phenylalanine, methionine, and glycine-containing dipeptides. Furthermore, when the effect of preventing cysteine ​​depletion is to be obtained, cysteine ​​is particularly used as the amino acid source.

[0028] The amino acid source may be a single amino acid source or a combination of two or more amino acid sources. For example, a cysteine ​​source may be used in combination with one or more other amino acid sources. Specifically, the amino acid source may be a cysteine ​​source in combination with one or more amino acid sources selected from a glycine source, an asparagine source, a glutamic acid source, a lysine source, a phenylalanine source, a methionine source, an ornithine source, and a tyrosine source. Specifically, the amino acid source may be a cysteine ​​source in combination with one or more amino acid sources selected from a glycine source, an asparagine source, a glutamic acid source, a lysine source, a phenylalanine source, and a methionine source.

[0029] Amino acids may be, for example, in the L-form. That is, for example, alanine, valine, leucine, isoleucine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, histidine, lysine, arginine, serine, threonine, aspartic acid, glutamic acid, asparagine, glutamine, proline, and ornithine may be L-alanine, L-valine, L-leucine, L-isoleucine, L-cysteine, L-methionine, L-phenylalanine, L-tyrosine, L-tryptophan, L-histidine, L-lysine, L-arginine, L-serine, L-threonine, L-aspartic acid, L-glutamic acid, L-asparagine, L-glutamine, L-proline, and L-ornithine, respectively. Also, for example, glycyl tyrosine may be glycyl-L-tyrosine.

[0030] When an amino acid source can form a salt, the amino acid source may be used in its free form, its salt, or a combination thereof. That is, unless otherwise specified, the term "amino acid source" may refer to the amino acid source in its free form, its salt, or a combination thereof. For example, the term "cysteine" may refer to cysteine ​​in its free form, its salt, or a combination thereof, unless otherwise specified. "Free form" refers to a form that does not form a salt. The salt is not particularly limited as long as it can be used in animal cell culture. For example, salts of acidic groups such as carboxyl groups include ammonium salts, salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, aluminum salts, zinc salts, salts with organic amines such as triethylamine, ethanolamine, morpholine, pyrrolidine, piperidine, piperazine, and dicyclohexylamine, and salts with basic amino acids such as arginine and lysine. Salts of basic groups such as amino groups include salts with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and hydrobromic acid; salts with organic carboxylic acids such as acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, tannic acid, butyric acid, hybenzic acid, pamoic acid, enanthic acid, decanoic acid, teoclic acid, salicylic acid, lactic acid, oxalic acid, mandelic acid, and malic acid; and salts with organic sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. One type of salt may be used, or two or more types of salts may be used in combination. For example, salts of cysteine, lysine, or ornithine particularly include salts with inorganic acids. For example, salts of cysteine, lysine, or ornithine, more particularly include hydrochlorides. For example, salts of tyrosine or glutamic acid particularly include salts with alkali metals. Salts of tyrosine or glutamic acid include, more particularly, sodium salts. Sodium salts of tyrosine include disodium salts. Sodium salts of glutamic acid include monosodium salts.Furthermore, when an amino acid source can form a salt, the amino acid source may be used as an anhydrate, a hydrate, or a combination thereof. That is, the term "amino acid source" (e.g., "amino acid source in free form" or "salt of amino acid source") may encompass anhydrate and hydrate unless otherwise specified. For example, the term "cysteine" (e.g., "cysteine ​​in free form" or "salt of cysteine") may encompass anhydrate and hydrate unless otherwise specified. For example, hydrates of cysteine, asparagine, or glutamic acid include monohydrates. Furthermore, for example, hydrates of glycyltyrosine include dihydrates. The amino acid source may be in a form appropriate for the use mode, such as ions, when using the composition of the present invention (e.g., when culturing animal cells).

[0031] A particular example of a cysteine ​​source (specifically, cysteine) is L-cysteine. Examples of L-cysteine ​​include free L-cysteine ​​and L-cysteine ​​hydrochloride. Examples of free L-cysteine ​​include free L-cysteine ​​anhydrate. Examples of L-cysteine ​​hydrochloride include L-cysteine ​​hydrochloride monohydrate. That is, examples of cysteine ​​sources include free L-cysteine ​​anhydrate and L-cysteine ​​hydrochloride monohydrate. Examples of cysteine ​​sources include free L-cysteine ​​anhydrate. When property (B) is selected, the cysteine ​​source is free L-cysteine ​​anhydrate.

[0032] Glycine sources (specifically, glycine or dipeptides containing glycine as a constituent element) particularly include glycine and glycyl-L-tyrosine. Glycine particularly includes free glycine. Glycyl-L-tyrosine particularly includes free glycyl-L-tyrosine. Free glycyl-L-tyrosine particularly includes free glycyl-L-tyrosine dihydrate.

[0033] The asparagine source (specifically, asparagine) particularly includes L-asparagine. The L-asparagine particularly includes free L-asparagine. The free L-asparagine particularly includes free L-asparagine monohydrate.

[0034] The glutamic acid source (specifically, glutamic acid) particularly includes L-glutamic acid. The L-glutamic acid particularly includes monosodium L-glutamate. The monosodium L-glutamate particularly includes monosodium L-glutamate monohydrate.

[0035] The lysine source (specifically, lysine) particularly includes L-lysine, which particularly includes L-lysine hydrochloride.

[0036] The phenylalanine source (specifically, phenylalanine) particularly includes L-phenylalanine, and the L-phenylalanine particularly includes free L-phenylalanine.

[0037] The methionine source (specifically, methionine) particularly includes L-methionine, and the L-methionine particularly includes free L-methionine.

[0038] The ornithine source (specifically ornithine) particularly includes L-ornithine, which particularly includes L-ornithine hydrochloride.

[0039] Examples of tyrosine sources (specifically, tyrosine or dipeptides containing tyrosine as a constituent element) include, in particular, L-tyrosine and glycyl-L-tyrosine. Examples of L-tyrosine include, in particular, free L-tyrosine and L-tyrosine disodium. Examples of L-tyrosine disodium include, in particular, L-tyrosine disodium dihydrate. Examples of glycyl-L-tyrosine include, in particular, free glycyl-L-tyrosine. Examples of free glycyl-L-tyrosine include, in particular, free glycyl-L-tyrosine dihydrate.

[0040] That is, the composition of the present invention may have, for example, one or more properties selected from the group consisting of the following properties (1) to (10): (1) the cysteine ​​is free L-cysteine ​​or L-cysteine ​​hydrochloride; (2) the glycine is free glycine or free glycyl-L-tyrosine; (3) the asparagine is free L-asparagine; (4) the glutamic acid is monosodium L-glutamate; (5) the lysine is L-lysine hydrochloride; (6) the phenylalanine is free L-phenylalanine; (7) the methionine is free L-methionine; (8) the ornithine is L-ornithine hydrochloride; (9) the tyrosine is L-tyrosine disodium; (10) the glycine-containing dipeptide is free glycyl-L-tyrosine.

[0041] Furthermore, the composition of the present invention may have one or more properties selected from the group consisting of the following properties (1a) to (10a): (1a) the cysteine ​​is free L-cysteine ​​anhydrate or L-cysteine ​​hydrochloride monohydrate; (2a) the glycine is free glycine or free glycyl-L-tyrosine; (3a) the asparagine is free L-asparagine monohydrate; (4a) the glutamic acid is L-glutamic acid monosodium monohydrate; (5a) the lysine is L-lysine hydrochloride; (6a) the phenylalanine is free L-phenylalanine; (7a) the methionine is free L-methionine; (8a) the ornithine is L-ornithine hydrochloride; (9a) the tyrosine is L-tyrosine disodium dihydrate; (10a) The glycine-containing dipeptide is glycyl-L-tyrosine dihydrate in the free form.

[0042] The properties (1a) to (10a) can be examples of the properties (1) to (10), respectively.

[0043] The amino acid source may be commercially available or may be obtained by appropriate production. The method for producing the amino acid source is not particularly limited. The amino acid source can be produced, for example, by chemical synthesis, enzymatic reaction, fermentation, extraction, or a combination thereof. Specifically, the amino acid source can be produced, for example, by culturing a microorganism capable of producing the amino acid source and recovering the amino acid source from the culture medium or cells. The amino acid source may be purified to a desired degree or not. That is, a purified product may be used as the amino acid source, or a material containing the amino acid source may be used. For example, the amino acid source may contain an amino acid source in an amount of 1% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, 90% (w / w) or more, or 95% (w / w) or more.

[0044] α-Ketoglutaric acid may be used in its free form, as a salt, or in a combination thereof. That is, unless otherwise specified, the term "α-ketoglutaric acid" may refer to α-ketoglutaric acid in its free form, its salt, or a combination thereof. The same description of salts of α-ketoglutaric acid with respect to the acidic group in the salts of amino acid sources can be applied mutatis mutandis to salts of α-ketoglutaric acid. Salts of α-ketoglutaric acid particularly include salts with alkali metals and alkaline earth metals. Salts of α-ketoglutaric acid more particularly include salts with alkali metals such as sodium salts and potassium salts. Salts of α-ketoglutaric acid more particularly include sodium salts. Sodium salts of α-ketoglutaric acid include monosodium salts and disodium salts. Sodium salts of α-ketoglutaric acid particularly include monosodium salts. Potassium salts of α-ketoglutaric acid include monopotassium salts and dipotassium salts. Furthermore, α-ketoglutaric acid may be used as an anhydrate, a hydrate, or a combination thereof. That is, the term "α-ketoglutaric acid" (e.g., "free α-ketoglutaric acid" or "salt of α-ketoglutaric acid") may encompass anhydrate and anhydrate unless otherwise specified. Hydrates of α-ketoglutaric acid include monohydrate and dihydrate. Specific examples of α-ketoglutaric acid (particularly salts of α-ketoglutaric acid) include monosodium α-ketoglutarate anhydrate and disodium α-ketoglutarate dihydrate. Note that when property (A) is selected, α-ketoglutaric acid is a salt of α-ketoglutaric acid. α-ketoglutaric acid may be in a form appropriate for the use mode, such as ions, when using the composition of the present invention (e.g., during the cultivation of animal cells).

