Culture medium composition

Abstract

The present invention provides: a culture medium composition for promoting the production of a virus having a target gene during the culture of mammalian cells that produce the virus having the target gene introduced therein, said culture medium composition containing β-alanine; and a culture medium composition for culturing mammalian cells that produce a virus or virus-like particles, said culture medium composition containing β-alanine.

Description

Medium composition

[0001] The present invention relates to a medium composition containing β-alanine for promoting the production of a virus carrying a gene of interest or for increasing the proportion of viruses carrying a gene of interest among all viruses produced during the cultivation of virus-producing cells into which a gene of interest has been introduced, and for culturing cells that produce viruses or virus-like particles and contain β-alanine.

[0002] To meet the needs for human gene therapy, vaccine production, etc., rapid and stable production technologies for viruses are essential. In particular, the cultivation of cells that produce viruses (hereinafter also referred to as virus-producing cells) is extremely important.

[0003] When designing and optimizing media for virus-producing cell culture, the components of the media are an important issue, as they affect cell growth and proliferation.

[0004] In addition, in the case of media for culturing virus-producing cells, the components contained in the media also affect the virus production ability within the cells. Furthermore, when producing viruses using cells transfected with a gene that produces a cancer-suppressing protein, the components contained in the media also affect the proportion of viruses carrying that gene among all the viruses produced. However, the components known to date have not necessarily produced satisfactory results. For example, when producing adeno-associated viruses using cells transfected with a gene, the proportion of adeno-associated viruses carrying that gene among all the adeno-associated viruses is said to be approximately 5% to 30% (Non-Patent Document 1).

[0005] Molecular Therapy Methods & Clinical Development; 2021 June 11; Vol. 21; p. 642-655

[0006] An object of the present invention is to provide a medium composition and the like for improving the production amount of a virus carrying a gene of interest or the ratio of a virus carrying a gene of interest to all viruses produced when virus-producing cells are used to produce the virus, and for promoting the production of a virus or virus-like particle when cells are used to produce the virus or virus-like particle.

[0007] As a result of extensive research into the above-mentioned problems, the present inventors discovered that the addition of β-alanine to human embryonic kidney 293 (HEK293) cells improves their ability to produce adeno-associated viruses carrying a gene of interest, and further research based on this finding led to the completion of the present invention.

[0008] [1] A medium composition containing β-alanine for promoting the production of a virus carrying a target gene during the culture of a virus-producing mammalian cell into which the target gene has been introduced. [2] The medium composition according to [1] above, wherein the mammalian cell is a cell selected from the group consisting of HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, and HeLa cells. [3] The medium composition according to [1] above, wherein the mammalian cell is a HEK293 cell. [4] The medium composition according to any of [1] to [3] above, wherein the virus-producing mammalian cell is an adeno-associated virus-producing mammalian cell. [5] The medium composition according to any of [1] to [4] above, wherein the concentration of β-alanine in the medium composition is 0.1 μM to 800 μM. [6] The medium composition according to any of [1] to [5] above, further comprising a polyamine or a salt thereof. [7] The medium composition according to [6] above, wherein the polyamine or a salt thereof is spermidine or a salt thereof. [8] The medium composition according to [6] or [7] above, wherein the concentration of the polyamine or its salt in the medium composition is 0.1 μM to 700 μM. [9] A medium composition comprising a polyamine or its salt, for promoting production of a virus carrying a gene of interest during the cultivation of virus-producing mammalian cells into which the gene of interest has been introduced.

[10] The medium composition according to [9] above, wherein the polyamine or its salt is spermidine or its salt.

[11] The medium composition according to [9] or

[10] above, wherein the concentration of the polyamine or its salt in the medium composition is 0.1 μM to 700 μM.

[12] A medium additive comprising β-alanine, for promoting production of a virus carrying a gene of interest during the cultivation of virus-producing mammalian cells into which the gene of interest has been introduced.

[13] A medium additive comprising a polyamine or its salt, for promoting production of a virus carrying a gene of interest during the cultivation of virus-producing mammalian cells into which the gene of interest has been introduced.

[14] Use of β-alanine in the production of a medium composition for promoting the production of a virus carrying a target gene during the cultivation of a mammalian cell that produces the virus and into which the target gene has been introduced.

[15] Use of a polyamine or a salt thereof in the manufacture of a medium composition for promoting production of a virus carrying a gene of interest during the cultivation of virus-producing mammalian cells into which a gene of interest has been introduced.

[16] A method for promoting production of a virus carrying a gene of interest, comprising the step of culturing virus-producing mammalian cells into which a gene of interest has been introduced in a medium containing β-alanine.

[17] A method for promoting production of a virus carrying a gene of interest, comprising the step of culturing virus-producing mammalian cells into which a gene of interest has been introduced in a medium containing polyamine or a salt thereof.

[18] A medium composition containing β-alanine for increasing the proportion of viruses carrying a gene of interest among all viruses produced during the cultivation of virus-producing mammalian cells into which a gene of interest has been introduced.

[19] The medium composition according to

[18] above, wherein the mammalian cells are cells selected from the group consisting of HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, and HeLa cells.

[20] The medium composition according to

[18] above, wherein the mammalian cells are HEK293 cells.

[21] The medium composition according to any of

[18] to

[20] above, wherein the virus-producing mammalian cells are adeno-associated virus-producing mammalian cells.

[22] The medium composition according to any of

[18] to

[21] above, wherein the concentration of β-alanine in the medium composition is 0.1 μM to 800 μM.

[23] The medium composition according to any of

[18] to

[22] above, further comprising a polyamine or a salt thereof.

[24] The medium composition according to

[23] above, wherein the polyamine or a salt thereof is spermidine or a salt thereof.

[25] The medium composition according to

[23] or

[24] above, wherein the concentration of polyamine or a salt thereof in the medium composition is 0.1 μM to 140 μM.

[26] A medium composition for increasing the ratio of viruses carrying a gene of interest among all viruses produced during the culture of virus-producing mammalian cells into which a gene of interest has been introduced, the medium composition comprising a polyamine or a salt thereof.

[27] The medium composition according to

[26] above, wherein the polyamine or a salt thereof is spermidine or a salt thereof.

[28] The medium composition according to

[26] or

[27] above, wherein the concentration of the polyamine or a salt thereof in the medium composition is 0.1 μM to 140 μM.

[29] A medium additive comprising β-alanine, for increasing the proportion of viruses carrying a gene of interest among all viruses produced during the cultivation of virus-producing mammalian cells into which a gene of interest has been introduced.

[30] A medium additive comprising a polyamine or a salt thereof, for increasing the proportion of viruses carrying a gene of interest among all viruses produced during the cultivation of virus-producing mammalian cells into which a gene of interest has been introduced.

[31] Use of β-alanine in the production of a medium composition for increasing the proportion of viruses carrying a gene of interest among all viruses produced during the cultivation of virus-producing mammalian cells into which a gene of interest has been introduced.

[32] Use of a polyamine or a salt thereof in the production of a medium composition for increasing the proportion of viruses carrying a gene of interest among all viruses produced during the cultivation of virus-producing mammalian cells into which a gene of interest has been introduced.

[33] A method for increasing the proportion of viruses carrying a gene of interest among all viruses produced during the cultivation, comprising the step of culturing virus-producing mammalian cells into which a gene of interest has been introduced in a medium containing β-alanine.

[34] A method for increasing the proportion of viruses carrying a gene of interest among all viruses produced during culturing, the method comprising the step of culturing virus-producing mammalian cells into which a gene of interest has been introduced in a medium containing a polyamine or a salt thereof.

[35] A method for producing a virus carrying a gene of interest, the method comprising the step of culturing virus-producing mammalian cells into which a gene of interest has been introduced in a medium containing β-alanine.

[36] A method for producing a virus carrying a gene of interest, the method comprising the step of culturing virus-producing mammalian cells into which a gene of interest has been introduced in a medium containing β-alanine, the method promoting the production of a virus carrying a gene of interest, or increasing the proportion of viruses carrying a gene of interest among all viruses produced during culturing virus-producing mammalian cells into which a gene of interest has been introduced.

[37] A method for producing a virus carrying a gene of interest, the method comprising the step of culturing virus-producing mammalian cells into which a gene of interest has been introduced in a medium containing a polyamine or a salt thereof.

[38] A method for producing a virus carrying a gene of interest, comprising the step of culturing virus-producing mammalian cells into which a gene of interest has been introduced in a medium containing a polyamine or a salt thereof, thereby promoting the production of the virus carrying the gene of interest or increasing the ratio of viruses carrying the gene of interest among all viruses produced during the culture of virus-producing mammalian cells into which a gene of interest has been introduced in the medium.

[39] A medium composition for culturing virus- or virus-like particle-producing mammalian cells, comprising β-alanine.

[40] The medium composition according to

[39] above, wherein the mammalian cells are cells selected from the group consisting of HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, and HeLa cells.

[41] The medium composition according to

[39] above, wherein the mammalian cells are HEK293 cells.

[42] The medium composition according to any of

[39] to

[41] above, wherein the virus- or virus-like particle-producing mammalian cells are adeno-associated virus-producing mammalian cells.

[43] The medium composition according to any one of

[39] to

[42] above, wherein the concentration of β-alanine in the medium composition is 0.1 μM to 800 μM.

[44] The medium composition according to any one of

[39] to

[43] above, further comprising a polyamine or a salt thereof.

