Culture medium for culturing virus-producing cells

Abstract

The present invention addresses the problem of providing: for the culturing of virus-producing cells, a culture medium for improving the virus productivity of the cells and an agent for producing said culture medium; and a method for culturing virus-producing cells using said culture medium. A culture medium for culturing virus-producing cells according to the present invention contains at least one selected from the group consisting of L-aspartic acid and glutathione or at least one selected from the group consisting of potassium, magnesium, iron, L-aspartic acid, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, glutathione, a glutamine source, and copper, and preferably contains all of the above in predetermined concentrations. The culture medium has excellent virus productivity.

Description

Virus-producing cell culture medium

[0001] The present invention relates to cell culture technology, particularly to a culture medium for virus-producing cells, a culture method, etc.

[0002] Rapid and stable production technologies for viruses and viral vectors are essential to meet the needs of human gene therapy and vaccine production. The cultivation of cells that produce viruses and viral vectors (hereinafter also referred to as "virus-producing cells") is particularly important. When designing and optimizing culture media for virus-producing cells, the components of the media are a key issue. The components of the media affect cell growth and proliferation. Additionally, media for virus-producing cells also affect the intracellular viral proliferation capacity (i.e., virus production capacity). Therefore, it is necessary to select appropriate components. For example, nutrients, vitamins, minerals, amino acids, and sugars are important for cell proliferation. Furthermore, the concentration of each component in the media affects cell growth and virus production. Therefore, by appropriately adjusting the concentration of each component, cell proliferation and virus productivity can be improved (Non-Patent Documents 1-4). On the other hand, some components may be harmful to cells at high concentrations, so a balance must be considered.

[0003] Liste-Calleja L, J Biosci Bioeng. 2014;117(4):471-7Petiot E, Vaccine. 2015;33(44):5974-81Cervera L, BMC Proc. 2011;5 Suppl 8(Suppl 8):P126Shen CF, Vaccine. 2012;30(2):300-6

[0004] An object of the present invention is to provide a medium that improves virus productivity in virus-producing cells when they are cultured, an agent for producing the medium, and a method for culturing virus-producing cells using the medium.

[0005] In view of the above problems, the present inventors have focused on medium components that improve virus productivity and conducted extensive research. As a result, they have discovered that a medium containing specific components at specific concentrations can improve virus productivity in virus-producing cells. They have also succeeded in preparing a medium additive (supplement) suitable for producing the medium, thereby completing the present invention. Specifically, the present invention is as follows.

[0006] [1] A medium additive for use in culturing virus-producing cells, comprising at least one selected from the group consisting of L-aspartic acid and glutathione, wherein the final concentrations of each component when added to a medium are within the following ranges: (4) L-aspartic acid: 40 to 790 mg / L (10) glutathione: 0.5 to 5 mg / L. [2] The agent according to [1], comprising L-aspartic acid and glutathione, wherein the final concentrations of each component when added to a medium are within the following ranges: (4) L-aspartic acid: 40 to 790 mg / L (10) glutathione: 0.5 to 5 mg / L. [3] The agent according to [1] or [2], further comprising at least one selected from the group consisting of potassium, magnesium, iron, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, and a glutamine source, wherein the final concentrations of each component when added to a culture medium are within the following ranges: (1) potassium: 8.65 to 86.5 mg / L (2) magnesium: 0.56 to 5.6 mg / L (3) iron: 3.19 to 31.9 mg / L (5) L-tryptophan: 48 to 480 mg / L (6) folic acid: 0.618 to 6.18 mg / L (7) riboflavin: 0.18 to 1.8 mg / L (8) ethanolamine: 10.2 to 102 mg / L (9) buffer: 90 to 900 mg / L (11) glutamine source: 17.54 to 175.4 mg / L. [4] The agent according to any one of [1] to [3], further comprising potassium, magnesium, iron, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, and a glutamine source, wherein the final concentrations of each component when added to a culture medium are within the following ranges: (1) potassium: 8.65 to 86.5 mg / L (2) magnesium: 0.56 to 5.6 mg / L (3) iron: 3.19 to 31.9 mg / L (5) L-tryptophan: 48 to 480 mg / L (6) folic acid: 0.618 to 6.18 mg / L (7) riboflavin: 0.18 to 1.8 mg / L (8) ethanolamine: 10.2 to 102 mg / L (9) buffer: 90 to 900 mg / L (11) glutamine source: 17.54 to 175.4 mg / L. [5] The agent according to [3] or [4], wherein the glutamine source is at least one selected from the group consisting of L-glutamine, alanylglutamine, and glycylglutamine.[6] A medium composition comprising a basal medium containing the medium additive according to any one of [1] to [5]. [7] A medium composition for use in culturing virus-producing cells, comprising at least one component selected from the group consisting of L-aspartic acid and glutathione, wherein the concentrations of each component in the medium composition are within the following ranges: (4) L-aspartic acid: 250 to 1000 mg / L (10) glutathione: 1.5 to 6 mg / L. [8] The medium composition according to [7], comprising L-aspartic acid and glutathione, wherein the concentrations of each component in the medium composition are within the following ranges: (4) L-aspartic acid: 250 to 1000 mg / L (10) glutathione: 1.5 to 6 mg / L. [9] The medium composition according to [7] or [8], further comprising at least one selected from the group consisting of potassium, magnesium, iron, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, and a glutamine source, wherein the concentrations of each component in the medium composition are within the following ranges: (1) potassium: 290 to 772 mg / L (2) magnesium: 7 to 45 mg / L (3) iron: 17 to 123 mg / L (5) L-tryptophan: 48 to 480 mg / L (6) folic acid: 3 to 59.6 mg / L (7) riboflavin: 0.7 to 4.8 mg / L (8) ethanolamine: 10.2 to 102 mg / L (9) buffer: 90 to 8800 mg / L (11) glutamine source: 17.54 to 1169 mg / L (12) copper: 0.002 to 0.024 mg / L.