[0045] As α-ketoglutaric acid, commercially available products may be used, or those obtained by appropriate production may be used. The method for producing α-ketoglutaric acid is not particularly limited. α-ketoglutaric acid can be produced, for example, by chemical synthesis, enzymatic reaction, fermentation, extraction, or a combination thereof. Specifically, α-ketoglutaric acid can be produced, for example, by culturing a microorganism capable of producing α-ketoglutaric acid and recovering α-ketoglutaric acid from the culture medium or the bacterial cells. α-ketoglutaric acid may or may not be purified to a desired degree. That is, as α-ketoglutaric acid, a purified product may be used, or a material containing α-ketoglutaric acid may be used. As the α-ketoglutaric acid, for example, a material having an α-ketoglutaric acid content of 1% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, 90% (w / w) or more, or 95% (w / w) or more may be used.

[0046] The composition of the present invention may consist of an active ingredient, or may contain ingredients other than the active ingredient. Ingredients other than the active ingredient are also referred to as "additional ingredients."

[0047] The additional components are not particularly limited as long as they do not impair the object of the present invention. The additional components can be appropriately selected depending on various conditions, such as the type of animal cells and the mode of use of the composition of the present invention. Examples of the additional components include medium components.

[0048] Culture medium components include a carbon source, an amino acid source, vitamins, inorganic components, a pH buffer, growth factors, serum, serum albumin, a selective agent, and a gene expression inducer. Examples of carbon sources include sugars such as glucose. The amino acid source is as described above. The additional amino acid source may or may not cause caking when mixed with free α-ketoglutaric acid. The additional amino acid source may be one that is not selected as the active amino acid source. For example, if cysteine ​​is selected as the active amino acid source, one or more amino acid sources other than cysteine ​​may be selected as additional components. Examples of additional amino acid sources include a tyrosine source. Vitamins include vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin E, and vitamin K, as well as their precursors. Inorganic components include sodium, potassium, calcium, magnesium, phosphorus, and various trace elements (e.g., Co, Cu, F, Fe, Mn, Mo, Ni, Se, Si, Ni, Bi, V, and Zn). pH buffers include sodium bicarbonate, phosphate, and HEPES. Growth factors include insulin, IGF-1, and FGF.

[0049] If the additional component can form a salt, the additional component may be used in its free form, as a salt, or a combination thereof. In other words, unless otherwise specified, the term "additional component" may refer to the additional component in its free form, its salt, or a combination thereof. With regard to the salt of the additional component, the descriptions regarding the salt of the amino acid source and the salt of α-ketoglutaric acid may be applied mutatis mutandis. Furthermore, if the additional component can form a hydrate, the additional component may be used in its anhydrate form, as well as a combination thereof. In other words, the term "additional component" (e.g., "additional component in its free form" or "salt of the additional component") may encompass anhydrates and hydrates, unless otherwise specified. With regard to the hydrate of the additional component, the descriptions regarding the hydrate of the amino acid source and the hydrate of α-ketoglutaric acid may be applied mutatis mutandis. When used, the additional component may be in a form appropriate for the mode of use, such as an ion.

[0050] In one embodiment, the composition of the present invention may have property (A) to prevent caking of the composition of the present invention that may occur due to the presence of additional ingredients, such as an amino acid source.

[0051] As the additional component, one component may be used, or two or more components may be used in combination.

[0052] The composition of the present invention can be produced, for example, by appropriately mixing the active ingredients and, optionally, additional ingredients. That is, examples of methods for producing the composition of the present invention include methods for producing the composition of the present invention that include mixing the active ingredients (i.e., mixing the amino acid source and α-ketoglutaric acid). Each active ingredient may or may not be pre-mixed with the additional ingredient before mixing with other active ingredients. That is, in the expression "mixing active ingredients," each active ingredient may be pre-mixed with the additional ingredient. Furthermore, the additional ingredient may be further mixed after mixing of the active ingredients.

[0053] The composition of the present invention may be formulated as appropriate, for example. When formulating, additives may be used as appropriate. Examples of additives include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, and surfactants. The additives can be selected as appropriate depending on various conditions, such as the shape of the composition of the present invention.

[0054] The composition of the present invention may be in a solid form, such as a powder, flake, tablet, etc. The composition of the present invention may be particularly powdered, for example, when at least an anti-caking effect is to be obtained.

[0055] The content and content ratio of each component (i.e., the active ingredient and optional additional ingredients) in the composition of the present invention are not particularly limited as long as the stability-improving effect is obtained. The content and content ratio of each component in the composition of the present invention can be appropriately set depending on various conditions such as the type of animal cell and the mode of use of the composition of the present invention.

[0056] The total content of the active ingredients in the composition of the present invention is greater than 0% (w / w) and less than 100% (w / w). The total content of the active ingredients in the composition of the present invention may be, for example, 1% (w / w) or more, 2% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, 20% (w / w) or more, 30% (w / w) or more, 50% (w / w) or more, or 70% (w / w) or more, or 100% (w / w) or less, 99.9% (w / w) or less, 90% (w / w) or less, 70% (w / w) or less, 50% (w / w) or less, 30% (w / w) or less, 20% (w / w) or less, 10% (w / w) or less, or 5% (w / w) or less, or any compatible combination thereof. The total content of the active ingredients in the composition of the present invention may be, for example, 1 to 10% (w / w), 10 to 30% (w / w), 30 to 50% (w / w), 50 to 70% (w / w), 70 to 90% (w / w), or 70 to 100% (w / w). The total content of the active ingredients in the composition of the present invention may be, for example, 1 to 100% (w / w), 5 to 90% (w / w), or 10 to 70% (w / w).

[0057] The content of α-ketoglutaric acid in the composition of the present invention may be, for example, in terms of molar ratio, 0.5 times or more, 1 time or more, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5 times or more, 5.5 times or more, 6 times or more, 6.5 times or more, 7 times or more, 7.5 times or more, 8 times or more, 8.5 times or more, 9 times or more, It may be 9.5 times or more, or 10 times or more, or 20 times or less, 15 times or less, 12 times or less, 10 times or less, 9.5 times or less, 9 times or less, 8.5 times or less, 8 times or less, 7.5 times or less, 7 times or less, 6.5 times or less, 6 times or less, 5.5 times or less, 5 times or less, 4.5 times or less, 4 times or less, 3.5 times or less, 3 times or less, 2.5 times or less, or 2 times or less, or any compatible combination thereof. Specifically, the content of α-ketoglutaric acid in the composition of the present invention may be, for example, in molar ratio, 0.5 to 2 times, 1 to 2 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 3 times, 3 to 3.5 times, 3.5 to 4 times, 4 to 4.5 times, 4.5 to 5 times, 5 to 5.5 times, 5.5 to 6 times, 6 to 6.5 times, 6.5 to 7 times, 7 to 7.5 times, 7.5 to 8 times, 8 to 8.5 times, 8.5 to 9 times, 9 to 9.5 times, 9.5 to 10 times, 10 to 12 times, 10 to 15 times, or 10 to 20 times the content of the amino acid source (e.g., cysteine) in the composition of the present invention. Specifically, the content of α-ketoglutaric acid in the composition of the present invention may be, for example, 1 to 20 times, 2 to 15 times, 3 to 10 times, or 4 to 7 times the molar ratio of the content of the amino acid source (e.g., cysteine) in the composition of the present invention. When two or more amino acid sources are used as active ingredients, the "content of amino acid sources" referred to here means the total content of those amino acid sources. However, when two or more amino acid sources are used as active ingredients, the content of α-ketoglutaric acid in the composition of the present invention can be set independently of the content of those amino acid sources within the ranges of the α-ketoglutaric acid content exemplified above.

[0058] The content of each active ingredient in the composition of the present invention can be set, for example, so as to obtain the above-mentioned ratio of the total content of the active ingredients in the composition of the present invention to the content of the active ingredient.

[0059] The content of each component (i.e., the active component and any additional component) in the composition of the present invention can be set, for example, so as to obtain the concentration of each component in the medium in the method of the present invention described below.

[0060] The active ingredients are contained in the composition of the present invention in a mixed state. The additional ingredients may be contained in the composition of the present invention in a mixed state with the active ingredient, or may be contained in the composition of the present invention separately from the active ingredient. When the composition of the present invention contains two or more additional ingredients, the additional ingredients may be contained in the composition of the present invention in a mixed state, or may be contained separately, or in any combination. For example, the composition of the present invention may be provided as a set consisting of a package of the active ingredient and a package of the additional ingredient. In such cases, the ingredients contained in the set can be used together as appropriate during use. The composition of the present invention may also be provided in a form in which a moisture absorbent is enclosed within the package (specifically, within at least the package of the active ingredient). The composition of the present invention (specifically, at least the active ingredient) may also be provided in a form in which it is enclosed in a packaging material with moisture-absorbing function.

[0061] When a material containing an active ingredient is used, the amount of the active ingredient (e.g., content (concentration) or amount used) is calculated based on the amount of the active ingredient itself in the material. When the active ingredient forms a salt or hydrate, the amount of the active ingredient (e.g., content (concentration) or amount used) is calculated based on the mass of the salt or hydrate converted to the mass of an equimolar free anhydrate.

[0062] <2> Method of the Present Invention The method of the present invention is a method comprising utilizing the composition of the present invention.

[0063] The composition of the present invention can be used, for example, for culturing animal cells. That is, the method of the present invention may be a method for culturing animal cells, which comprises culturing animal cells using the composition of the present invention.

[0064] In one aspect, a target substance may be produced by culturing animal cells. That is, when animal cells have the ability to produce a target substance, the target substance can be produced by culturing the cells. That is, one aspect of the method of the present invention (specifically, a method for culturing animal cells) may be a method for producing a target substance, which includes culturing animal cells capable of producing the target substance using the composition of the present invention, and recovering the target substance.