[45] The medium composition according to

[44] above, wherein the polyamine or a salt thereof is spermidine or a salt thereof.

[46] The medium composition according to

[44] or

[45] above, wherein the concentration of the polyamine or a salt thereof in the medium composition is 0.1 μM to 700 μM.

[47] A medium composition for culturing mammalian cells that produce viruses or virus-like particles, comprising a polyamine or a salt thereof.

[48] A medium additive for culturing mammalian cells that produce viruses or virus-like particles, comprising β-alanine.

[49] A medium additive for culturing mammalian cells that produce viruses or virus-like particles, comprising a polyamine or a salt thereof.

[50] Use of β-alanine in the production of a medium composition for culturing mammalian cells that produce viruses or virus-like particles.

[51] Use of a polyamine or a salt thereof in the manufacture of a medium composition for culturing mammalian cells that produce viruses or virus-like particles.

[52] A method for culturing viruses or virus-like particles, comprising the step of culturing mammalian cells that produce the cells in a medium containing β-alanine.

[53] A method for culturing viruses or virus-like particles, comprising the step of culturing mammalian cells that produce the cells in a medium containing a polyamine or a salt thereof.

[54] A method for producing the viruses or virus-like particles, comprising the step of culturing mammalian cells that produce the viruses or virus-like particles in a medium containing β-alanine.

[55] A method for producing the viruses or virus-like particles, comprising the step of culturing mammalian cells that produce the viruses or virus-like particles in a medium containing a polyamine or a salt thereof.

[0009] According to the present invention, when a virus carrying a target gene is produced using virus-producing mammalian cells, it is possible to improve the production amount of the virus carrying the target gene or the ratio of the virus carrying the target gene to all the viruses produced.

[0010] FIG. 1 shows the results of examining the effect of β-alanine on AAV8 genome titer in Example 1. The vertical axis represents the relative AAV8 genome titer, with the AAV8 genome titer obtained when AAV8 was produced using a medium without β-alanine being set at 100%. On the horizontal axis, "Viral Production Medium" represents the case where AAV8 was produced using a medium without β-alanine, and "Viral Production Medium + β-alanine" represents the case where AAV8 was produced using a medium supplemented with β-alanine to a final concentration of 100 μM. FIG. 2 shows the results of examining the effect of β-alanine on the full / empty capsid ratio (full capsid ratio) in Example 1. The vertical axis represents the relative full capsid ratio, with the full capsid ratio obtained when AAV8 was produced using a medium not supplemented with β-alanine set to 100%. On the horizontal axis, "Viral Production Medium" represents the case where AAV8 was produced using a medium not supplemented with β-alanine, and "Viral Production Medium + β-alanine" represents the case where AAV8 was produced using a medium supplemented with β-alanine to a final concentration of 100 μM. Figure 3 shows the results of examining the effect of β-alanine on the AAV8 genome titer in Example 2. The vertical axis represents the relative AAV8 genome titer, with the AAV8 genome titer obtained when AAV8 was produced using a medium not supplemented with β-alanine set to 100%. The horizontal axis indicates "Viral Production Medium" when AAV8 was produced using a medium without β-alanine, while "+100 μM β-alanine," "+300 μM β-alanine," "+1 mM β-alanine," "+1.5 mM β-alanine," and "+2 mM β-alanine" indicate when AAV8 was produced using a medium supplemented with β-alanine to final concentrations of 100 μM, 300 μM, 1 mM, 1.5 mM, and 2 mM, respectively. FIG. 4 shows the results of examining the effect of β-alanine on capsid number (virus number) in Example 3. The vertical axis indicates the relative capsid number, with the capsid number obtained when AAV8 was produced using a medium without β-alanine being set at 100%.The horizontal axis "Viral Production Medium" represents the case where AAV8 was produced using a medium to which β-alanine was not added, and "+300 μM β-alanine" represents the case where AAV8 was produced using a medium to which β-alanine was added so that the final concentration was 300 μM. FIG. 5 shows the results of examining the effect of β-alanine on the full capsid ratio in Example 3. The vertical axis represents the relative full capsid ratio, when the full capsid ratio obtained when AAV8 was produced using a medium to which β-alanine was not added was set to 100%. The horizontal axis "Viral Production Medium" represents the case where AAV8 was produced using a medium to which β-alanine was not added, and "+300 μM β-alanine" represents the case where AAV8 was produced using a medium to which β-alanine was added so that the final concentration was 300 μM. 6 shows the results of examining the effect of spermidine trihydrochloride on the AAV8 genome titer in Example 4. The vertical axis represents the relative AAV8 genome titer, when the AAV8 genome titer obtained when AAV8 is produced in a medium not supplemented with spermidine trihydrochloride is set at 100%. On the horizontal axis, "Viral Production Medium" represents the case where AAV8 was produced using a medium to which spermidine trihydrochloride was not added, "Viral Production Medium + 30 μM spermidine" represents the case where AAV8 was produced using a medium to which spermidine trihydrochloride was added to a final concentration of 30 μM, and "Viral Production Medium + 100 μM spermidine" represents the case where AAV8 was produced using a medium to which spermidine trihydrochloride was added to a final concentration of 100 μM. Figure 7 shows the results of examining the effect of spermidine trihydrochloride on the full capsid ratio in Example 4. The vertical axis represents the relative full capsid ratio, with the full capsid ratio obtained when AAV8 is produced using a medium not supplemented with spermidine trihydrochloride set at 100%.On the horizontal axis, "Viral Production Medium" represents the case where AAV8 was produced using a medium to which spermidine trihydrochloride was not added, "Viral Production Medium + 30 μM spermidine" represents the case where AAV8 was produced using a medium to which spermidine trihydrochloride was added to a final concentration of 30 μM, and "Viral Production Medium + 100 μM spermidine" represents the case where AAV8 was produced using a medium to which spermidine trihydrochloride was added to a final concentration of 100 μM. Figure 8 shows the results of examining the effect of spermidine trihydrochloride and / or β-alanine on the AAV8 genome titer in Example 5. The vertical axis represents the relative AAV8 genome titer, when the AAV8 genome titer obtained when AAV8 was produced using a medium containing neither spermidine trihydrochloride nor β-alanine was set at 100%. On the horizontal axis, "Viral Production Medium" represents the case where AAV8 was produced using a medium to which neither spermidine trihydrochloride nor β-alanine was added. "Viral Production Medium + 200 μM spermidine" represents the case where AAV8 was produced using a medium to which spermidine trihydrochloride was added to a final concentration of 200 μM. "Viral Production Medium + 300 μM β-alanine" represents the case where AAV8 was produced using a medium to which β-alanine was added to a final concentration of 300 μM. "β-alanine" represents the case where AAV8 was produced using a medium supplemented with spermidine trihydrochloride to a final concentration of 200 μM and β-alanine to a final concentration of 300 μM.

[0011] 1. Medium Composition 1 The present invention provides a medium composition containing β-alanine for promoting the production of a virus carrying a target gene during the cultivation of a virus-producing mammalian cell into which the target gene has been introduced (hereinafter also referred to as "medium composition 1 of the present invention").

[0012] β-alanine may exist in the form of a solvate such as a hydrate, and in the present invention, these solvates are also referred to as β-alanine.

[0013] The β-alanine used in the medium composition 1 of the present invention may be synthesized by a method known per se, or may be obtained as a commercially available product.

[0014] The concentration of β-alanine in medium composition 1 of the present invention is not particularly limited as long as the effects of the present invention are achieved, but is usually 0.1 μM to 800 μM, preferably 1 μM to 700 μM, more preferably 3 μM to 600 μM, even more preferably 10 μM to 500 μM, and particularly preferably 30 μM to 400 μM.

[0015] As used herein, the term "target gene" refers to a heterologous polynucleotide that can be introduced into a cell or organism via a viral vector or the like, and may be any polynucleotide, such as a gene encoding a polypeptide or protein, or a polynucleotide that can be transcribed into an inhibitory polynucleotide (e.g., siRNA, miRNA, shRNA, etc.), and is preferably intended for the treatment of a disease in an animal, including a human.

[0016] Examples of viruses include adenoviruses, adeno-associated viruses (AAVs), retroviruses, lentiviruses, and Sendai viruses, as well as orthomyxoviruses, paramyxoviruses, reoviruses, picornaviruses, flaviviruses, arenaviruses, herpesviruses, and poxviruses, and recombinant viruses thereof. Among these, adenoviruses and AAVs are preferred, and AAVs are more preferred. Examples of AAV include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, AAVDJ8, AAV-B1, AAVM41, AAVrhlO, AAVrh74, and chimeras thereof, with AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9 being more preferred, and AAV2 and AAV8 being even more preferred.

[0017] The mammalian cells for producing the virus are not particularly limited as long as they are capable of producing a virus carrying the desired gene of interest, and cells from humans, monkeys, rodents, etc. can be used. Specific examples include HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, HeLa cells, CV-1 cells, LLC-MK2 cells, MDBK cells, WI-38 cells, MRC5 cells (human fibroblasts), BHK21 cells, etc., and preferred are HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, HeLa cells, etc., all of which are commercially available. When the virus is adenovirus or AAV, HEK293 cells, Vero cells, A549 cells, etc. are more preferably used, and HEK293 cells are even more preferably used.