[10] The medium composition according to any one of [7] to [9], further comprising potassium, magnesium, iron, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, and a glutamine source, wherein the concentrations of each component in the medium composition are within the following ranges: (1) potassium: 290 to 772 mg / L (2) magnesium: 7 to 45 mg / L (3) iron: 17 to 123 mg / L (5) L-tryptophan: 48 to 480 mg / L (6) folic acid: 3 to 59.6 mg / L (7) riboflavin: 0.7 to 4.8 mg / L (8) ethanolamine: 10.2 to 102 mg / L (9) buffer: 90 to 8800 mg / L (11) glutamine source: 17.54 to 1169 mg / L (12) copper: 0.002 to 0.024 mg / L.

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

[10] , wherein the glutamine source is at least one selected from the group consisting of L-glutamine, alanylglutamine, and glycylglutamine.

[12] A method for producing a medium composition for use in culturing virus-producing cells, comprising at least one selected from the group consisting of L-aspartic acid and glutathione, the method comprising adjusting the concentrations of each component in the medium composition to be within the following concentration ranges: (4) L-aspartic acid: 250 to 1000 mg / L (10) glutathione: 1.5 to 6 mg / L.

[13] The method according to

[12] , wherein the medium composition for culturing virus-producing cells, comprising L-aspartic acid and glutathione, the method comprising adjusting the concentrations of each component in the medium composition to be within the following concentration ranges: (4) L-aspartic acid: 250 to 1000 mg / L (10) glutathione: 1.5 to 6 mg / L.

[14] The method according to

[12] or

[13] , wherein the medium composition further comprises at least one selected from the group consisting of potassium, magnesium, iron, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, and a glutamine source, and the concentrations of each component in the medium composition are within the following ranges: (1) potassium: 290 to 772 mg / L (2) magnesium: 7 to 45 mg / L (3) iron: 17 to 123 mg / L (5) L-tryptophan: 48 to 480 mg / L (6) folic acid: 3 to 59.6 mg / L (7) riboflavin: 0.7 to 4.8 mg / L (8) ethanolamine: 10.2 to 102 mg / L (9) buffer: 90 to 8800 mg / L (11) glutamine source: 17.54 to 1169 mg / L (12) Copper: 0.002 to 0.024 mg / L.