[0065] The target substance is not particularly limited as long as it can be produced using animal cells. Examples of the target substance include proteins and viruses. A protein produced as a target substance is also referred to as a "target protein." A virus produced as a target substance is also referred to as a "target virus."

[0066] The animal cells are not particularly limited. They can be appropriately selected depending on various conditions, such as the intended use of the animal cells. For example, when animal cells are used to produce a target protein, the animal cells are not particularly limited as long as they can express the target protein. Animal cells are also referred to as "hosts," "expression hosts," or "host cells." Examples of animals include mammals, birds, amphibians, and insects. Examples of animals include mammals in particular. Examples of mammals include rodents, primates, and various other mammals. Examples of rodents include hamsters, mice, rats, and guinea pigs. Examples of hamsters include Chinese hamsters. Examples of primates include humans, monkeys, and chimpanzees. Examples of monkeys include African green monkeys. Examples of other mammals include dogs. Examples of birds include chickens. Examples of amphibians include African clawed frogs. Examples of insects include the fall armyworm (Spodoptera frugiperda). Furthermore, the tissues or cells from which the animal cells are derived are not particularly limited. Examples of tissues or cells from which animal cells are derived include ovaries, kidneys, adrenal glands, tongue epithelium, olfactory epithelium, pineal gland, thyroid gland, melanocytes, skin, spleen, liver, lungs, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testes, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood (including umbilical cord blood), bone marrow, heart, eyes, brain, and nervous tissue. Animal cells may or may not be differentiated. Specific examples of animal cells include germ cells, somatic cells, stem cells, and progenitor cells. Germ cells include sperm and eggs. Somatic cells include fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes, osteocytes, pericytes, dendritic cells, adipocytes, mesenchymal cells, epithelial cells, epidermal cells (e.g., keratinocytes, corneocytes, etc.), endothelial cells, vascular endothelial cells, hepatic parenchymal cells, chondrocytes, cumulus cells, nerve cells, glial cells, oligodendrocytes, microglia, astrocytes, cardiac cells, esophageal cells, muscle cells (e.g., smooth muscle cells, skeletal muscle cells), pancreatic beta cells, melanocytes, and mononuclear cells.Stem cells include adult stem cells such as hematopoietic stem cells, satellite cells, neural stem cells, mesenchymal stem cells, mammary stem cells, olfactory mucosa stem cells, neural crest stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, germline stem cells, intestinal stem cells, and hair follicle stem cells; pluripotent stem cells such as embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, and induced pluripotent stem cells (iPS cells); and cancer stem cells. Progenitor cells include satellite cells, pancreatic progenitor cells, vascular progenitor cells, endothelial progenitor cells, and hematopoietic progenitor cells (e.g., CD34-positive cells derived from umbilical cord blood). Chinese hamster cells include Chinese hamster ovary-derived cell lines (CHO). Specific examples of CHO include CHO-DG44, CHO-K1, CHO DUX (DHFR-), CHO-S, and CHO-MK. Mouse cells include NS0 cells derived from mouse myeloma. Human cells include human embryonic kidney (HEK)-derived cell lines. Specific examples of HEK include HEK293 and HEK293T. Canine cells include MDCK cells derived from canine kidneys. African green monkey cells include African green monkey kidney-derived cell lines (COS). Specific examples of COS include COS-1. Xenopus laevis cells include Xenopus laevis oocytes. Fall armyworm cells include Sf9 cells, Sf21 cells, and SF+ cells derived from the ovaries of Fall armyworm.

[0067] "Animal cells capable of producing a target substance" refers to animal cells capable of producing a target substance. Specifically, "animal cells capable of producing a target substance" may refer to animal cells capable of producing a target substance (e.g., expressing a target protein) and accumulating the target substance in the culture medium to an extent that the target substance can be recovered when cultured in a medium. "Accumulation in the culture medium" may specifically refer to accumulation in the medium, on the cell surface, intracellularly, or a combination thereof. Accumulation of a target substance outside the cell (e.g., in the medium or on the cell surface) is also referred to as "secretion" or "secretory production" of the target substance. In other words, animal cells may have the ability to secrete and produce a target substance (the ability to secrete and produce a target substance). The amount of target substance accumulated in the culture medium may be, for example, 10 μg / L or more, 1 mg / L or more, 100 mg / L or more, or 1 g / L or more. Animal cells may be capable of producing one target substance, or two or more target substances.

[0068] The animal cells may be those that inherently have the ability to produce a target substance, or may be those that have been modified to have the ability to produce a target substance. Furthermore, the animal cells may be those that have been modified so that their inherent ability to produce a target substance is enhanced. Animal cells that have the ability to produce a target substance can be obtained, for example, by imparting the ability to produce a target substance to the animal cells described above, or by enhancing the ability of the animal cells described above to produce a target substance. For example, the ability to produce a target protein can be imparted or enhanced by introducing a gene encoding the target protein. A gene encoding a target protein is also referred to as a "target protein gene." Furthermore, for example, the ability to produce a target virus can be imparted by infecting animal cells with the target virus.

[0069] The target protein is not particularly limited as long as it can be expressed in an animal cell as a host. The protein may be derived from the host or may be a heterologous protein. A "heterologous protein" refers to a protein that is exogenous to the host producing the protein (i.e., the animal cell capable of producing the target protein). The target protein may be, for example, a naturally occurring protein, a modified version of a naturally occurring protein, or a protein with an artificially designed amino acid sequence. The target protein may be, for example, a protein derived from a microorganism, a plant, an animal, or a virus. The target protein may particularly be a human protein. The target protein may be a monomeric protein or a multimeric protein. The target protein may be a secreted protein or a non-secreted protein. The term "protein" also includes so-called peptides, such as oligopeptides and polypeptides.

[0070] Specific examples of target proteins include enzymes, physiologically active proteins, receptor proteins, antigenic proteins, and other proteins.

[0071] Examples of the enzyme include cellulase, xylanase, transglutaminase, protein glutaminase, protein asparaginase, isomaltodextranase, protease, endopeptidase, exopeptidase, aminopeptidase, carboxypeptidase, collagenase, chitinase, γ-glutamylvaline synthetase, glutamic acid-cysteine ​​ligase, and glutathione synthetase.

[0072] Physiologically active proteins include growth factors, hormones, cytokines, antibody-related molecules, and antibody mimetics.

[0073] Growth factors include epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), transforming growth factor (TGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), and acidic fibroblast growth factor (AGF). These include aFGF or FGF1, basic fibroblast growth factor (bFGF or FGF2), fibroblast growth factor (FGF-4), keratinocyte growth factor (KGF-1 or FGF7, KGF-2 or FGF10), hepatocyte growth factor (HGF), stem cell factor (SCF), and activin. Activins include activin A, C, and E.

[0074] Examples of hormones include insulin, glucagon, somatostatin, human growth hormone (hGH), parathyroid hormone (PTH), calcitonin, and exenatide.

[0075] Cytokines include interleukins, interferons, and tumor necrosis factors (TNFs).

[0076] Furthermore, the physiologically active protein may be the entire protein or a portion thereof. Examples of the portion of the protein include a physiologically active portion. Specific examples of the physiologically active portion include teriparatide, a physiologically active peptide consisting of the N-terminal 34 amino acid residues of the mature form of parathyroid hormone (PTH).

[0077] The term "antibody-related molecule" refers to a protein containing a molecular species consisting of a single domain selected from the domains constituting a complete antibody or a combination of two or more domains. The domains constituting a complete antibody include the heavy chain domains VH, CH1, CH2, and CH3, and the light chain domains VL and CL. An antibody-related molecule may be a monomeric or multimeric protein, as long as it contains the above-mentioned molecular species. When the antibody-related molecule is a multimeric protein, it may be a homomultimer consisting of a single type of subunit, or a heteromultimer consisting of two or more types of subunits. Specific examples of antibody-related molecules include complete antibodies, Fab, F(ab'), F(ab')2, Fc, dimers consisting of a heavy chain (H chain) and a light chain (L chain), Fc fusion proteins, heavy chains (H chains), light chains (L chains), single-chain Fvs (scFvs), sc(Fv)2, disulfide-linked Fvs (sdFvs), diabodies, and VHH fragments (nanobodies (registered trademark)). More specifically, antibody-related molecules include trastuzumab, adalimumab, nivolumab, VHH antibody N15, and VHH antibody 9g8. Fc fusion proteins include fusion proteins of various target proteins and Fc regions, as exemplified herein. Specific examples of Fc fusion proteins include fusion proteins of Notch ligands and Fc regions, as described below. Fusion proteins of Notch ligands and Fc regions include DLL4-Fc (i.e., fusion proteins of DLL4 and Fc regions).

[0078] "Antibody mimetics" may refer to organic compounds that can specifically bind to antigens but are not structurally related to antibodies. Specific examples of antibody mimetics include the Z domain of Protein A (Affibody). More specific examples of antibody mimetics include ZHER2 affibody.

[0079] Receptor proteins include receptor proteins for physiologically active proteins and other physiologically active substances. Other physiologically active substances include neurotransmitters such as dopamine. Receptor proteins may also be orphan receptors for which the corresponding ligand is unknown.

[0080] The antigen protein is not particularly limited as long as it can induce an immune response. The antigen protein can be appropriately selected depending on, for example, the target of the expected immune response. The antigen protein can be used, for example, as a vaccine.

[0081] Other proteins include liver-type fatty acid-binding protein (LFABP), fluorescent proteins, immunoglobulin-binding proteins, albumin, fibroin-like proteins, and extracellular proteins. Fluorescent proteins include green fluorescent protein (GFP). Immunoglobulin-binding proteins include protein A, protein G, and protein L. Albumins include human serum albumin. Notch ligands include DLL1, DLL3, DLL4, Jagged-1, and Jagged-2. Notch ligands may be configured as fusion proteins with an Fc region, for example. Fibroin-like proteins include those disclosed in WO2017 / 090665 and WO2017 / 171001.