[0018] The method for introducing a gene of interest into virus-producing mammalian cells is not particularly limited and can be performed using gene transfer methods well known to those skilled in the art. More specifically, examples include chemical methods using transfection reagents such as cationic lipids, cationic polymers (e.g., polyetherimide (PEI)), calcium phosphate, etc.; physical methods such as lipofection, electroporation, microinjection, sonoporation, and laser irradiation; and infection methods (biological methods) using viral vectors (e.g., adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and Sendai virus vectors). Chemical methods using transfection reagents are preferred, and chemical methods using cationic lipids or cationic polymers are more preferred. The gene of interest can be prepared by methods well known to those skilled in the art, but it can also be obtained as a commercially available product.

[0019] The culture may be either adherent culture or suspension culture, but suspension culture is preferred. As used herein, "adherent culture" refers to culturing cells by adhering them to a culture substrate, specifically a method of growing cells while adhering to the surface of the culture substrate and while adhering cells to each other. Examples of culture substrates include, but are not limited to, multiwell plates, culture dishes, petri dishes, culture flasks, microcarriers, hollow fibers, and the like. The culture may be static culture on a substrate. As used herein, "suspension culture" refers to a cell culture method in which cells are not adhered to the culture vessel. Suspension culture may or may not involve external pressure or vibration on the liquid medium, or shaking or rotation in the liquid medium.

[0020] The culture vessel used for culturing is not particularly limited as long as it is capable of culturing the target cells, and examples include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, petri dishes, tubes, trays, culture bags, roller bottles, bioreactors, etc.

[0021] The culture vessel may be cell-adhesive or cell-non-adhesive, and is appropriately selected depending on the purpose. A cell-adhesive culture vessel may be coated with any cell-supporting substrate, such as an extracellular matrix (ECM), for the purpose of improving adhesion of cells to the surface of the culture vessel. The cell-supporting substrate may be any substance intended for cell adhesion.

[0022] "Promoting the production of viruses carrying a gene of interest" refers to increasing the genome titer of the gene of interest in all viruses produced during the culture of virus-producing mammalian cells into which the gene of interest has been introduced. That is, it refers to (a) improving the full capsid ratio regardless of whether the capsid number (virus number) increases or decreases, thereby increasing the overall genome titer, or (b) increasing the capsid number regardless of whether the full capsid ratio is high or low, thereby increasing the overall genome titer.

[0023] Medium composition 1 of the present invention may further contain a polyamine or a salt thereof, and is more preferably a medium composition containing β-alanine and a polyamine or a salt thereof.

[0024] The polyamine is not particularly limited as long as it is a compound having two or more amino groups, such as primary amino groups, secondary amino groups, or tertiary amino groups, in the molecule, and includes aliphatic polyamines including cyclic polyamines and aromatic polyamines having one or more aromatic rings. Among these, aliphatic polyamines including cyclic polyamines are preferred. Furthermore, the polyamine may have a substituent other than the amino group, such as halogen, hydroxy, carboxy, carbamoyl, alkoxy having 1 to 6 carbon atoms, alkanoyl having 1 to 6 carbon atoms, or alkoxycarbonyl having 1 to 6 carbon atoms, but is preferably a polyamine having no substituent other than the amino group.

[0025] More specific examples of polyamines include linear or branched aliphatic polyamines having 2 to 20 carbon atoms and two or more primary and / or secondary amino groups in the molecule, linear or branched aliphatic polyamines having 3 to 18 carbon atoms are preferred, linear aliphatic polyamines having 3 to 15 carbon atoms are more preferred, linear aliphatic polyamines having 4 to 12 carbon atoms are even more preferred, and spermidine, spermine, and putrescine are even more preferred, with spermidine being particularly preferred. That is, medium composition 1 of the present invention is particularly preferably a medium composition containing β-alanine and spermidine or a salt thereof.

[0026] Examples of the salts of polyamines include acid addition salts and amino acid addition salts.

[0027] Examples of acid addition salts include inorganic acid salts such as hydrochloride, sulfate, hydrobromide, nitrate, and phosphate, and organic acid salts such as acetate, mesylate, succinate, maleate, fumarate, citrate, and tartrate, and examples of amino acid addition salts include addition salts of aspartic acid, glutamic acid, etc. Among these, inorganic acid salts such as hydrochloride and phosphate are preferred, and hydrochloride is more preferred.

[0028] When it is desired to obtain a salt of a polyamine, if the polyamine is obtained in the form of a salt, it can be purified as is. Alternatively, if the polyamine is obtained in the free form, it can be isolated and purified by dissolving or suspending the polyamine in an appropriate solvent and adding an acid or the like.

[0029] Furthermore, polyamines or salts thereof may exist in the form of solvates such as hydrates, and in the present invention, these solvates are also included in the term polyamines or salts thereof.

[0030] The polyamine or its salt used in the medium composition 1 of the present invention may be synthesized by a method known per se, or may be obtained as a commercially available product.

[0031] When medium composition 1 of the present invention contains a polyamine or a salt thereof, the concentration of the polyamine or a salt thereof in medium composition 1 of the present invention is not particularly limited as long as the effects of the present invention are achieved, but is usually 0.1 μM to 700 μM, preferably 0.3 μM to 500 μM, more preferably 1 μM to 400 μM, even more preferably 10 μM to 300 μM, and particularly preferably 30 μM to 200 μM.

[0032] Furthermore, medium composition 1 of the present invention may or may not further contain a pyruvate dehydrogenase (PDH) kinase inhibitor.

[0033] PDH kinase is an enzyme that phosphorylates and inactivates PDH, and regulates its activity in an antagonistic manner to PDH phosphatase. PDH kinase inhibitors are organic molecules that prevent PDH kinase from phosphorylating specific serine residues on the E1 (alpha subunit) of the PDH complex, which is involved in regulating PDH activity.

[0034] Various compounds are known as PDH kinase inhibitors, such as dichloroacetic acid or a salt thereof, AZD7545, VER-246608, PDHK-IN-5, and Leelamin, but dichloroacetic acid or a salt thereof is preferred, and sodium salt of dichloroacetic acid is more preferred.

[0035] When medium composition 1 of the present invention contains a PDH kinase inhibitor, the concentration of the PDH kinase inhibitor in medium composition 1 of the present invention is generally 0.1 mM to 1,000 mM, preferably 0.3 mM to 300 mM, more preferably 0.5 mM to 100 mM, and even more preferably 1 mM to 20 mM.

[0036] Medium composition 1 of the present invention may further contain other components that are favorable for virus production, as long as the effects of the present invention are not impaired. Other components include, for example, sugars such as glucose, fructose, sucrose, and maltose; amino acids other than β-alanine; proteins such as albumin and transferrin; peptides such as glycylglycylglycine and soybean peptide; serum; vitamins such as choline, vitamin A, B vitamins (thiamine, pyridoxine, cyanocobalamin, biotin, pantothenic acid, nicotinamide, etc.), vitamin C, and vitamin E; fatty acids such as oleic acid, arachidonic acid, and linoleic acid; lipids such as cholesterol; inorganic salts such as sodium chloride, calcium chloride, and sodium dihydrogen phosphate; trace elements such as zinc and selenium; buffers such as N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) and N-[tris(hydroxymethyl)methyl]glycine (Tricine); antibiotics such as amphotericin B, kanamycin, gentamicin, streptomycin, and penicillin; Type I collagen, Type II collagen, and the like. Examples of other components include cell adhesion factors and extracellular matrix components such as collagen, fibronectin, laminin, poly-L-lysine, and poly-D-lysine; cytokines and growth factors such as interleukin, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), transforming growth factor (TGF)-α, transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), and activin A; and hormones such as dexamethasone, hydrocortisone, estradiol, progesterone, glucagon, and insulin. Appropriate components can be selected and used depending on the type of cells to be cultured, the type of virus to be produced, and the like. The above-mentioned other components may be contained alone or in combination of two or more.

[0037] The other components can be contained in the medium composition 1 of the present invention at a concentration of, for example, a total amount of 0.001% by weight to 99.9% by weight, preferably 0.01% by weight to 99% by weight, and more preferably 0.1% by weight to 95% by weight.

[0038] Furthermore, medium composition 1 of the present invention may contain serum or may be serum-free. The serum is not particularly limited as long as it is derived from an animal and does not inhibit cell growth, but preferred examples include mammalian serum (e.g., fetal bovine serum, human serum, etc.), and more preferred is fetal bovine serum. The serum concentration may be within a known concentration range. Medium composition 1 of the present invention is preferably serum-free.

[0039] The medium composition 1 of the present invention can be prepared, for example, by adding β-alanine directly to a basal medium, and this embodiment is preferred because of its simplicity. When β-alanine is in the form of a solution in the form of the medium additive 1 of the present invention described below, the medium composition 1 of the present invention can be obtained by adding 1 / 10,000 to 1 / 5, preferably 1 / 5,000 to 1 / 8, and more preferably 1 / 1,000 of the amount of the medium additive 1 of the present invention to a basal medium.

[0040] The basal medium refers to a medium containing carbon sources, nitrogen sources, inorganic salts, etc., which are essential for cell culture. There are no particular limitations on the medium as long as it can achieve the effects of the present invention, and it may be selected appropriately depending on the cells to be cultured.