[15] The method according to any one of

[12] to

[14] , wherein the medium composition further comprises potassium, magnesium, iron, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, and a glutamine source, and the concentrations of each component in the medium composition are within the following ranges: (1) potassium: 290 to 772 mg / L (2) magnesium: 7 to 45 mg / L (3) iron: 17 to 123 mg / L (5) L-tryptophan: 48 to 480 mg / L (6) folic acid: 3 to 59.6 mg / L (7) riboflavin: 0.7 to 4.8 mg / L (8) ethanolamine: 10.2 to 102 mg / L (9) buffer: 90 to 8800 mg / L (11) glutamine source: 17.54 to 1169 mg / L (12) copper: 0.002 to 0.024 mg / L.

[16] The method according to

[14] or

[15] , wherein the glutamine source is at least one selected from the group consisting of L-glutamine, alanylglutamine, and glycylglutamine.

[17] A method for culturing virus-producing cells, comprising a step of culturing virus-producing cells in the medium composition according to any one of [7] to

[11] .

[18] A method for producing a virus, comprising culturing virus-infected virus-producing cells in the medium composition according to any one of [7] to

[11] .

[0007]

[01] A medium additive for use in culturing virus-producing cells, comprising at least one selected from the group consisting of potassium, magnesium, iron, L-aspartic acid, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, glutathione, and a glutamine source, wherein the final concentrations of each component when added to a medium are within the following ranges: (1) potassium: 8.65 to 86.5 mg / L (2) magnesium: 0.56 to 5.6 mg / L (3) iron: 3.19 to 31.9 mg / L (4) L-aspartic acid: 171 to 1710 mg / L (5) L-tryptophan: 48 to 480 mg / L (6) folic acid: 0.618 to 6.18 mg / L (7) riboflavin: 0.18 to 1.8 mg / L (8) ethanolamine: 10.2 to 102 mg / L (9) Buffer: 90-900 mg / L (10) Glutathione: 0.6-6 mg / L (11) Glutamine source: 17.54-175.4 mg / L.

[02] A medium additive for use in culturing virus-producing cells, comprising potassium, magnesium, iron, L-aspartic acid, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, glutathione, and a glutamine source, wherein the final concentrations of each component when added to a medium are within the following ranges: (1) potassium: 8.65 to 86.5 mg / L (2) magnesium: 0.56 to 5.6 mg / L (3) iron: 3.19 to 31.9 mg / L (4) L-aspartic acid: 171 to 1710 mg / L (5) L-tryptophan: 48 to 480 mg / L (6) folic acid: 0.618 to 6.18 mg / L (7) riboflavin: 0.18 to 1.8 mg / L (8) ethanolamine: 10.2 to 102 mg / L (9) Buffer: 90 to 900 mg / L (10) Glutathione: 0.6 to 6 mg / L (11) Glutamine source: 17.54 to 175.4 mg / L.

[03] The agent according to

[01] or

[02] , wherein the glutamine source is at least one selected from the group consisting of L-glutamine, alanylglutamine, and glycylglutamine.

[04] A medium composition comprising a basal medium containing the medium additive according to any one of

[01] to

[03] .[05A] The medium composition according to

[04] , comprising at least one selected from the group consisting of potassium, magnesium, iron, L-aspartic acid, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, glutathione, a glutamine source, and copper, in the following concentration ranges: (1) potassium: 290 to 772 mg / L (2) magnesium: 7 to 45 mg / L (3) iron: 17 to 123 mg / L (4) L-aspartic acid: 220 to 3894 mg / L (5) L-tryptophan: 48 to 480 mg / L (6) folic acid: 3 to 59.6 mg / L (7) riboflavin: 0.7 to 4.8 mg / L (8) ethanolamine: 10.2 to 102 mg / L (9) buffer: 90 to 8800 mg / L (10) glutathione: 2 to 6 mg / L (11) Glutamine source: 17.54 to 1169 mg / L (12) Copper: 0.002 to 0.024 mg / L. [05B] The medium composition according to

[04] , comprising potassium, magnesium, iron, L-aspartic acid, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, glutathione, a glutamine source, and copper in the following concentration ranges: (1) potassium: 290 to 772 mg / L (2) magnesium: 7 to 45 mg / L (3) iron: 17 to 123 mg / L (4) L-aspartic acid: 220 to 3894 mg / L (5) L-tryptophan: 48 to 480 mg / L (6) folic acid: 3 to 59.6 mg / L (7) riboflavin: 0.7 to 4.8 mg / L (8) ethanolamine: 10.2 to 102 mg / L (9) buffer: 90 to 8800 mg / L (10) glutathione: 2 to 6 mg / L (11) Glutamine source: 17.54 to 1169 mg / L (12) Copper: 0.002 to 0.024 mg / L

[06] The medium composition according to [05A] or [05B], wherein the glutamine source is at least one selected from the group consisting of L-glutamine, alanylglutamine, and glycylglutamine.