[0082] Extracellular proteins include fibronectin, vitronectin, collagen, osteopontin, laminin, and their partial sequences. Laminin is a protein with a heterotrimeric structure consisting of an α chain, a β chain, and a γ chain. Examples of laminins include mammalian laminins. The subunit chains of laminin (i.e., α chains, β chains, and γ chains) include five types of α chains (α1-α5), three types of β chains (β1-β3), and three types of γ chains (γ1-γ3). Laminin forms various isoforms depending on the combination of these subunit chains. Specific examples of laminins include laminin 111, laminin 121, laminin 211, laminin 213, laminin 221, laminin 311, laminin 321, laminin 332, laminin 411, laminin 421, laminin 423, laminin 511, laminin 521, and laminin 523. Laminin subsequences include laminin E8, the E8 fragment of laminin. Laminin E8 is a heterotrimeric protein consisting of an α-chain E8 fragment (α-chain E8), a β-chain E8 fragment (β-chain E8), and a γ-chain E8 fragment (γ-chain E8). The subunit chains of laminin E8 (i.e., α-chain E8, β-chain E8, and γ-chain E8) are collectively referred to as the "E8 subunit chain." Examples of E8 subunit chains include E8 fragments of the laminin subunit chains listed above. Laminin E8 is composed of various isoforms, depending on the combination of these E8 subunit chains. Specific examples of laminin E8 include laminin 111E8, laminin 121E8, laminin 211E8, laminin 221E8, laminin 332E8, laminin 421E8, laminin 411E8, laminin 511E8, and laminin 521E8. The numbers in the name of laminin E8 indicate, from left to right, the α chain, β chain, and γ chain. For example, "laminin 511E8" refers to the E8 fragment of laminin 511, specifically a protein with a heterotrimeric structure consisting of the E8 fragment of the α5 chain (α5 chain E8), the E8 fragment of the β1 chain (β1 chain E8), and the E8 fragment of the γ1 chain (γ1 chain E8).

[0083] "α chain E8" may refer to the region near the C-terminus of the α chain, specifically a C-terminal fragment excluding globular domains 4 and 5 of the α chain, or more specifically a fragment of 780 to 830 (e.g., 790 to 800) amino acid residues at the C-terminus, excluding globular domains 4 and 5 of the α chain. "β chain E8" may refer to a C-terminal fragment of the β chain, specifically a fragment of 220 to 230 amino acid residues at the C-terminus of the β chain. "γ chain E8" may refer to a C-terminal fragment of the γ chain, specifically a fragment of 240 to 250 amino acid residues at the C-terminus of the γ chain. Examples of human α5 chain E8 include the region from amino acid numbers 2534 to 3327 in GenBank Accession No. NP_005551. Examples of human β1 chain E8 include the region from amino acid numbers 1561 to 1786 in GenBank Accession No. NP_002282. An example of human γ1 chain E8 is the region from amino acid numbers 1364 to 1609 of GenBank Accession No. NP_002284.

[0084] The target protein may be, for example, a protein having a known or naturally occurring amino acid sequence of the above-mentioned protein. Alternatively, the target protein may be, for example, a variant of a protein having a known or naturally occurring amino acid sequence of the above-mentioned protein. Examples of variants include proteins having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added at one or several positions in the known or naturally occurring amino acid sequence. Specifically, "one or several" may mean, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3. Examples of variants include proteins having an amino acid sequence that is, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identical to the entire known or naturally occurring amino acid sequence. It should be noted that a protein identified by its biological species of origin is not limited to the protein itself found in that biological species, but also includes proteins having the amino acid sequence of a protein found in that biological species and variants thereof. Variants may or may not be found in that biological species. That is, for example, a "human-derived protein" is not limited to the protein itself found in humans, but includes proteins having the amino acid sequence of a protein found in humans and variants thereof.

[0085] The "identity" between amino acid sequences refers to the identity between amino acid sequences calculated using blastp with default scoring parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment).

[0086] The target protein gene is not particularly limited as long as it encodes the target protein described above. The target protein gene may be, for example, a gene having a known or natural nucleotide sequence of a gene encoding the protein described above. The target protein gene may also be, for example, a variant of a gene having a known or natural nucleotide sequence of a gene encoding the protein described above. The target protein gene may be modified, for example, to encode a protein having a variant sequence such as those exemplified above. The target protein gene may be one in which any codon has been replaced with an equivalent codon. The target protein gene may be modified, for example, to have an optimal codon depending on the codon usage frequency of the host cell.

[0087] In the present invention, the term "gene" is not limited to DNA and may include any polynucleotide as long as it encodes a corresponding expression product. In other words, a "target protein gene" may refer to any polynucleotide that encodes a target protein. A target protein gene may be DNA, RNA, or a combination thereof. A target protein gene may be single-stranded or double-stranded. A target protein gene may be single-stranded DNA or single-stranded RNA. A target protein gene may be double-stranded DNA, double-stranded RNA, or a hybrid strand consisting of a DNA strand and an RNA strand. A target protein gene may contain both DNA residues and RNA residues in a single polynucleotide strand. A target protein gene may or may not contain an intron. The form of the target protein gene can be selected appropriately depending on various conditions, such as the means for expressing the target protein.

[0088] Unless otherwise specified, the expression "having an (amino acid or nucleotide) sequence" means "including the (amino acid or nucleotide) sequence" and also includes the case where "consisting of the (amino acid or nucleotide) sequence."

[0089] The target protein is expressed from the target protein gene. That is, animal cells capable of producing the target protein have the target protein gene. Specifically, animal cells capable of producing the target protein have the target protein gene in an expressible manner. It is sufficient for the animal cells capable of producing the target protein to have the target protein gene until the target protein is expressed to a desired extent. That is, the animal cells capable of producing the target protein may or may not have the target protein gene after the target protein is expressed. It is noted that "expression of the target protein gene" and "expression of the target protein" may be used synonymously.

[0090] A target protein gene can be obtained by cloning from an organism that has the target protein gene. Nucleic acids such as genomic DNA or cDNA containing the gene can be used for cloning. Alternatively, a target protein gene can be obtained by chemical synthesis (Gene, 60(1), 115-127 (1987)).

[0091] The obtained target protein gene can be used as is or after appropriate modification. That is, by modifying the target protein gene, its variants can be obtained. Gene modification can be performed by known techniques. For example, a desired mutation can be introduced into a target site in DNA by site-directed mutagenesis. Examples of site-directed mutagenesis include PCR-based methods (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and phage-based methods (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)). Alternatively, a variant of a target protein gene can be obtained directly by chemical synthesis.

[0092] The form in which a target protein gene is introduced into a host cell is not particularly limited. The target protein gene may be retained in the host cell in an expressible manner. Specifically, for example, when the target protein gene is introduced in a form requiring transcription of DNA or the like, the target protein gene may be retained in the host cell in an expressible manner under the control of a promoter that functions in the host cell. In the host cell, the target protein gene may be present extrachromosomally or may be introduced onto the chromosome. When two or more genes are introduced, each gene may be retained in the host cell in an expressible manner.

[0093] The promoter for expressing the target protein gene is not particularly limited as long as it functions in the host cell. A "promoter functional in the host cell" refers to a promoter that has promoter activity in the host cell. The promoter may be a promoter native to the host cell or a heterologous promoter. The promoter may be the native promoter of the target protein gene or a promoter of another gene. The promoter may be stronger than the native promoter of the target protein gene. Examples of promoters that function in animal cells include the SV40 promoter, EF1a promoter, RSV promoter, CMV promoter, and SRalpha promoter. Furthermore, highly active forms of native promoters may be obtained and used by using various reporter genes. Methods for evaluating promoter strength and examples of strong promoters are described in, for example, Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

[0094] A target protein gene can be introduced into a host cell using, for example, a vector containing the gene. A vector containing a target protein gene is also referred to as a "target protein gene expression vector." A target protein gene expression vector can be constructed, for example, by ligating a DNA fragment containing the target protein gene to a vector. The target protein gene can be introduced into a host cell by introducing the expression vector into the host cell. The vector may contain a marker such as a drug resistance gene. The vector may also contain an expression regulatory sequence such as a promoter for expressing the inserted gene. The vector can be selected appropriately depending on various conditions, such as the type of host cell and the mode of introduction of the target protein gene. Vectors that can be used for gene introduction into animal cells include plasmid vectors and viral vectors. Examples of viral vectors include retroviral vectors and adenoviral vectors. Examples of plasmid vectors include pcDNA series vectors (pcDNA3.1, etc.; Thermo Fisher Scientific), pBApo-CMV series vectors (Takara Bio), and pCI-neo (Promega). Depending on the type and configuration of the vector, the vector can be integrated into the chromosome of the host cell, can autonomously replicate extrachromosomally, or can be temporarily maintained extrachromosomally in the host cell. For example, vectors having a viral replication origin such as the SV40 replication origin can autonomously replicate extrachromosomally in animal cells. Specifically, for example, the pcDNA series vectors have the SV40 replication origin and can autonomously replicate extrachromosomally in host cells (e.g., COS-1 and HEK293T) that express the SV40 large T antigen.

[0095] Alternatively, a target protein gene can be introduced into a host cell by, for example, introducing a nucleic acid fragment containing the gene into the host cell. Such nucleic acid fragments include linear DNA and linear RNA. Examples of linear RNA include mRNA and cRNA.

[0096] The method for introducing nucleic acids such as vectors and nucleic acid fragments into host cells can be selected appropriately depending on various conditions such as the type of host cell. Methods for introducing nucleic acids such as vectors and nucleic acid fragments into animal cells include the DEAE-dextran method, calcium phosphate method, lipofection, electroporation, and microinjection. Furthermore, when the vector is a viral vector, the vector can be introduced into the host cell by infecting the host cell with the vector (virus).