[0041] Examples of basal media include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A medium, Minimum Essential Medium (MEM), Eagle's Minimum Essential Medium (EMEM), and alpha Modified Eagle's Minimum Essential Medium (alpha Modified Eagle's Minimum Essential Medium). Examples of the medium include αMEM, Roswell Park Memorial Institute (RPMI) 1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, Fischer's Medium, and a mixture of these media. The basal medium may be prepared by a method known per se, or may be prepared using Viral Production Medium (Thermo Fisher Scientific: A4817901), HyClone HyCell TransFx-H transfection media (Cytiva: SH30939.01), BalanCD® HEK293 (Fujifilm Wako Pure Chemical Industries, Ltd.: 551-34231), EX-CELL® 293 (Merck: 14571C), FreeStyle™ 293 Expression Medium (Thermo Fisher Scientific: The medium may be commercially available, such as PEG-400 (available from Thermo Fisher Scientific: 12338018), CDM4HEK293 (available from Cytiva: SH30858.02), or Expi293™ Expression Medium (available from Thermo Fisher Scientific: A1435101), or may be one currently under development.

[0042] When medium composition 1 of the present invention contains a polyamine or a salt thereof, a PDH kinase inhibitor, serum, and / or other components, medium composition 1 of the present invention can be produced by adding β-alanine, and the polyamine or a salt thereof, the PDH kinase inhibitor, serum, and / or other components to the basal medium as appropriate and mixing them directly, or by other production methods commonly used in the field of medium compositions. The order in which these components are added is not particularly limited and can be determined appropriately depending on the intended use, etc.

[0043] The medium composition 1 of the present invention may be provided in a liquid state, or may be prepared in a more concentrated state than the concentration at the time of use, or in a solid state such as a freeze-dried powder, and then diluted with a solvent such as water, or dissolved or dispersed in a solvent such as water, at the time of use.

[0044] By culturing virus-producing mammalian cells into which a target gene has been introduced in medium composition 1 of the present invention, the production of the virus carrying the target gene can be promoted.

[0045] The present invention also provides use of β-alanine in the production of a medium composition for promoting the production of a virus carrying a target gene during the culture of a virus-producing mammalian cell into which the target gene has been introduced. The respective terms, etc., are as explained above.

[0046] The present invention also provides a medium composition containing a polyamine or a salt thereof for promoting the production of a virus carrying a gene of interest during the cultivation of virus-producing mammalian cells into which a gene of interest has been introduced, and the use of a polyamine or a salt thereof in the production of a medium composition for promoting the production of a virus carrying a gene of interest during the cultivation of virus-producing mammalian cells into which a gene of interest has been introduced. The respective terms, etc. are as explained above.

[0047] 2. Medium Additive 1 The present invention also provides a medium additive containing β-alanine for promoting the production of a virus carrying a target gene during the cultivation of virus-producing mammalian cells into which the target gene has been introduced (hereinafter also referred to as "medium additive 1 of the present invention").

[0048] The "β-alanine," "target gene," "virus," "virus-producing mammalian cells," "culture," and "promotion of production of virus carrying a target gene," as well as the method for introducing a target gene into virus-producing mammalian cells, are as described above for medium composition 1 of the present invention.

[0049] The concentration of β-alanine in medium additive 1 of the present invention is not particularly limited as long as the effects of the present invention are achieved, but β-alanine is contained in medium additive 1 so that the final concentration in the basal medium when medium additive 1 is added to the basal medium is typically 0.1 μM to 800 μM, preferably 1 μM to 700 μM, more preferably 3 μM to 600 μM, even more preferably 10 μM to 500 μM, and particularly preferably 30 μM to 400 μM. For example, when medium additive 1 of the present invention is in the form of a solution, the concentration of β-alanine in medium additive 1 is also not particularly limited as long as the effects of the present invention are achieved, but is typically 10 μM to 800 mM, preferably 30 μM to 700 mM, more preferably 50 μM to 600 mM, even more preferably 100 μM to 500 mM, and particularly preferably 300 μM to 400 mM.

[0050] The "basal medium" is as explained above for medium composition 1 of the present invention.

[0051] Culture medium additive 1 of the present invention may further contain a polyamine or a salt thereof, and is more preferably a culture medium additive containing β-alanine and a polyamine or a salt thereof, and particularly preferably a culture medium additive containing β-alanine and spermidine or a salt thereof.

[0052] The "polyamine or its salt" is as explained above for medium composition 1 of the present invention.

[0053] When medium additive 1 of the present invention contains a polyamine or a salt thereof, the concentration of the polyamine or a salt thereof in medium additive 1 of the present invention is not particularly limited as long as the effects of the present invention are exhibited, but the polyamine or a salt thereof is contained in medium additive 1 so that the final concentration in the basal medium when medium additive 1 is added to the basal medium is typically 0.1 μM to 700 μM, preferably 0.3 μM to 500 μM, more preferably 1 μM to 400 μM, even more preferably 10 μM to 300 μM, and particularly preferably 30 μM to 200 μM. For example, when medium additive 1 of the present invention is in the form of a solution, the concentration of the polyamine or a salt thereof in medium additive 1 is not particularly limited as long as the effects of the present invention are exhibited, but is typically 10 μM to 700 mM, preferably 30 μM to 500 mM, more preferably 50 μM to 400 mM, even more preferably 100 μM to 300 mM, and particularly preferably 300 μM to 200 mM.

[0054] Furthermore, the medium additive 1 of the present invention may or may not contain a PDH kinase inhibitor.

[0055] The "PDH kinase inhibitor" is as explained above for medium composition 1 of the present invention.

[0056] When medium additive 1 of the present invention contains a PDH kinase inhibitor, the PDH kinase inhibitor is contained in medium additive 1 so that the final concentration in the basal medium when medium additive 1 is added to the basal medium is generally 0.1 mM to 1,000 mM, preferably 0.3 mM to 300 mM, more preferably 0.5 mM to 100 mM, and even more preferably 1 mM to 20 mM. For example, when medium additive 1 of the present invention is in the form of a solution, the concentration of the PDH kinase inhibitor in medium additive 1 is generally 1 mM to 10,000 mM, preferably 3 mM to 3,000 mM, more preferably 10 mM to 2,000 mM, and even more preferably 20 mM to 1,000 mM.

[0057] The medium additive 1 of the present invention may contain other components that are favorable for virus production, as long as the effects of the present invention are not impaired. Examples of other components include nutrients, vitamins, minerals, amino acids other than β-alanine, sugars, etc. that are typically added to culture media. The above-mentioned other components may be contained alone or in combination of two or more.

[0058] The content of the above-mentioned other components in the culture medium additive 1 of the present invention is not particularly limited, but the total amount is preferably 0.000001 to 99.99% by weight, more preferably 0.00001 to 99.9% by weight, even more preferably 0.00001 to 99% by weight, even more preferably 0.0001 to 95% by weight, and particularly preferably 0.0001 to 90% by weight.

[0059] When medium additive 1 of the present invention is β-alanine itself, it can be used as is. When it contains a polyamine or a salt thereof, a PDH kinase inhibitor, and / or other components as described above, it can be produced by adding the polyamine or a salt thereof, the PDH kinase inhibitor, and / or other components to β-alanine as appropriate and mixing them as is, or by other production methods commonly used in the field of medium additives. The order in which these components are added is not particularly limited and can be determined appropriately depending on the intended use, etc. Furthermore, medium additive 1 of the present invention may be in the form of a solution, or the solution may be freeze-dried or otherwise processed to form a solid powder.

[0060] Culture medium additive 1 of the present invention can usually be used by adding it to a basal medium. Furthermore, for example, in cases where virus-producing mammalian cells are cultured in a medium that does not contain one or more selected from β-alanine and polyamines or salts thereof to grow the cells to a certain extent, the cell number is adjusted as necessary, a gene of interest is introduced into the cells, and the cells are subsequently cultured in the same medium, culture medium additive 1 of the present invention can be added to the medium before (e.g., several hours before), during, or after (e.g., several hours after) the gene of interest is introduced into the virus-producing mammalian cells. For example, when culture medium additive 1 of the present invention is in the form of a solution, medium additive 1 is added in an amount of 1 / 10,000 to 1 / 5, preferably 1 / 5,000 to 1 / 8, and more preferably 1 / 1,000, to a basal medium or a medium that does not contain one or more selected from β-alanine and polyamines or salts thereof.

[0061] By culturing virus-producing mammalian cells into which a gene of interest has been introduced and which produce the virus, in a basal medium or a medium that does not contain one or more selected from β-alanine and polyamines or salts thereof, to which medium additive 1 of the present invention has been added, the production of the virus carrying the gene of interest can be promoted.

[0062] The present invention also provides a medium additive for promoting production of a virus carrying a target gene during the culture of a mammalian cell that produces the virus and into which the target gene has been introduced, the medium additive comprising the polyamine or a salt thereof. The respective terms and the like are as explained above.

[0063] 3. Method for promoting production of a virus carrying a gene of interest The present invention also provides a method for promoting production of a virus carrying a gene of interest (hereinafter also referred to as the "promoting method of the present invention"), which comprises the step of culturing virus-producing mammalian cells into which a gene of interest has been introduced, in a medium containing β-alanine.

[0064] The terms "β-alanine," "target gene," "virus," "virus-producing mammalian cells," "culture," and "promotion of production of virus carrying a target gene," as well as the method for introducing a target gene into virus-producing mammalian cells, are as explained above for medium composition 1 of the present invention. Furthermore, "medium containing β-alanine" refers to a medium having the same composition as medium composition 1 of the present invention.