[07] A medium composition for use in culturing virus-producing cells, comprising at least one component selected from the group consisting of potassium, magnesium, iron, L-aspartic acid, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, glutathione, a glutamine source, and copper, wherein the concentrations of each component in the medium composition are within the following ranges: (1) potassium: 290 to 772 mg / L (2) magnesium: 7 to 45 mg / L (3) iron: 17 to 123 mg / L (4) L-aspartic acid: 220 to 3894 mg / L (5) L-tryptophan: 48 to 480 mg / L (6) folic acid: 3 to 59.6 mg / L (7) riboflavin: 0.7 to 4.8 mg / L (8) ethanolamine: 10.2 to 102 mg / L (9) buffer: 90 to 8800 mg / L (10) Glutathione: 2-6 mg / L (11) Glutamine source: 17.54-1169 mg / L (12) Copper: 0.002-0.024 mg / L.

[08] The medium composition according to

[07] , wherein the concentrations of each component in the medium composition are within the following ranges: (1) potassium: 290 to 370 mg / L (2) magnesium: 15 to 25 mg / L (3) iron: 18 to 30 mg / L (4) L-aspartic acid: 500 to 1000 mg / L (5) L-tryptophan: 100 to 200 mg / L (6) folic acid: 5 to 20 mg / L (7) riboflavin: 1 to 2 mg / L (8) ethanolamine: 30 to 50 mg / L (9) buffer: 1000 to 3000 mg / L (10) glutathione: 2 to 4 mg / L (11) glutamine source: 145 to 1030 mg / L (12) copper: 0.004 to 0.015 mg / L.

[09] A medium composition for use in culturing virus-producing cells, comprising potassium, magnesium, iron, L-aspartic acid, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, glutathione, a glutamine source, and copper, wherein the concentrations of each component in the medium composition are within the following ranges: (1) potassium: 290 to 772 mg / L (2) magnesium: 7 to 45 mg / L (3) iron: 17 to 123 mg / L (4) L-aspartic acid: 220 to 3894 mg / L (5) L-tryptophan: 48 to 480 mg / L (6) folic acid: 3 to 59.6 mg / L (7) riboflavin: 0.7 to 4.8 mg / L (8) ethanolamine: 10.2 to 102 mg / L (9) buffer: 90 to 8800 mg / L (10) Glutathione: 2-6 mg / L (11) Glutamine source: 17.54-1169 mg / L (12) Copper: 0.002-0.024 mg / L.

[010] The medium composition according to

[09] , wherein the concentrations of each component in the medium composition are within the following ranges: (1) potassium: 290 to 370 mg / L (2) magnesium: 15 to 25 mg / L (3) iron: 18 to 30 mg / L (4) L-aspartic acid: 500 to 1000 mg / L (5) L-tryptophan: 100 to 200 mg / L (6) folic acid: 5 to 20 mg / L (7) riboflavin: 1 to 2 mg / L (8) ethanolamine: 30 to 50 mg / L (9) buffer: 1000 to 3000 mg / L (10) glutathione: 2 to 4 mg / L (11) glutamine source: 145 to 1030 mg / L (12) copper: 0.004 to 0.015 mg / L.

[011] A method for producing a medium composition for use in culturing virus-producing cells, comprising adjusting at least one selected from the group consisting of potassium, magnesium, iron, L-aspartic acid, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, glutathione, a glutamine source, and copper to the following concentration ranges: (1) potassium: 290 to 772 mg / L (2) magnesium: 7 to 45 mg / L (3) iron: 17 to 123 mg / L (4) L-aspartic acid: 220 to 3894 mg / L (5) L-tryptophan: 48 to 480 mg / L (6) folic acid: 3 to 59.6 mg / L (7) riboflavin: 0.7 to 4.8 mg / L (8) ethanolamine: 10.2 to 102 mg / L (9) buffer: 90 to 8800 mg / L (10) Glutathione: 2-6 mg / L (11) Glutamine source: 17.54-1169 mg / L (12) Copper: 0.002-0.024 mg / L.