[0097] Alternatively, cells inherently containing a target protein gene may be modified to increase the expression of the target protein gene. "Increased gene expression" means that the expression level of the gene per cell is increased compared to unmodified cells. "Unmodified cells" herein refer to control cells that have not been modified to increase the expression of the target gene. Examples of unmodified cells include wild-type cells and the original cells. Techniques for increasing the expression of a target protein gene include increasing the copy number of the target protein gene and improving the transcription efficiency or translation efficiency of the target protein gene. The copy number of the target protein gene can be increased by introducing the target protein gene into host cells. Introduction of the target protein gene can be performed as described above. The introduced target protein gene may be derived from the host cell or from a heterologous source. The transcription efficiency or translation efficiency of the target protein gene can be improved by modifying the expression regulatory sequence of the gene, such as a promoter. For example, the transcription efficiency of the target protein gene can be improved by replacing the promoter of the target protein gene with a stronger promoter.

[0098] In the method for culturing animal cells, animal cells are cultured using the composition of the present invention. That is, the animal cell culture is carried out using the composition of the present invention. The composition of the present invention may be used, for example, as a medium for culturing animal cells. That is, for example, when the composition of the present invention is a medium, animal cells may be cultured in the composition of the present invention (i.e., medium). That is, "culturing animal cells using the composition of the present invention" may mean, for example, culturing animal cells in the composition of the present invention, which is a medium. Furthermore, the composition of the present invention may be used, for example, as a medium additive for culturing animal cells. That is, for example, when the composition of the present invention is a medium additive, the composition of the present invention (i.e., medium additive) may be added to a medium, and animal cells may be cultured in the medium to which the composition of the present invention has been added. That is, "culturing animal cells using the composition of the present invention" may mean, for example, adding the composition of the present invention, which is a medium additive, to a medium, and culturing animal cells in the medium to which the composition of the present invention has been added.

[0099] "Culturing animal cells" is not limited to those aimed at the proliferation of animal cells, but may also include those not aimed at the proliferation of animal cells, such as the maintenance of animal cells or the production of target substances using animal cells.

[0100] The medium composition and culture conditions are not particularly limited as long as the purpose of animal cell culture is achieved, except that the culture is performed using the composition of the present invention. For example, when the purpose is to grow animal cells, the medium composition and culture conditions can be configured to grow the animal cells. Furthermore, when the purpose is to maintain animal cells, the medium composition and culture conditions can be configured to maintain the animal cells (i.e., to allow the animal cells to survive). Furthermore, when the purpose is to produce a target substance such as a target protein, the medium composition and culture conditions can be configured to produce the target substance (e.g., to express the target protein if the target substance is a target protein). When the purpose is not to grow animal cells, the animal cells may or may not grow during culture. Even when the purpose is not to grow animal cells, the animal cells may typically grow during culture. The medium composition and culture conditions can be appropriately set depending on various conditions, such as the type of animal cells. Except for using the composition of the present invention, the culture can be performed using, for example, a conventional medium and conventional conditions used for animal cell culture, either directly or with appropriate modifications.

[0101] The culture may be divided into a seed culture and a main culture. When the culture is divided into a seed culture and a main culture, the composition of the present invention may be used in either or both of the seed culture and the main culture. When the culture is divided into a seed culture and a main culture, the composition of the present invention may be used, particularly at least in the main culture. When the culture is divided into a seed culture and a main culture, descriptions of the culture (e.g., "culture period (period of culture)" and "start of culture") may apply to either the seed culture or the main culture unless otherwise specified. The culture conditions for the seed culture and the main culture may or may not be the same. Furthermore, when the purpose is to produce a target substance such as a target protein, the target substance only needs to be produced during at least the period of the main culture. For example, animal cells may be sufficiently grown in a seed culture, and then the target substance such as a target protein may be produced in a main culture.

[0102] Culture can be performed by batch culture, fed-batch culture, continuous culture, or a combination thereof. Continuous cultures include perfusion culture and chemostat culture. The medium used at the start of culture is also called the "initial medium" or "basal medium." The medium supplied to a culture system (e.g., the initial medium) in fed-batch culture is also called the "feed medium." The medium supplied to a culture system (e.g., the initial medium) in continuous culture (not limited to perfusion culture) is also called the "perfusion medium." Supplying a feed medium or perfusion medium to a culture system in fed-batch or continuous culture is also simply called "medium supply." Medium supply may be performed throughout the entire culture period or only for a portion of the culture period. Medium supply may be performed continuously or intermittently. During culture (particularly continuous culture such as perfusion culture), the culture medium may be withdrawn. The culture medium may be withdrawn throughout the entire culture period or only during a portion of the culture period. The culture medium may be withdrawn continuously or intermittently. The culture medium withdrawal and the medium supply may or may not be carried out simultaneously. When culture is carried out in separate stages of seed culture and main culture, the culture forms of the seed culture and the main culture may or may not be the same.

[0103] The culture can be carried out, for example, using a liquid medium.

[0104] The media used for culture, for example, the basal medium, the feed medium, and the perfusion medium, can be selected independently.

[0105] The medium used for the culture may be a commercially available medium or an appropriately prepared medium. Examples of commercially available media include animal cell culture media such as D-MEM (Dulbecco's Modified Eagle Medium), CELLiST Basal Media BASAL3, BASAL4P, and BASAL10 (Ajinomoto Co., Inc.), Opti-MEM (Thermo Fisher Scientific), RPMI 1640 (Thermo Fisher Scientific), CD293 (Thermo Fisher Scientific), CHO-S-SFMII (Thermo Fisher Scientific), CHO-SF (Sigma-Aldrich), EX-CELL CD CHO (Sigma-Aldrich), EX-CELL™302 (Sigma-Aldrich), IS CHO-CD (Irvine Scientific), and IS CHO-CDXP (Irvine Scientific).

[0106] The medium used for culture may be, for example, the composition of the present invention (specifically, the composition of the present invention that is a medium). That is, when the composition of the present invention is a medium, the composition of the present invention may be used for culture as is, or may be appropriately prepared as a liquid medium of a desired composition. For example, the composition of the present invention may be diluted with an aqueous medium such as water or an aqueous buffer solution to prepare a liquid medium and use it for culture. The composition of the present invention may be used as one or more media selected from a basal medium, a feed medium, and a perfusion medium. That is, for example, in the case of fed-batch culture, the composition of the present invention may be used as one or both of the basal medium and the feed medium. Furthermore, for example, in the case of continuous culture, the composition of the present invention may be used as one or both of the basal medium and the perfusion medium.

[0107] Furthermore, the medium used for culture may be, for example, a medium supplemented with the composition of the present invention (specifically, the composition of the present invention as a medium additive). The medium to which the composition of the present invention is added may be a commercially available medium or an appropriately prepared medium. The composition of the present invention may be added to one or more media selected from a basal medium, a feed medium, and a perfusion medium. That is, for example, in the case of fed-batch culture, the composition of the present invention may be added to one or both of the basal medium and the feed medium. Furthermore, for example, in the case of continuous culture, the composition of the present invention may be added to one or both of the basal medium and the perfusion medium.

[0108] The medium used for culture may contain, for example, an amino acid source and / or α-ketoglutaric acid. For example, when the medium used for culture is the composition of the present invention, or when the medium used for culture is a medium supplemented with the composition of the present invention, the medium used for culture contains an amino acid source and α-ketoglutaric acid. The medium used for culture may also contain, for example, various medium components. The medium components are as described above in the description of the composition of the present invention. The medium exemplified above may be used for culture after, for example, adding components such as an amino acid source, α-ketoglutaric acid, and various medium components as appropriate.

[0109] Various components such as amino acid sources, α-ketoglutaric acid, and medium components may all be contained in the initial medium, feed medium, perfusion medium, or a combination thereof. That is, various components such as amino acid sources may be supplied to the medium alone or in any combination during the culture process. These components may all be supplied once, multiple times, or continuously. The compositions (e.g., the types and / or concentrations of the components) of the initial medium, feed medium, and perfusion medium may or may not be the same. That is, the types of components contained in the initial medium may or may not be the same as the types of components contained in the feed medium or perfusion medium. Furthermore, the concentrations of each component contained in the initial medium may or may not be the same as the concentrations of each component contained in the feed medium or perfusion medium. For example, when a feed medium is used for perfusion culture, the compositions of the initial medium and the feed medium may be the same. Two or more feed mediums or perfusion media with different compositions (e.g., types and / or concentrations of components contained therein) may also be used. For example, when multiple feed medium or perfusion medium supplies are intermittently performed, the composition of the feed medium or perfusion medium may or may not be the same for each supply. Furthermore, various components such as amino acid sources may be supplied to the medium in a form not contained in the feed medium or perfusion medium, such as a powder. For example, various components such as amino acid sources may be supplied to the medium by adding the composition of the present invention.

[0110] The seeding amount of animal cells at the start of culture is, for example, 1 × 10 in terms of the number of viable cells. ３ cells / mL or more, 1×10 ４ cells / mL or more, 1×10 ５ cells / mL or more, 1×10 ６ cells / mL or more, or 1 x 10 ７ cells / mL or more, and may be 1 x 10 ８ cells / mL or less, 1×10 ７ cells / mL or less, 1×10 ６ cells / mL or less, 1×10 ５ cells / mL or less, or 1 x 10 ４The seeding amount of animal cells at the start of culture may be, for example, 1 x 10 cells / mL in terms of the number of viable cells. ３ ~1 x 10 ４ cells / mL, 1×10 ４ ~1 x 10 ５ cells / mL, 1×10 ５ ~1 x 10 ６ cells / mL, 1×10 ６ ~1 x 10 ７ cells / mL, or 1 x 10 ７ ~1 x 10 ８ The seeding amount of animal cells at the start of culture may be, for example, 1 × 10 cells / mL in terms of the number of viable cells. ３ ~1 x 10 ８ cells / mL, 1×10 ４ ~1 x 10 ７ cells / mL, or 1 x 10 ５ ~1 x 10 ６ The viable cell count may be measured using, for example, a viable cell autoanalyzer, Vi-CELL TM Measurement can be performed using XR (Beckman Coulter).