[0065] Mammalian cells transfected with a target gene and capable of producing a virus can be cultured under the same conditions as those used to culture normal mammalian cells, such as 95% humidity and CO ２ An example of such a culture is at a concentration of 5% to 10% (v / v), but the culture is not limited to these conditions. Culture can be carried out at, for example, 30°C to 37°C, but may be carried out at a temperature outside the above range as long as the desired cell growth and virus production into which the target gene has been introduced are achieved. The culture period is not particularly limited, and is usually 1 hour to 14 days, preferably 5 hours to 7 days, more preferably 12 hours to 150 hours, and even more preferably 48 hours to 120 hours.

[0066] Furthermore, in the promotion method of the present invention, the culture of virus-producing mammalian cells into which a gene of interest has been introduced can be carried out by any of the following methods: (1) Virus-producing mammalian cells are cultured in a medium containing one or more selected from β-alanine and polyamines or salts thereof to grow the cells to a certain extent, and after adjusting the cell number as necessary, the gene of interest is introduced into the cells, and the cells are subsequently cultured in the same medium; (2) Virus-producing mammalian cells are cultured in a medium not containing one or more selected from β-alanine and polyamines or salts thereof to grow the cells to a certain extent, and after adjusting the cell number as necessary, the medium is replaced with a medium containing one or more selected from β-alanine and polyamines or salts thereof, the gene of interest is then introduced into the cells, and the cells are subsequently cultured in the same medium (the order of medium replacement and gene of interest introduction may be reversed). (3) Virus-producing mammalian cells are cultured in a medium that does not contain one or more selected from β-alanine and polyamines or salts thereof to grow the cells to a certain extent, and after adjusting the cell number as necessary, a gene of interest is introduced into the cells, which are then cultured in the same medium. Here, one or more selected from β-alanine and polyamines or salts thereof are added to the medium before (e.g., several hours before), during, or after (e.g., several hours after) the gene of interest is introduced into the virus-producing mammalian cells.

[0067] The present invention also provides a method for promoting production of a virus carrying a gene of interest, comprising the step of culturing virus-producing mammalian cells into which the gene of interest has been introduced in a medium containing a polyamine or a salt thereof.

[0068] 4. Medium Composition 2 The present invention also provides a medium composition containing β-alanine for increasing the proportion of viruses carrying a gene of interest among all viruses produced during the culture of virus-producing mammalian cells into which a gene of interest has been introduced (hereinafter also referred to as "medium composition 2 of the present invention").

[0069] The "β-alanine," "target gene," "virus," "virus-producing mammalian cells," and "culture," as well as the method for introducing the target gene into the virus-producing mammalian cells, are as described above for medium composition 1 of the present invention.

[0070] "Improving the ratio of viruses carrying the target gene among all viruses produced" refers to increasing the ratio of viruses (capsids) carrying the target gene among all viruses (capsids) produced during the culture of virus-producing mammalian cells into which the target gene has been introduced.

[0071] The concentration of β-alanine in medium composition 2 of the present invention is not particularly limited as long as the effects of the present invention are achieved, but is usually 0.1 μM to 800 μM, preferably 1 μM to 700 μM, more preferably 3 μM to 600 μM, even more preferably 10 μM to 500 μM, and particularly preferably 30 μM to 400 μM.

[0072] Unless otherwise specified below, medium composition 2 of the present invention may contain the same polyamine or a salt thereof, PDH kinase inhibitor, serum, and / or other components as described above for medium composition 1 of the present invention, so long as the effects of the present invention are not impaired. Of these, medium composition 2 of the present invention is more preferably a medium composition containing β-alanine and a polyamine or a salt thereof, and particularly preferably a medium composition containing β-alanine and spermidine or a salt thereof.

[0073] When medium composition 2 of the present invention contains a polyamine or a salt thereof, the concentration of the polyamine or a salt thereof in medium composition 2 of the present invention is not particularly limited as long as the effects of the present invention are exhibited, but is usually 0.1 μM to 180 μM, preferably 0.3 μM to 150 μM, more preferably 1 μM to 130 μM, even more preferably 10 μM to 120 μM, and particularly preferably 30 μM to 100 μM.

[0074] The production method, form, and method of use of medium composition 2 are the same as those described above for medium composition 1 of the present invention.

[0075] By culturing virus-producing mammalian cells into which a target gene has been introduced in medium composition 2 of the present invention, the proportion of viruses carrying the target gene among all the viruses produced can be increased.

[0076] The present invention also provides use of β-alanine in the production of a medium composition for increasing the proportion of viruses carrying a gene of interest among all viruses produced during the culture of virus-producing mammalian cells into which a gene of interest has been introduced. The respective terms, etc., are as explained above.

[0077] The present invention also provides a medium composition containing a polyamine or a salt thereof for increasing the proportion of viruses carrying a gene of interest among all viruses produced during the culture of virus-producing mammalian cells into which a gene of interest has been introduced, and use of a polyamine or a salt thereof in the production of a medium composition for increasing the proportion of viruses carrying a gene of interest among all viruses produced during the culture of virus-producing mammalian cells into which a gene of interest has been introduced. The respective terms, etc. are as explained above.

[0078] 5. Medium Additive 2 The present invention also provides a medium additive containing β-alanine for increasing the proportion of viruses carrying a gene of interest among all viruses produced during the cultivation of virus-producing mammalian cells into which the gene of interest has been introduced (hereinafter also referred to as "medium additive 2 of the present invention").

[0079] The "β-alanine," "target gene," "virus," "virus-producing mammalian cells," and "culture," as well as the method for introducing the target gene into the virus-producing mammalian cells, are as explained above for medium composition 1 of the present invention, and the "improvement of the proportion of viruses carrying the target gene among all viruses produced" is as explained above for medium composition 2 of the present invention.

[0080] The concentration of β-alanine in medium additive 2 of the present invention is not particularly limited as long as the effects of the present invention are achieved, but β-alanine is included in medium additive 2 so that the final concentration in the basal medium when medium additive 2 is added to the basal medium is typically 0.1 μM to 800 μM, preferably 1 μM to 700 μM, more preferably 3 μM to 600 μM, even more preferably 10 μM to 500 μM, and particularly preferably 30 μM to 400 μM. For example, when medium additive 2 of the present invention is in the form of a solution, the concentration of β-alanine in medium additive 2 is also not particularly limited as long as the effects of the present invention are achieved, but is typically 10 μM to 800 mM, preferably 30 μM to 700 mM, more preferably 50 μM to 600 mM, even more preferably 100 μM to 500 mM, and particularly preferably 300 μM to 400 mM.

[0081] The "basal medium" is as explained above for medium composition 1 of the present invention.

[0082] As long as the effects of the present invention are not impaired, medium additive 2 of the present invention may contain the same polyamine or a salt thereof, PDH kinase inhibitor, and / or other components as described above for medium additive 1 of the present invention, in the same amounts as described above for medium additive 1 of the present invention. Among these, medium additive 2 of the present invention is more preferably a medium additive containing β-alanine and a polyamine or a salt thereof, and particularly preferably a medium additive containing β-alanine and spermidine or a salt thereof.

[0083] When medium additive 2 of the present invention contains a polyamine or a salt thereof, the concentration of the polyamine or a salt thereof in medium additive 2 of the present invention is not particularly limited as long as the effects of the present invention are exhibited, but the polyamine or a salt thereof is contained in medium additive 2 so that the final concentration in the basal medium when medium additive 2 is added to the basal medium is typically 0.1 μM to 180 μM, preferably 0.3 μM to 150 μM, more preferably 1 μM to 130 μM, even more preferably 10 μM to 120 μM, and particularly preferably 30 μM to 100 μM. For example, when medium additive 2 of the present invention is in the form of a solution, the concentration of the polyamine or a salt thereof in medium additive 2 is not particularly limited as long as the effects of the present invention are exhibited, but is typically 10 μM to 600 mM, preferably 30 μM to 500 mM, more preferably 50 μM to 400 mM, even more preferably 100 μM to 300 mM, and particularly preferably 300 μM to 200 mM.

[0084] The production method, form and method of use of the medium additive 2 of the present invention are the same as those explained above for the medium additive 1 of the present invention.

[0085] By adding medium additive 2 of the present invention to a basal medium and culturing virus-producing mammalian cells into which a target gene has been introduced, the proportion of viruses carrying the target gene among all the viruses produced can be increased.

[0086] The present invention also provides a medium additive containing a polyamine or a salt thereof for increasing the proportion of viruses carrying a target gene among all viruses produced during the culture of virus-producing mammalian cells into which a target gene has been introduced. The respective terms, etc., are as explained above.

[0087] 6. Method for increasing the proportion of viruses carrying a gene of interest The present invention also provides a method for increasing the proportion of viruses carrying a gene of interest among all viruses produced during culture, the method comprising the step of culturing virus-producing mammalian cells into which a gene of interest has been introduced in a medium containing β-alanine.

[0088] The "target gene," "virus," "virus-producing mammalian cells," "culture," and "β-alanine," as well as the method for introducing the target gene into the virus-producing mammalian cells, are as explained above for medium composition 1 of the present invention; the "improvement of the ratio of viruses carrying the target gene among all viruses produced" is as explained above for medium composition 2 of the present invention; and the "culture" method is as explained above for the promotion method of the present invention. Furthermore, "medium containing β-alanine" refers to a medium having the same composition as medium composition 2 of the present invention.