[012] A method for producing a medium composition for use in culturing virus-producing cells, comprising adjusting potassium, magnesium, iron, L-aspartic acid, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, glutathione, a glutamine source, and copper to the following concentration ranges: (1) potassium: 290 to 772 mg / L (2) magnesium: 7 to 45 mg / L (3) iron: 17 to 123 mg / L (4) L-aspartic acid: 220 to 3894 mg / L (5) L-tryptophan: 48 to 480 mg / L (6) folic acid: 3 to 59.6 mg / L (7) riboflavin: 0.7 to 4.8 mg / L (8) ethanolamine: 10.2 to 480 mg / L (9) buffer: 90 to 8800 mg / L (10) glutathione: 2 to 6 mg / L (11) Glutamine source: 17.54 to 1169 mg / L (12) Copper: 0.002 to 0.024 mg / L.

[013] A method for culturing virus-producing cells, comprising culturing virus-producing cells in the medium composition according to any one of

[04] to

[010] .

[014] A method for producing a virus, comprising culturing virus-infected virus-producing cells in the medium composition according to any one of

[04] to

[010] .

[0008] According to the present invention, it is possible to provide a medium composition for culturing virus-producing cells that has excellent virus productivity, and a medium additive suitable for producing the medium. When virus-producing cells are cultured in the medium composition of the present invention, the amount of virus produced per cell is significantly increased. Therefore, more efficient virus production becomes possible. The resulting virus can function as a viral vector useful for gene therapy and vaccine production.

[0009] FIG. 1 is a graph showing the results of examining the proliferation ability of virus-producing cells in media containing L-aspartic acid and glutathione (reduced). The results are for conditions where the L-aspartic acid concentration is high. The average value of n=2 is shown. FIG. 2 is a graph showing the results of examining the proliferation ability of virus-producing cells in media containing L-aspartic acid and glutathione (reduced). The results are for conditions where the L-aspartic acid concentration is low. The average value of n=2 is shown. FIG. 3 is a graph showing the results of examining the proliferation ability of virus-producing cells in media containing L-aspartic acid and glutathione (reduced). The results are for conditions where the basal medium is changed. The average value of n=2 is shown. FIG. 4 is a graph showing the results of examining the proliferation ability of virus-producing cells in various media. The results are for conditions where the cell density and the concentration of virus used for infection are low. In the graph, "Supplement" means AJI Supplement. FIG. 5 is a graph showing the results of examining the virus productivity of virus-producing cells in various media. These are results obtained under conditions where the cell density and the concentration of virus used for infection were low. The values ​​are shown as mean values ​​± standard deviations (n ​​= 3). In the graph, "Supplement" refers to AJI Supplement. Figure 6 is a graph showing the results of examining the proliferation ability of virus-producing cells in various media. The results are obtained under conditions where the cell density and the concentration of virus used for infection were high. The values ​​are shown as mean values ​​± standard deviations (n ​​= 3). In the graph, "Supplement" refers to AJI Supplement. Figure 7 is a graph showing the results of examining the virus production ability of virus-producing cells in various media. The results are obtained under conditions where the cell density and the concentration of virus used for infection were high. The values ​​are shown as mean values ​​± standard deviations (n ​​= 3). In the graph, "Supplement" refers to AJI Supplement.

[0010] The present invention is described below. Terms used herein have the meanings commonly used in the art unless otherwise specified. 1. Medium Additive The present invention provides a medium additive for use in culturing virus-producing cells (hereinafter also referred to as the "medium additive of the present invention"). The medium additive of the present invention can generally be added to a basal medium (described below). For example, when the medium additive is in the form of a solution, it is added to the basal medium in an amount of 1 / 20 to 1 / 5, preferably 1 / 15 to 1 / 8, and more preferably 1 / 10. One embodiment of the medium additive of the present invention comprises at least one member selected from the group consisting of L-aspartic acid and glutathione, and preferably both L-aspartic acid and glutathione. Another embodiment of the medium additive of the present invention comprises at least one member selected from the group consisting of potassium, magnesium, iron, L-aspartic acid, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, glutathione, and a glutamine source. Typically, two, three, four, five, six, seven, eight, nine, or ten types are included, and preferably all eleven types are included. In another embodiment of the present invention, the medium additive includes magnesium, folic acid, riboflavin, ethanolamine, a buffer, and glutathione. In another embodiment of the present invention, the medium additive includes potassium and iron. The medium additive may be a solution obtained by dissolving each desired component in an appropriate solvent (e.g., water), or may be a solid powder obtained by lyophilizing the solution. The medium additive may also be a simple mixture of solid components. Each component will be described below.