[0111] The culture may be carried out under, for example, 5 to 15% CO ２ CO etc. ２The cultivation may be carried out under a pH-containing atmosphere. The pH of the medium may be, for example, near neutral. "Near neutral" may mean, for example, pH 6 to 8, pH 6.5 to 7.5, or pH 6.8 to 7.2. The pH of the medium can be adjusted as needed during cultivation. The pH of the medium can be adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide. The cultivation temperature may be, for example, 30 to 38°C. The culture period may be, for example, 0.5 days or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 12 days or more, 15 days or more, or 20 days or more, or 60 days or less, 50 days or less, 40 days or less, 30 days or less, 25 days or less, 20 days or less, 15 days or less, 12 days or less, 10 days or less, 9 days or less, 8 days or less, or 7 days or less, or any compatible combination thereof. Specifically, the culture period may be, for example, 1 to 60 days, 3 to 25 days, or 5 to 20 days. During culture, expression of a gene, such as a target protein gene, may be induced as appropriate.

[0112] The concentration of the amino acid source in the medium may be, for example, 0.1 mM or more, 0.3 mM or more, 0.5 mM or more, 0.7 mM or more, 1 mM or more, 1.5 mM or more, 2 mM or more, 3 mM or more, 4 mM or more, or 5 mM or more, or 30 mM or less, 25 mM or less, 20 mM or less, 15 mM or less, 10 mM or less, 7 mM or less, 5 mM or less, 4 mM or less, 3 mM or less, 2 mM or less, 1.5 mM or less, or 1 mM or less, or a compatible combination thereof. The concentration of the amino acid source in the medium may be, for example, 0.1 to 1 mM, 0.3 to 1 mM, 0.5 to 1 mM, 0.7 to 1 mM, 1 to 1.5 mM, 1.5 to 2 mM, 2 to 3 mM, 3 to 4 mM, 4 to 5 mM, 5 to 7 mM, 5 to 10 mM, 5 to 15 mM, 5 to 20 mM, 5 to 25 mM, or 5 to 30 mM. The concentration of the amino acid source in the medium may be, for example, 0.1 to 10 mM, 0.3 to 5 mM, 0.5 to 3 mM, or 1 to 2 mM, or may be 0.1 to 30 mM, 0.3 to 25 mM, or 0.5 to 20 mM. When two or more amino acid sources are used as active ingredients, the term "amino acid source concentration" used herein refers to the total concentration of those amino acid sources. However, when two or more amino acid sources are used as active ingredients, the concentrations of those amino acid sources can be independently set within the ranges of the amino acid source concentrations exemplified above. Furthermore, the descriptions regarding the amino acid source concentrations exemplified above also apply mutatis mutandis to the concentrations of the amino acid sources that are additional components. Specifically, for example, the concentration of the cysteine ​​source in the medium may be 0.1 to 10 mM, 0.3 to 5 mM, 0.5 to 3 mM, or 1 to 2 mM. Specifically, for example, the concentration of the tyrosine source in the medium may be 0.1 to 30 mM, 0.3 to 25 mM, or 0.5 to 20 mM.

[0113] The concentration of α-ketoglutaric acid in the medium may be, for example, 0.1 mM or more, 0.3 mM or more, 0.5 mM or more, 0.7 mM or more, 1 mM or more, 1.5 mM or more, 2 mM or more, 3 mM or more, 4 mM or more, 5 mM or more, 7 mM or more, or 10 mM or more, or 50 mM or less, 30 mM or less, 20 mM or less, 15 mM or less, 10 mM or less, 7 mM or less, 5 mM or less, 4 mM or less, 3 mM or less, 2 mM or less, 1.5 mM or less, or 1 mM or less, or a compatible combination thereof. The α-ketoglutaric acid concentration in the medium may be, for example, 0.1 to 1 mM, 0.3 to 1 mM, 0.5 to 1 mM, 0.7 to 1 mM, 1 to 1.5 mM, 1.5 to 2 mM, 2 to 3 mM, 3 to 4 mM, 4 to 5 mM, 5 to 7 mM, 7 to 10 mM, 10 to 15 mM, 10 to 20 mM, 10 to 30 mM, or 10 to 50 mM. The α-ketoglutaric acid concentration in the medium may be, for example, 0.1 to 30 mM, 0.3 to 20 mM, 0.5 to 15 mM, or 1 to 10 mM.

[0114] Each of the various components, such as the amino acid source, may be contained in the medium throughout the entire culture period, or only during a portion of the culture period. In other words, "culture is carried out in a medium containing a certain component" means that the component is contained in the medium during at least a portion of the culture period, and does not necessarily have to be contained in the medium throughout the entire culture period. Each of the various components, such as the amino acid source, may be contained in the medium at the start of culture, or may be supplied to the medium after the start of culture. Furthermore, each of the various components, such as the amino acid source, may be contained in the medium at the start of culture, and further supplied to the medium after the start of culture (e.g., after consumption of the active ingredient).

[0115] Each of the various components, such as the amino acid source, may be contained in the medium at the concentrations exemplified above throughout the entire culture period, or may be contained in the medium at the concentrations exemplified above only during a portion of the culture period. In other words, "culture is carried out in a medium containing a certain component at a certain concentration" means that the concentration of the component in the medium is within that range for at least a portion of the culture period, but does not necessarily have to be within that range throughout the entire culture period. Each of the various components, such as the amino acid source, may be contained in the medium at the concentrations exemplified above, for example, at the start of culture, or may be supplied to the medium after the start of culture to achieve the concentrations exemplified above. Furthermore, each of the various components, such as the amino acid source, may be contained in the medium at the concentrations exemplified above at the start of culture, and further supplied to the medium after the start of culture (e.g., after the component has been consumed) to achieve the concentrations exemplified above.

[0116] The length of the "partial culture period" is not particularly limited as long as it allows animal cells to be cultured. The length of the "partial culture period" can be appropriately set depending on various conditions, such as the type of component, the type of animal cells, the length of the culture period, and the desired amount of target substance production. The "partial culture period" may be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more of the total culture period. Furthermore, the "partial culture period" may be, for example, 0.5 days or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 12 days or more, or 15 days or more.

[0117] Furthermore, the concentrations of various components, such as amino acid sources, in the medium may be set to the concentrations exemplified above, for example, as average values ​​throughout a specific period during culture. That is, "culture is carried out in a medium containing a certain component at a certain concentration" may mean that the average value of the concentration of the component in the medium throughout a specific period during culture is within that concentration range. The "average value of the concentration of a certain component in the medium throughout a specific period during culture" is not particularly limited as long as it allows for understanding fluctuations in the concentration of the component during a specific period during culture, and may mean, for example, the average value of the concentration of the component in the medium measured every 60 minutes, every 30 minutes, every 20 minutes, or every 10 minutes throughout a specific period during culture. The "specific period during culture" may refer to the entire culture period or a portion of the culture period. The "partial culture period" is as described above.

[0118] Each of the various components, such as the amino acid source, may be supplied to the medium throughout the entire culture period, or may be supplied to the medium only during a portion of the culture period. The "part of the culture period" is as described above. Each of the various components, such as the amino acid source, may be supplied to the medium continuously or intermittently, for example. Each of the various components, such as the amino acid source, may be supplied to the medium every day, or every few days, for example.

[0119] The concentrations of various components, such as amino acid sources, in the feed medium or perfusion medium may be within the range of the concentrations of the components in the media exemplified above, or may be, for example, 1-fold or more, 1.1-fold or more, 1.3-fold or more, 1.5-fold or more, 2-fold or more, 3-fold or more, 5-fold or more, 7-fold or more, 10-fold or more, 15-fold or more, or 20-fold or less, or 100-fold or less, 70-fold or less, 50-fold or less, 30-fold or less, 20-fold or less, 15-fold or less, 10-fold or less, 7-fold or less, 5-fold or less, 3-fold or less, 2-fold or less, or any combination thereof that is consistent therewith. The concentrations of various components such as amino acid sources in the feed medium or perfusion medium may be, for example, 1 to 2 times, 1.1 to 2 times, 1.3 to 2 times, 1.5 to 2 times, 2 to 3 times, 3 to 5 times, 5 to 7 times, 7 to 10 times, 10 to 15 times, 15 to 20 times, 20 to 30 times, 20 to 50 times, 20 to 70 times, or 20 to 100 times the concentrations of the components in the above-exemplified media. The concentrations of various components such as amino acid sources in the feed medium or perfusion medium may be, for example, 1 to 100 times, 2 to 50 times, or 5 to 20 times the concentrations of the components in the above-exemplified media. Furthermore, the concentration of the amino acid source (e.g., cysteine ​​concentration) in the feed medium or perfusion medium may be, for example, 1 mM or more, 2 mM or more, 5 mM or more, 10 mM or more, 15 mM or more, 20 mM or more, 30 mM or more, 40 mM or more, 50 mM or more, or 60 mM or more, or may be a saturation concentration or less, 100 mM or less, 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, 50 mM or less, 40 mM or less, 30 mM or less, or 20 mM or less, or any compatible combination thereof. The concentration of the amino acid source (e.g., cysteine ​​concentration) in the feed medium or perfusion medium may be, for example, 1 to 20 mM, 2 to 20 mM, 5 to 20 mM, 10 to 20 mM, 15 to 20 mM, 20 to 30 mM, 30 to 40 mM, 40 to 50 mM, 50 to 60 mM, 60 to 70 mM, 60 to 80 mM, 60 to 90 mM, 60 to 100 mM, or 60 mM to saturation. The concentration of the amino acid source (e.g., cysteine ​​concentration) in the feed medium or perfusion medium may be, for example, 5 to 100 mM, 10 to 90 mM, or 20 to 80 mM.When two or more amino acid sources are used as active ingredients, the "amino acid source concentration" herein refers to the total concentration of those amino acid sources. However, when two or more amino acid sources are used as active ingredients, the concentrations of those amino acid sources can be independently set within the ranges of amino acid source concentrations exemplified above. Furthermore, the description of the amino acid source concentrations exemplified above can also be applied mutatis mutandis to the concentration of the amino acid source that is an additional ingredient.