[0089] The present invention also provides a method for increasing the proportion of viruses carrying a gene of interest among all viruses produced during culture, the method comprising culturing virus-producing mammalian cells into which a gene of interest has been introduced in a medium containing a polyamine or a salt thereof.

[0090] 7. Method for Producing a Virus Carrying a Gene of Interest The present invention also provides a method for producing a virus carrying a gene of interest (hereinafter also referred to as "Production Method 1 of the present invention"), which comprises the step of culturing virus-producing mammalian cells into which a gene of interest has been introduced, in a medium containing β-alanine.

[0091] The terms "β-alanine," "target gene," "virus," "virus-producing mammalian cells," and "culture," as well as the method for introducing a target gene into virus-producing mammalian cells, are as explained above for medium composition 1 of the present invention, and the "culture" method is as explained above for promotion method 1 of the present invention. Furthermore, "a medium containing β-alanine" refers to a medium having a composition similar to that of medium composition 1 of the present invention or medium composition 2 of the present invention.

[0092] In Production Method 1 of the present invention, virus-producing mammalian cells into which a gene of interest has been introduced are cultured in a medium containing β-alanine, and the virus carrying the gene of interest can then be purified by a method known per se. For example, the cultured cells are collected from the resulting culture medium and disrupted, and the resulting cell lysate containing the virus is then subjected to appropriate steps such as filtration, ultracentrifugation, chromatography, and ultrafiltration, thereby purifying the virus carrying the gene of interest.

[0093] Production method 1 of the present invention can promote the production of viruses carrying a gene of interest, or can increase the ratio of viruses carrying the gene of interest among all viruses produced when virus-producing mammalian cells into which a gene of interest has been introduced are cultured. That is, the present invention also provides a method for producing a virus carrying a gene of interest, which includes the step of culturing virus-producing mammalian cells into which a gene of interest has been introduced in a medium containing β-alanine, thereby promoting the production of viruses carrying the gene of interest, or increasing the ratio of viruses carrying the gene of interest among all viruses produced when virus-producing mammalian cells into which a gene of interest has been introduced are cultured.

[0094] The present invention also provides a method for producing a virus carrying a gene of interest, comprising the step of culturing virus-producing mammalian cells into which a gene of interest has been introduced in a medium containing a polyamine or a salt thereof. The present invention further provides a method for producing a virus carrying a gene of interest, comprising the step of culturing virus-producing mammalian cells into which a gene of interest has been introduced in a medium containing a polyamine or a salt thereof, thereby promoting the production of a virus carrying a gene of interest or improving the proportion of viruses carrying the gene of interest among all viruses produced during the culture of virus-producing mammalian cells into which a gene of interest has been introduced. The respective terms, etc. are as explained above.

[0095] 8. Medium Composition 3 The present invention also provides a medium composition containing β-alanine for culturing mammalian cells that produce viruses or virus-like particles (hereinafter also referred to as "medium composition 3 of the present invention").

[0096] "β-alanine," "virus," and "culture" are as explained above for medium composition 1 of the present invention.

[0097] Virus-like particles are molecules that resemble viruses but do not contain genetic material and are not infectious.

[0098] The virus or virus-like particle is preferably a virus.

[0099] The mammalian cells that produce viruses or virus-like particles are the same as the "virus-producing mammalian cells" explained above for medium composition 1 of the present invention.

[0100] The concentration of β-alanine in medium composition 3 of the present invention and the method of production, form and method of use of the medium composition are the same as those explained above for medium composition 1 of the present invention.

[0101] Medium composition 3 of the present invention may contain the same polyamine or a salt thereof, PDH kinase inhibitor, serum and / or other components as described above for medium composition 1 of the present invention, in the same amounts as described above for medium composition 1 of the present invention, as long as the effects of the present invention are not impaired.

[0102] The present invention also provides use of β-alanine in the manufacture of a medium composition for culturing mammalian cells that produce viruses or virus-like particles.

[0103] The present invention also provides a medium composition for culturing mammalian cells that produce viruses or virus-like particles, comprising a polyamine or a salt thereof, and use of a polyamine or a salt thereof in the production of a medium composition for culturing mammalian cells that produce viruses or virus-like particles. The respective terms, etc. are as explained above.

[0104] 9. Medium Additive 3 The present invention also provides a medium additive for culturing mammalian cells that produce viruses or virus-like particles, which contains β-alanine (hereinafter also referred to as "medium additive 3 of the present invention").

[0105] "β-alanine," "virus," and "culture" are as explained above for medium composition 1 of the present invention, and "virus-like particles" are as explained above for medium composition 3 of the present invention.

[0106] The virus or virus-like particle is preferably a virus.

[0107] The mammalian cells that produce viruses or virus-like particles are the same as the "virus-producing mammalian cells" explained above for medium composition 1 of the present invention.

[0108] Culture medium additive 3 of the present invention may contain the same polyamine or a salt thereof, PDH kinase inhibitor and / or other components as described above for culture medium additive 1 of the present invention, in the same amounts as described above for culture medium additive 1 of the present invention, as long as the effects of the present invention are not impaired.

[0109] The concentration of β-alanine in medium additive 3 of the present invention and the method of production, form and method of use of the medium additive are the same as those explained above for medium additive 1 of the present invention.

[0110] The present invention also provides a medium additive for culturing mammalian cells that produce viruses or virus-like particles, comprising a polyamine or a salt thereof. The respective terms, etc., are as explained above.

[0111] 10. Cell Culturing Method The present invention also provides a method for culturing cells (hereinafter also referred to as the "culturing method of the present invention") that comprises a step of culturing mammalian cells that produce viruses or virus-like particles in a medium containing β-alanine.

[0112] "β-alanine," "virus," and "culture" are as explained above for medium composition 1 of the present invention, and "virus-like particles" are as explained above for medium composition 3 of the present invention. Furthermore, "medium containing β-alanine" refers to a medium having the same composition as medium composition 3 of the present invention.

[0113] The virus or virus-like particle is preferably a virus.

[0114] The mammalian cells that produce viruses or virus-like particles are the same as the "virus-producing mammalian cells" explained above for medium composition 1 of the present invention.

[0115] Mammalian cells that produce viruses or virus-like particles can be cultured under the same conditions as those used to culture normal mammalian cells, such as 95% humidity and CO ２ An example of such a culture is at a concentration of 5% to 10% (v / v), but the culture is not limited to these conditions. Culture can be carried out at, for example, 30°C to 37°C, but may be carried out at a temperature outside the above range as long as the desired cell growth and virus or virus-like particle production are achieved. The culture period is not particularly limited and is usually 1 hour to 14 days, preferably 5 hours to 7 days, more preferably 12 hours to 150 hours, and even more preferably 48 hours to 120 hours. Furthermore, β-alanine may be added at any point during the culture period, and the culture may be continued.

[0116] The present invention also provides a method for culturing a mammalian cell that produces a virus or virus-like particle, the method comprising culturing the cell in a medium containing a polyamine or a salt thereof.

[0117] 11. Method for Producing Virus or Virus-Like Particle The present invention also provides a method for producing the virus or virus-like particle (hereinafter also referred to as "Production Method 2 of the Present Invention"), which comprises the step of culturing mammalian cells that produce the virus or virus-like particle in a medium containing β-alanine.

[0118] "β-alanine," "virus," and "culture" are as explained above for medium additive 1 of the present invention, "virus-like particles" are as explained above for medium composition 3 of the present invention, and the "culture" method is as explained above for the culture method of the present invention. Furthermore, "a medium containing β-alanine" means a medium having the same composition as medium composition 3 of the present invention.

[0119] The virus or virus-like particle is preferably a virus.

[0120] The mammalian cells that produce viruses or virus-like particles are the same as the "virus-producing mammalian cells" explained above for medium composition 1 of the present invention.

[0121] In Production Method 2 of the present invention, mammalian cells that produce viruses or virus-like particles are cultured in a medium containing β-alanine, and the viruses or virus-like particles can then be purified by a method known per se. For example, the cultured cells are collected from the resulting culture medium and disrupted, and the resulting cell lysate containing the viruses or virus-like particles can be subjected to appropriate steps such as filtration, ultracentrifugation, chromatography, and ultrafiltration to purify the viruses or virus-like particles.

[0122] The present invention also provides a method for producing a virus or virus-like particle, the method comprising culturing a mammalian cell that produces the virus or virus-like particle in a medium containing a polyamine or a salt thereof. The respective terms and conditions are as explained above.

[0123] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples in any way.