[0120] The concentrations of various components, such as amino acid sources, can be measured by known methods used for detecting or identifying compounds. Examples of such methods include HPLC, UPLC, LC / MS, GC / MS, and NMR. These methods can also be used to confirm the production of target substances. These methods may be used alone or in combination of two or more.

[0121] Animal cells can be cultured as described above. When the animal cells have the ability to produce a target substance, such as a target protein, culturing the animal cells as described above produces the target substance (e.g., the target protein is expressed), thereby obtaining a culture containing the target substance. Specifically, the target substance, such as the target protein, may accumulate in the medium, on the cell surface, inside the cells, or a combination thereof.

[0122] Below, the procedures for confirming the production of a target protein, recovering it, purifying it, and the like will be described with particular reference to the case of producing a target protein; however, such procedures can also be carried out appropriately for target substances other than the target protein.

[0123] The production of a target protein can be confirmed by known methods used for detecting or identifying proteins. Such methods include, for example, SDS-PAGE, Western blotting, mass spectrometry, N-terminal amino acid sequence analysis, and enzyme activity measurement. These methods may be used alone or in combination of two or more.

[0124] The target protein can be collected as appropriate. Specifically, the target protein can be collected as an appropriate fraction containing the target protein. Examples of such fractions include cultures, culture supernatants, cultured cells, and processed cultured cell products (disrupted cells, lysates, extracts (cell-free extracts), etc.). Cultured cells may be obtained in the form of immobilized cells immobilized on a carrier such as acrylamide or carrageenan.

[0125] The protein of interest may be further purified to a desired extent.

[0126] When the target protein accumulates in the medium, the target protein can be purified from the supernatant after removing solid matter such as cells from the culture by centrifugation or the like.

[0127] When a target protein accumulates intracellularly, the target protein can be purified from the treated product after subjecting the cells to treatment such as disruption, lysis, or extraction. The cells can be recovered from the culture by centrifugation or the like. Treatment such as cell disruption, lysis, or extraction can be carried out by known methods. Examples of such methods include ultrasonic disruption, the Dynomill method, bead disruption, French press disruption, and lysozyme treatment. These methods may be used alone or in appropriate combination of two or more.

[0128] When a target protein accumulates on the cell surface, the target protein can be solubilized and then purified from the solubilized product. Solubilization can be performed by known methods. Examples of such methods include increasing the salt concentration and using a surfactant. These methods may be used alone or in combination of two or more.

[0129] Purification of the target protein (e.g., from the supernatant, treated product, or solubilized product as described above) can be carried out by known methods used for protein purification. Such methods include, for example, ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, and isoelectric precipitation. These methods may be used alone or in appropriate combination of two or more.

[0130] The target protein may be obtained in a free state, or in the form of an immobilized enzyme immobilized on a solid phase such as a resin.

[0131] The recovered target protein may be formulated as appropriate. The dosage form is not particularly limited and can be appropriately selected depending on various conditions, such as the intended use of the target protein. Examples of dosage forms include liquids, suspensions, powders, tablets, pills, and capsules. Pharmacologically acceptable additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, odorants, fragrances, diluents, and surfactants can be used in the formulation.

[0132] <4> Use of Active Ingredient The present invention also discloses the use of the active ingredient in the above-exemplified uses. That is, the present invention discloses, for example, the use of the active ingredient to improve the stability of a composition, or the use of the active ingredient in producing a composition for animal cell culture. In other words, the present invention discloses methods for achieving the above-exemplified uses using the active ingredient. That is, the present invention discloses, for example, a method for improving the stability of a composition, which includes mixing the active ingredient, or a method for producing a composition for animal cell culture, which includes mixing the active ingredient.

[0133] The present invention also discloses active ingredients for use in the above-exemplified applications, i.e., active ingredients for use in improving the stability of a composition and active ingredients for use in producing a composition for animal cell culture.

[0134] The present invention also discloses the use of each active ingredient in combination with other active ingredients. Each active ingredient may be used in combination with other active ingredients for the uses exemplified above. That is, the present invention discloses, for example, the use of a salt of α-ketoglutaric acid to improve the stability of a composition containing an amino acid source (e.g., cysteine), and the use of free L-cysteine ​​anhydrate to improve the stability of a composition containing α-ketoglutaric acid. The use of a salt of α-ketoglutaric acid to improve the stability of a composition containing an amino acid source (e.g., cysteine) particularly includes the use of a salt of α-ketoglutaric acid to prevent caking of a composition containing an amino acid source (e.g., cysteine), and the use of a salt of α-ketoglutaric acid to prevent a decrease in the cysteine ​​content in a composition containing cysteine. The use of free L-cysteine ​​anhydrate to improve the stability of a composition containing α-ketoglutaric acid particularly includes the use of free L-cysteine ​​anhydrate to prevent caking of a composition containing α-ketoglutaric acid. In other words, the present invention discloses methods for achieving the above-exemplified uses by using each active ingredient in combination with other active ingredients. Specifically, the present invention discloses, for example, a method for improving the stability of a composition containing an amino acid source (e.g., cysteine), which comprises mixing a salt of α-ketoglutaric acid with the amino acid source (e.g., cysteine), and a method for improving the stability of a composition containing α-ketoglutaric acid, which comprises mixing free L-cysteine ​​anhydrate with α-ketoglutaric acid. Methods for improving the stability of a composition containing an amino acid source (e.g., cysteine) particularly include methods for preventing the solidification of a composition containing an amino acid source (e.g., cysteine) and methods for preventing a decrease in the cysteine ​​content in a composition containing cysteine. Methods for improving the stability of a composition containing α-ketoglutaric acid particularly include methods for preventing the solidification of a composition containing α-ketoglutaric acid.

[0135] The present invention will now be described in more detail with reference to the following non-limiting examples.

[0136] Example 1: Prevention of caking of composition In this example, the effects on the properties of a mixed powder of L-cysteine ​​and α-ketoglutaric acid when the form of L-cysteine ​​and / or α-ketoglutaric acid was changed were evaluated.

[0137] (1) Preparation of mixed powders Mixed powders were obtained by mixing L-cysteine ​​and α-ketoglutaric acid in the forms shown in Table 1 at a molar ratio of 1:5.8. All mixed powders were prepared as white, smooth powders.

[0138] (2) Evaluation Experiments Each mixed powder and silica gel (manufactured by Shin-Etsu Chemical Industry Co., Ltd.) was stored in an incubator (manufactured by Espec Co., Ltd.; CSH-122HG) with the temperature controlled at 25°C or 40°C under light-shielded conditions, and the caking of the powder after storage for 3, 7, or 14 days was evaluated visually. In the table, "-" indicates that no caking was observed. In the table, "+" to "+++" indicate that caking was observed, with more "+"s indicating a greater degree of caking. The specific evaluation criteria are as follows: -: No caking (caked lumps) was present in the mixed powder. +: There were 1 to 9 caking objects (caked lumps) in the total mixed powder. ++: There were 10 or more caking objects (caked lumps) in the total mixed powder, and caking was clearly evident. +++: The mixed powder was caking overall, and the powder shape was not maintained.

[0139] (3) Evaluation Results The results are shown in Table 1 and Figures 1 and 2. Caking was observed after storage at 25°C for 3 days or more in the mixed powder of L-cysteine ​​hydrochloride monohydrate and free α-ketoglutaric acid, and in the mixed powder in which L-cysteine ​​hydrochloride monohydrate was replaced with L-cysteine ​​hydrochloride anhydrate. In contrast, no caking was observed after storage at 25°C for 3 days or more in the mixed powder in which L-cysteine ​​hydrochloride monohydrate was replaced with free L-cysteine ​​anhydrate and / or free α-ketoglutaric acid was replaced with monosodium α-ketoglutarate. Furthermore, no caking was observed after storage at 40°C for 3 days or more, where caking was observed in the other mixed powders.

[0140] From the above, it was revealed that the stability (specifically, caking resistance) of a mixed powder of L-cysteine ​​and α-ketoglutaric acid is improved by using free-form L-cysteine ​​anhydrate as the L-cysteine ​​and / or monosodium α-ketoglutarate as the α-ketoglutaric acid. In particular, it was revealed that the stability (specifically, caking resistance) of the mixed powder is significantly improved by using free-form L-cysteine ​​anhydrate and monosodium α-ketoglutarate.

[0141]

[0142] Example 2: Prevention of caking of composition In this example, the influence of changing the form of α-ketoglutaric acid on the properties of mixed powders of various amino acid sources and α-ketoglutaric acid was evaluated.

[0143] (1) Powder Preparation Various amino acid sources and α-ketoglutaric acid (free α-ketoglutaric acid or monosodium α-ketoglutarate) were mixed at a molar ratio of 1:1.5 to obtain mixed powders. The mixed powder of glycyl-L-tyrosine dihydrate and free α-ketoglutaric acid, and the mixed powder of L-lysine hydrochloride and free α-ketoglutaric acid, showed caking immediately after preparation. All other mixed powders were prepared as white, smooth powders. A photograph of the mixed powders immediately after preparation is shown in Figure 3.

[0144] (2) Evaluation Experiments Each mixed powder and silica gel (manufactured by Shin-Etsu Chemical Industry Co., Ltd.) was stored in an incubator (manufactured by Espec Corporation; CSH-122HG) with the temperature controlled at 4°C, 25°C, or 40°C under light-shielded conditions, and the caking of the powder was evaluated visually after storage for 3, 7, or 14 days. In the table, "-" indicates that no caking was observed. In the table, "+" to "+++" indicate that caking was observed, with more "+"s indicating a greater degree of caking. The specific evaluation criteria are as follows: -: No caking (caked lumps) was present in the mixed powder. +: There were 1 to 9 caking objects (caked lumps) in the total mixed powder. ++: There were 10 or more caking objects (caked lumps) in the total mixed powder, and caking was clearly evident. +++: The mixed powder was caking overall, and the powder shape was not maintained.