[0124] In the following Examples 1 to 5, the AAV production ability of human embryonic kidney cells 293 (HEK293) using β-alanine or spermidine trihydrochloride was evaluated. The reagents, cells, and media used were as follows: β-alanine: β-alanine (Fujifilm Wako Pure Chemical Industries, Ltd., 014-01062) Spermidine trihydrochloride: spermidine trihydrochloride (Merck, S2501) HEK293: Viral Production Cells 2.0 (Thermo Fisher Scientific, A49784) HEK293 medium: Viral Production Medium (Thermo Fisher Scientific: A4817901)

[0125] Example 1: AAV production in HEK293 cells with the addition of β-alanine using a suspension culture system Viral Production Cells 2.0 were seeded into 30 mL of Viral Production Medium (VIOLAMO, SEF125V) containing GlutaMAX™ Supplement (Thermo Fisher Scientific: 35050061) added to a final concentration of 4 mM, and the cells were incubated at 37°C and 8% CO. ２ The cells were cultured with agitation in an incubator set at 4°C for two passages. Then, 0.6 x 10 cells were cultured in (1) a medium prepared by adding GlutaMAX™ Supplement to 30 mL of Viral Production Medium to a final concentration of 4 mM, or (2) a medium prepared by adding GlutaMAX™ Supplement to 30 mL of Viral Production Medium to a final concentration of 4 mM and β-alanine to a final concentration of 100 μM. ６ The cells were seeded at a cell density of 100 cells / mL and cultured with stirring for 3 days. On the third day, the number of viable cells was measured using a viable cell autoanalyzer, Vi-CELL BLU (manufactured by Becman). Then, when the number of viable cells was 3 x 10 ６The cells were diluted with fresh medium to a concentration of 1000 cells / mL, and 3 mL of the solution was seeded into a 6-well suspension culture plate (Sumitomo Bakelite Co., Ltd.: MS-8006R). Then, using a cationic lipid-based AAV-MAX Transfection Kit (Thermo Fisher Scientific: A50515), AAVpro® Packaging Plasmid (AAV8) (Takara Bio Inc.: 6681) and pAAV-ZsGreen1 Vector (Takara Bio Inc.: 6231) were transfected. After 3 days of culture, the cells were diluted with fresh medium to a concentration of 1000 cells / mL, and 3 mL of the solution was seeded into a 6-well suspension culture plate (Sumitomo Bakelite Co., Ltd.: MS-8006R). Then, using a cationic lipid-based AAV-MAX Transfection Kit (Thermo Fisher Scientific: A50515), the cells were transfected with AAVpro® Packaging Plasmid (AAV8) (Takara Bio Inc.: 6681) and pAAV-ZsGreen1 Vector (Takara Bio Inc.: 6231). After 3 days of culture, the cells were titrated using AAVpro® Titration Kit (for Real Time PCR) Ver. AAV8 vector was extracted using PCR kit No. 2 (Takara Bio Inc.: 6233), and the AAV8 genome titer was measured using a 7500 Fast Real-Time PCR System (Thermo Fisher Scientific: 4357362). Furthermore, the number of virus particles was measured using an AAV-8 Titration ELISA Kit (PROGEN Biotechnik: PRAAV8). From this, the full / empty capsid ratio (full capsid ratio) was calculated.

[0126] The effect of β-alanine on AAV production in HEK293 was examined in series, and the results are shown in Figures 1 and 2. The AAV8 genome titer (relative) is shown in Figure 1, and the full capsid ratio (relative) is shown in Figure 2. It was confirmed that the addition of β-alanine improved the AAV8 genome titer and the full capsid ratio.

[0127] Example 2 AAV Production in HEK293 Cells with the Addition of β-Alanine Using a Suspension Culture System The effect of adding β-alanine on promoting AAV production in HEK293 cells was investigated under the same conditions as in Example 1 using (1) a medium in which GlutaMAX™ Supplement was added to 30 mL of Viral Production Medium to a final concentration of 4 mM, and (2) a medium in which GlutaMAX™ Supplement was added to 30 mL of Viral Production Medium to a final concentration of 4 mM and β-alanine to final concentrations of 100 μM, 300 μM, 1 mM, 1.5 mM, and 2 mM.

[0128] The effect of β-alanine on AAV production in HEK293 was examined in a series (AAV8 genome titer (relative)) and the results are shown in Figure 3. As a result, an improvement in the AAV8 genome titer was confirmed when a medium containing β-alanine added to a final concentration of 100 μM or 300 μM was used.

[0129] Example 3 AAV Production in HEK293 Cells by Addition of β-Alanine Using a Suspension Culture System The effect of adding β-alanine on promoting AAV production in HEK293 cells was investigated under the same conditions as in Example 1 using (1) a medium in which GlutaMAX™ Supplement was added to 30 mL of Viral Production Medium to a final concentration of 4 mM, and (2) a medium in which GlutaMAX™ Supplement was added to 30 mL of Viral Production Medium to a final concentration of 4 mM and β-alanine to a final concentration of 300 μM.

[0130] The effect of β-alanine on AAV production in HEK293 was examined in a series of experiments, and the results are shown in Figures 4 and 5. The virus count (capsid count (relative)) is shown in Figure 4, and the full capsid ratio (relative) is shown in Figure 5. As a result, it was confirmed that the use of a medium supplemented with β-alanine to a final concentration of 300 µM resulted in an increase in the capsid number and an improvement in the full capsid ratio.

[0131] Example 4: AAV production in HEK293 cells with the addition of spermidine trihydrochloride using a suspension culture system Viral Production Cells 2.0 were seeded in 30 mL of Viral Production Medium (VIOLAMO, SEF125V) containing GlutaMAX™ Supplement (Thermo Fisher Scientific: 35050061) added to a final concentration of 4 mM, and the cells were incubated at 37°C and 8% CO. ２ The cells were then cultured with agitation in an incubator set at 4°C for two passages. Then, 0.6 x 10 cells were cultured in a medium prepared by adding GlutaMAX™ Supplement to 30 mL of Viral Production Medium to a final concentration of 4 mM, or in a medium prepared by adding GlutaMAX™ Supplement to 30 mL of Viral Production Medium to a final concentration of 4 mM and spermidine trihydrochloride to a final concentration of 30 μM or 100 μM. ６ The cells were seeded at a cell density of 100 cells / mL and cultured with stirring for 3 days. On the third day, the number of viable cells was measured using a viable cell autoanalyzer, Vi-CELL BLU (manufactured by Becman). Then, when the number of viable cells was 3 x 10 ６The cells were diluted with fresh medium to a concentration of 1000 cells / mL, and 3 mL of the solution was seeded into a 6-well suspension culture plate (Sumitomo Bakelite Co., Ltd.: MS-8006R). Then, using a cationic lipid-based AAV-MAX Transfection Kit (Thermo Fisher Scientific: A50515), AAVpro® Packaging Plasmid (AAV8) (Takara Bio Inc.: 6681) and pAAV-ZsGreen1 Vector (Takara Bio Inc.: 6231) were transfected. After 3 days of culture, the cells were diluted with fresh medium to a concentration of 1000 cells / mL, and 3 mL of the solution was seeded into a 6-well suspension culture plate (Sumitomo Bakelite Co., Ltd.: MS-8006R). Then, using a cationic lipid-based AAV-MAX Transfection Kit (Thermo Fisher Scientific Inc.: A50515), the cells were transfected with AAVpro® Packaging Plasmid (AAV8) (Takara Bio Inc.: 6681) and pAAV-ZsGreen1 Vector (Takara Bio Inc.: 6231). After 3 days of culture, the cells were titrated using AAVpro® Titration Kit (for Real Time PCR) Ver. AAV8 vectors were extracted using PCR kit 2 (Takara Bio Inc.: 6233), and the AAV8 genome titer was measured using a 7500 Fast Real-Time PCR System (Thermo Fisher Scientific: 4357362). Furthermore, the number of virus particles was measured using an AAV-8 Titration ELISA Kit (PROGEN Biotechnik: PRAAV8). From this, the full capsid ratio (full / (full + empty) ratio = virus carrying the gene of interest / total virus) was calculated.

[0132] The effect of spermidine trihydrochloride on AAV production in HEK293 was examined in a series of experiments, and the results are shown in Figures 6 and 7. The AAV8 genome titer (relative) is shown in Figure 6, and the full capsid ratio (relative) is shown in Figure 7. It was confirmed that the addition of spermidine trihydrochloride improved the AAV8 genome titer and the full capsid ratio.

[0133] Example 5: AAV production in HEK293 with the addition of spermidine trihydrochloride and β-alanine using a suspension culture system (1) A medium prepared by adding GlutaMAX™ Supplement to 30 mL of Viral Production Medium to a final concentration of 4 mM, (2) A medium prepared by adding GlutaMAX™ Supplement to 30 mL of Viral Production Medium to a final concentration of 4 mM and spermidine trihydrochloride to a final concentration of 200 μM, (3) A medium prepared by adding GlutaMAX™ Supplement to 30 mL of Viral Production Medium to a final concentration of 4 mM and spermidine trihydrochloride to a final concentration of 200 μM, (4) A medium prepared by adding GlutaMAX™ Supplement to 30 mL of Viral Production Medium to a final concentration of 4 mM and spermidine trihydrochloride to a final concentration of 200 μM, (5) A medium prepared by adding GlutaMAX™ Supplement to 30 mL of Viral Production Medium to a final concentration of 4 mM and spermidine trihydrochloride to a final concentration of 200 μM, (6) A medium prepared by adding GlutaMAX™ Supplement to 30 mL of Viral Production Medium to a final concentration of 4 mM and spermidine trihydrochloride to a final concentration of 200 μM, (7) A medium prepared by adding GlutaMAX™ Supplement to 30 mL of Viral Production Medium to a final concentration of 4 mM and spermidine trihydrochloride to a final concentration of 200 μM, (8) A medium prepared by adding GlutaMAX™ Supplement to 30 mL of Viral Production Medium to a final concentration of 4 mM and spermidine trihydrochloride to a final concentration of 200 μM, (9) A medium prepared by adding GlutaMAX™ Supplement to 30 mL of Viral Production Medium to a final concentration of 4 mM and spermidine trihydrochloride to a final concentration of 200 μM, (10) A medium prepared by adding GlutaMAX™ Supplement (3) A medium supplemented with GlutaMAX™ Supplement to a final concentration of 4 mM and β-alanine to a final concentration of 300 μM, and (4) a medium supplemented with 30 mL of Viral Production Medium to a final concentration of 4 mM, spermidine trihydrochloride to a final concentration of 200 μM, and β-alanine to a final concentration of 300 μM, were used to examine the effect of adding spermidine trihydrochloride and / or β-alanine on promoting AAV production in HEK293 under the same conditions as in Example 1.