[0145] (3) Evaluation Results The results are shown in Tables 2 and 3 and Figures 4 to 6. For many amino acid sources, caking of the mixed powder was prevented by changing free α-ketoglutaric acid to monosodium α-ketoglutarate. In particular, for free glycyl-L-tyrosine dihydrate, free L-phenylalanine, L-lysine hydrochloride, free L-methionine, free glycine, free L-asparagine monohydrate, L-cysteine ​​hydrochloride anhydrate, and free L-cysteine ​​anhydrate, changing free α-ketoglutaric acid to monosodium α-ketoglutarate prevented caking of the mixed powder.

[0146] From the above, it has become clear that the stability (specifically, caking resistance) of mixed powders can be improved by using monosodium α-ketoglutarate as α-ketoglutaric acid, not only for mixed powders of L-cysteine ​​and α-ketoglutaric acid, but also for mixed powders of many amino acid sources and α-ketoglutaric acid.

[0147]

[0148]

[0149] Example 3: Prevention of decrease in L-cysteine ​​content in a composition In this experimental example, the effect of changing the form of L-cysteine ​​and / or α-ketoglutaric acid on the change in L-cysteine ​​content in a mixed powder of L-cysteine ​​and α-ketoglutaric acid was evaluated.

[0150] (1) Preparation of Powder L-cysteine ​​(L-cysteine ​​hydrochloride monohydrate or free L-cysteine ​​anhydrate) and α-ketoglutaric acid (free α-ketoglutaric acid or monosodium α-ketoglutarate) were mixed in a molar ratio of 1:4.9 to obtain a mixed powder. All mixed powders were prepared as white, free-flowing powders.

[0151] (2) Evaluation Experiment Each mixed powder and silica gel (manufactured by Shin-Etsu Chemical Co., Ltd.) were stored in a light-shielded incubator (manufactured by Espec Co., Ltd.; CSH-122HG) with the temperature controlled at 25 ° C or 40 ° C, and the L-cysteine ​​content in the mixed powder was analyzed over time according to the method of WO2021 / 060517A.

[0152] (3) Evaluation Results The results are shown in Figures 7 to 10. In the mixed powder containing free α-ketoglutaric acid, a decrease in the L-cysteine ​​content was confirmed upon storage at 25°C or 40°C, regardless of whether the L-cysteine ​​was L-cysteine ​​hydrochloride monohydrate or free L-cysteine ​​anhydrate, and L-cysteine ​​became undetectable after storage at 40°C for 7 days or more. On the other hand, the decrease in the L-cysteine ​​content was significantly improved in the mixed powder containing free L-cysteine ​​and monosodium α-ketoglutarate.

[0153] From the above, it was revealed that the stability of a mixed powder of L-cysteine ​​and α-ketoglutaric acid (specifically, the stability of L-cysteine) is improved by using free L-cysteine ​​anhydrate as the L-cysteine ​​and monosodium α-ketoglutarate as the α-ketoglutaric acid.

[0154] According to the present invention, the stability of a composition containing an amino acid source and α-ketoglutaric acid can be improved. Furthermore, according to the present invention, the composition can be used to culture animal cells.

Claims

1. A composition for animal cell culture, comprising: containing an amino acid source and α-ketoglutaric acid; A composition having the following properties (A) and / or (B): (A) The α-ketoglutaric acid is a salt of α-ketoglutaric acid; (B) The amino acid source is free L-cysteine ​​anhydrate.

2. The composition of claim 1 having at least said property (A).

3. The composition of claim 1 which is a powder.

4. The composition of claim 1 , which is a culture medium or a culture medium additive.

5. 5. The composition of claim 4, wherein the medium is a basal medium, a feed medium, or a perfusion medium.

6. 2. The composition of claim 1, wherein the salt of α-ketoglutaric acid is an alkali metal salt or alkaline earth metal salt of α-ketoglutaric acid.

7. 2. The composition of claim 1, wherein the salt of α-ketoglutaric acid is a sodium salt or a potassium salt of α-ketoglutaric acid.

8. 2. The composition of claim 1, wherein the salt of α-ketoglutaric acid is monosodium α-ketoglutarate or disodium α-ketoglutarate.

9. 2. The composition of claim 1, wherein the amino acid source is cysteine, glycine, asparagine, glutamic acid, lysine, phenylalanine, methionine, ornithine, tyrosine, or a glycine-containing dipeptide.

10. The composition according to claim 9, having one or more properties selected from the group consisting of the following properties (1) to (10): (1) The cysteine ​​is free L-cysteine ​​or L-cysteine ​​hydrochloride; (2) The glycine is free glycine or free glycyl-L-tyrosine; (3) The asparagine is free L-asparagine; (4) The glutamic acid is monosodium L-glutamate; (5) The lysine is L-lysine hydrochloride; (6) The phenylalanine is free L-phenylalanine; (7) The methionine is free L-methionine; (8) The ornithine is L-ornithine hydrochloride; (9) The tyrosine is L-tyrosine disodium; (10) The glycine-containing dipeptide is free glycyl-L-tyrosine.

11. The composition according to claim 9 or 10, which satisfies one or more properties selected from the group consisting of the following properties (1a) to (10a): (1a) The cysteine ​​is free L-cysteine ​​anhydrate or L-cysteine ​​hydrochloride monohydrate; (2a) The glycine is free glycine or free glycyl-L-tyrosine; (3a) The asparagine is free L-asparagine monohydrate; (4a) The glutamic acid is monosodium L-glutamate monohydrate; (5a) The lysine is L-lysine hydrochloride; (6a) The phenylalanine is free L-phenylalanine; (7a) The methionine is free L-methionine; (8a) The ornithine is L-ornithine hydrochloride; (9a) The tyrosine is L-tyrosine disodium dihydrate; (10a) The glycine-containing dipeptide is glycyl-L-tyrosine dihydrate in the free form.

12. The composition of claim 1 , wherein the amino acid source is cysteine.

13. The composition according to claim 9 or 10, wherein the cysteine ​​is free L-cysteine ​​anhydrate or L-cysteine ​​hydrochloride monohydrate.

14. The composition according to claim 9 or 10, wherein the cysteine ​​is a free form L-cysteine ​​anhydrate.

15. 2. The composition according to claim 1, wherein the content of said α-ketoglutaric acid is 1 to 20 times the content of said amino acid source in terms of molar ratio.

16. A method for producing a target substance, comprising: Culturing animal cells capable of producing a target substance using the composition according to claim 1 or 2; and Recovering the target substance A method comprising:

17. The method of claim 16 , wherein the target substance is a protein or a virus.

18. A method for culturing animal cells, comprising: Culturing animal cells using the composition according to claim 1 or 2. A method comprising:

19. Use of a salt of α-ketoglutaric acid, 1. Use for preventing caking of a composition containing an amino acid source.

20. 20. The use of claim 19, wherein the amino acid source is cysteine, glycine, asparagine, glutamic acid, lysine, phenylalanine, methionine, ornithine, tyrosine, or a glycine-containing dipeptide.

21. The use according to claim 20, which satisfies one or more conditions selected from the following conditions (1) to (10): (1) The cysteine ​​is free L-cysteine ​​or L-cysteine ​​hydrochloride; (2) The glycine is free glycine or free glycyl-L-tyrosine; (3) The asparagine is free L-asparagine; (4) The glutamic acid is monosodium L-glutamate; (5) The lysine is L-lysine hydrochloride; (6) The phenylalanine is free L-phenylalanine; (7) The methionine is free L-methionine; (8) The ornithine is L-ornithine hydrochloride; (9) The tyrosine is L-tyrosine disodium; (10) The glycine-containing dipeptide is free glycyl-L-tyrosine.

22. The use according to claim 20 or 21, which satisfies one or more conditions selected from the following conditions (1a) to (10a): (1a) The cysteine ​​is free L-cysteine ​​anhydrate or L-cysteine ​​hydrochloride monohydrate; (2a) The glycine is free glycine or free glycyl-L-tyrosine; (3a) The asparagine is free L-asparagine monohydrate; (4a) The glutamic acid is monosodium L-glutamate monohydrate; (5a) The lysine is L-lysine hydrochloride; (6a) The phenylalanine is free L-phenylalanine; (7a) The methionine is free L-methionine; (8a) The ornithine is L-ornithine hydrochloride; (9a) The tyrosine is L-tyrosine disodium dihydrate; (10a) The glycine-containing dipeptide is glycyl-L-tyrosine dihydrate in the free form.

23. 21. The use according to claim 19 or 20, wherein the amino acid source is cysteine.

24. Use of a salt of α-ketoglutaric acid, Use for preventing a decrease in the cysteine ​​content in a composition containing cysteine.

25. 25. The use according to claim 19, 20, or 24, wherein the salt of α-ketoglutaric acid is an alkali metal salt or alkaline earth metal salt of α-ketoglutaric acid.

26. The use according to claim 19, 20 or 24, wherein the salt of α-ketoglutaric acid is a sodium salt or a potassium salt of α-ketoglutaric acid.

27. 25. The use according to claim 19, 20, or 24, wherein the salt of α-ketoglutaric acid is monosodium α-ketoglutarate or disodium α-ketoglutarate.

28. The use according to claim 20, 21, or 24, wherein the cysteine ​​is free L-cysteine ​​anhydrate or L-cysteine ​​hydrochloride monohydrate.

29. The use according to claim 20, 21, or 24, wherein the cysteine ​​is free L-cysteine ​​anhydrate.

30. The use according to claim 19, 20, or 24, wherein the content of the salt of α-ketoglutaric acid in the composition is 1 to 20 times the content of the amino acid source or the cysteine ​​in the composition in terms of molar ratio.

31. 1. A method for producing a composition for animal cell culture, comprising: The composition is a composition according to claim 1 or 2, mixing said amino acid source and said α-ketoglutaric acid.