[0134] The effects of spermidine trihydrochloride and / or β-alanine on AAV production in HEK293 were examined in a series, and the AAV8 genome titer (relative) is shown in Figure 8. It was confirmed that the addition of β-alanine and spermidine trihydrochloride individually improved the AAV8 genome titer, and that the addition of β-alanine in combination with spermidine trihydrochloride synergistically improved the AAV8 genome titer.

[0135] Example 6: Adenovirus production in HEK293 cells by adding β-alanine using a suspension culture system In the following example, the adenovirus production ability of human embryonic kidney cells 293 (HEK293) by β-alanine can be evaluated. The reagents, cells, and media used are as follows: β-alanine: β-alanine (Fujifilm Wako Pure Chemical Industries, Ltd., 014-01062) HEK293: suspension-type HEK293 2. sus (ATCC: CRL-1573.3) Adenovirus: Human adenovirus 32 (ATCC VR-625 ＴＭ ) HEK293 medium: 293 SFM II (Thermo Fisher Scientific), CDM4HEK293 (Cytiva)

[0136] (1) After thawing the frozen cells in an incubator set to approximately 37°C, 10 mL of glutamine-containing 293 SFM II was placed in a 15 mL centrifuge tube, and 1 mL of the thawed cell solution was added and suspended. (2) The cells were centrifuged (1000 rpm, 190 x g, 24°C) for 5 minutes using a centrifuge (AX-310, Tomy Seiko Co., Ltd.) and the supernatant was removed. (3) 1 mL of subculture medium was added to the 15 mL centrifuge tube and the cells were resuspended. The entire volume was transferred to a 50 mL tube, and 29 mL of glutamine-containing 293 SFM II was added and suspended. 5 mL of the cell suspension was placed in a petri dish and heated with CO ２ Incubator (Set temperature: 37°C, CO ２ (4) After culturing, collect the culture medium and transfer it to a 15 mL centrifuge tube. (5) Centrifuge for 5 minutes (1000 rpm, 190 × g, 24 °C) and remove the supernatant. (6) Add 1 mL of glutamine-containing 293 SFM II to the 15 mL centrifuge tube and resuspend. Count the cell number using a hemocytometer and count 3 × 10 cells. ５ Add glutamine-containing 293 SFM II to a concentration of 100 cells / mL. Add 5 mL of the adjusted cell suspension to a petri dish and incubate in CO ２ Cultivate in an incubator with shaking (50 rpm) for 3 to 4 days. (7) After culturing, repeat steps (4) to (6) once. (8) CO ２After culturing in an incubator for 3 to 4 days, collect the culture medium and transfer it to a 15 mL centrifuge tube. (9) Centrifuge for 5 minutes (1000 rpm, 190 × g, 24 °C) and remove the supernatant. (10) Add 1 mL of fresh culture medium to the 15 mL centrifuge tube and resuspend. Count the cell number using a hemocytometer and confirm that the number is 3 × 10 ５ Add 5 mL of the adjusted cell suspension to two dishes and incubate in CO ２ The cells are cultured in an incubator with shaking for 3 to 4 days. (11) After culturing, steps (8) to (10) are repeated three times. (12) After culturing for 3 days, the number of cells is counted and 1 x 10 ５ The virus solution was thawed from frozen storage and diluted to 1 x 10 cells / mL with CDM4HEK293 with or without β-alanine, and 5 mL was added to each petri dish. ５ For cells / mL, the virus solution is added to a petri dish so that the MOI is 0.1.

[0137] It can be confirmed that the addition of β-alanine improves the titer of adenovirus.

[0138] According to the present invention, when a virus carrying a target gene is produced using virus-producing mammalian cells, it is possible to improve the production amount of the virus carrying the target gene or the ratio of the virus carrying the target gene to all the viruses produced.

[0139] This application is based on Japanese Patent Application No. 2023-143947 (filing date: September 5, 2023) and Japanese Patent Application No. 2023-202980 (filing date: November 30, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A medium composition containing β-alanine for promoting the production of a virus carrying a target gene during the cultivation of a mammalian cell that produces the virus and into which the target gene has been introduced.

2. The medium composition of claim 1, wherein the mammalian cells are cells selected from the group consisting of HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, and HeLa cells.

3. The medium composition according to claim 1, wherein the mammalian cells are HEK293 cells.

4. The medium composition according to claim 1, wherein the virus-producing mammalian cells are adeno-associated virus-producing mammalian cells.

5. The medium composition according to any one of claims 1 to 4, wherein the concentration of β-alanine in the medium composition is 0.1 μM to 800 μM.

6. The medium composition according to any one of claims 1 to 4, further comprising a polyamine or a salt thereof.

7. The medium composition according to claim 6, wherein the polyamine or a salt thereof is spermidine or a salt thereof.

8. The medium composition according to claim 7, wherein the concentration of spermidine or a salt thereof in the medium composition is 0.1 μM to 700 μM.

9. A medium additive containing β-alanine for promoting the production of a virus carrying a target gene during the cultivation of a mammalian cell that produces the virus and into which the target gene has been introduced.

10. Use of β-alanine in the manufacture of a medium composition for promoting the production of a virus carrying a target gene during the cultivation of a mammalian cell that produces the virus and into which the target gene has been introduced.

11. A method for promoting production of a virus carrying a target gene, comprising the step of culturing virus-producing mammalian cells into which a target gene has been introduced in a medium containing β-alanine.

12. A medium composition containing β-alanine for increasing the ratio of viruses carrying a target gene among all viruses produced during the cultivation of virus-producing mammalian cells into which a target gene has been introduced.

13. The medium composition of claim 12, wherein the mammalian cells are cells selected from the group consisting of HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, and HeLa cells.

14. The medium composition of claim 12, wherein the mammalian cells are HEK293 cells.

15. The medium composition according to claim 12, wherein the virus-producing mammalian cells are adeno-associated virus-producing mammalian cells.

16. The medium composition according to any one of claims 12 to 15, wherein the concentration of β-alanine in the medium composition is 0.1 μM to 800 μM.

17. The medium composition according to any one of claims 12 to 15, further comprising a polyamine or a salt thereof.

18. The medium composition according to claim 17, wherein the polyamine or a salt thereof is spermidine or a salt thereof.

19. The medium composition according to claim 18, wherein the concentration of spermidine or a salt thereof in the medium composition is 0.1 μM to 140 μM.

20. A medium additive containing β-alanine for increasing the ratio of viruses carrying a target gene among all viruses produced during the cultivation of virus-producing mammalian cells into which a target gene has been introduced.

21. Use of β-alanine in the manufacture of a medium composition for increasing the ratio of viruses carrying a target gene among all viruses produced during the cultivation of mammalian cells that produce a virus into which a target gene has been introduced.

22. A method for increasing the proportion of viruses carrying a target gene among all viruses produced during culture, the method comprising the step of culturing virus-producing mammalian cells into which a target gene has been introduced in a medium containing β-alanine.

23. A method for producing a virus carrying a target gene, comprising the step of culturing virus-producing mammalian cells into which a target gene has been introduced in a medium containing β-alanine.

24. A method for producing a virus carrying a target gene, comprising the step of culturing virus-producing mammalian cells into which a target gene has been introduced in a medium containing a polyamine or a salt thereof.

25. A medium composition for culturing mammalian cells producing viruses or virus-like particles, comprising β-alanine.

26. The medium composition of claim 25, wherein the mammalian cells are cells selected from the group consisting of HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells, and HeLa cells.

27. The medium composition of claim 25, wherein the mammalian cells are HEK293 cells.

28. The medium composition of claim 25, wherein the mammalian cells that produce viruses or virus-like particles are mammalian cells that produce adeno-associated viruses.

29. The medium composition according to any one of claims 25 to 28, wherein the concentration of β-alanine in the medium composition is 0.1 μM to 800 μM.

30. The medium composition according to any one of claims 25 to 28, further comprising a polyamine or a salt thereof.

31. The medium composition according to claim 30, wherein the polyamine or a salt thereof is spermidine or a salt thereof.

32. The medium composition according to claim 31, wherein the concentration of spermidine or a salt thereof in the medium composition is 0.1 μM to 700 μM.

33. A medium additive for culturing mammalian cells producing viruses or virus-like particles, comprising β-alanine.

34. Use of β-alanine in the manufacture of a medium composition for the culture of mammalian cells producing viruses or virus-like particles.

35. A method for culturing a mammalian cell that produces a virus or a virus-like particle, the method comprising the step of culturing the cell in a medium containing β-alanine.

36. A method for producing a virus or virus-like particle, comprising the step of culturing a mammalian cell that produces the virus or virus-like particle in a medium containing β-alanine.