[0011] (1.1 Potassium) Potassium is a nutrient necessary for cell survival and proliferation and is necessary for maintaining cellular physiological functions, including the regulation of metabolic processes. It is also essential for controlling the cell cycle (Biotechnology and Bioengineering. 2018;115:921-931, Appl Microbiol Biotechnol (2015) 99:9935-9949). Potassium may be present in any form as long as it can function in the medium additive and in the medium composition obtained by adding the medium additive to a basal medium. Potassium is typically included in the medium additive as a potassium salt. Examples of potassium salts include, but are not limited to, potassium chloride, potassium hydroxide, potassium sulfate, potassium phosphate, dipotassium hydrogen phosphate, potassium carbonate, and potassium bicarbonate. Potassium chloride is preferred. The potassium salt may be anhydrous or hydrated. Potassium is included in the medium additive so that the final concentration when added to the basal medium is in the range of 8.65 to 86.5 mg / L, preferably 15 to 60 mg / L, and particularly preferably 20 to 40 mg / L. The potassium concentration in the medium additive is usually 86.5 to 865 mg / L, preferably 150 to 600 mg / L, and particularly preferably 200 to 400 mg / L. Too high a potassium concentration in the medium additive results in a too high final concentration when added to the basal medium, increasing osmotic pressure and reducing cell growth, which is undesirable. Too low a potassium concentration in the medium additive results in a too low final concentration when added to the basal medium, preventing the intracellular sodium-potassium pump from activating and preventing the production of ATP necessary for cell growth, which is undesirable. When potassium is provided as a potassium salt, the above concentration represents the concentration converted to the functional molecule (i.e., free potassium) only. Potassium concentration can be measured by any method known in the art, but is typically performed by ICP-MS.

[0012] (1.2 Magnesium) Magnesium acts as a cofactor for enzyme activity and regulates many enzymatic reactions within cells. It is particularly required for many enzymes in the glycolytic pathway, such as phosphofructokinase and pyruvate kinase. Magnesium also participates in the formation of ATP (adenosine triphosphate) and supports cellular energy metabolism. The form of magnesium is not limited as long as it can function in the medium additive and in the medium composition obtained by adding the medium additive to a basal medium. Magnesium salts are typically included in the medium additive as a magnesium salt. Examples of magnesium salts include, but are not limited to, magnesium sulfate, magnesium nitrate, magnesium chloride, and magnesium acetate. Magnesium sulfate is preferred. Magnesium salts may be anhydrous or hydrated. Magnesium is included in the medium additive so that the final concentration when added to the basal medium is in the range of 0.56 to 5.6 mg / L, preferably 1 to 4 mg / L, and particularly preferably 1.5 to 2.5 mg / L. The magnesium concentration in the medium additive is typically 5.6 to 56 mg / L, preferably 10 to 40 mg / L, and particularly preferably 15 to 25 mg / L. If the magnesium concentration in the medium additive is too high, the final concentration will be too high when added to a basal medium, and the presence of an excess of essential metals in the medium will inhibit cell growth, which is undesirable. If the magnesium concentration in the medium additive is too low, the final concentration will be low when added to a basal medium, and the essential metals required by cells will not be supplied, inhibiting cell growth, which is undesirable. When magnesium is provided as a magnesium salt, the above concentration represents the concentration converted into only the functional molecule (i.e., free magnesium). Magnesium concentration can be measured by any method known in the art, but is typically performed by ICP MS.

[0013] (1.3 Iron) Iron is involved in the structure of enzymes and proteins in the electron transport chain and is an essential element for maintaining the proper function of these proteins (Biotechnol. Prog. 2000, 16, 872-884). Iron ions contained in electron carriers such as cytochromes and iron-sulfur proteins play an important role in the electron transport chain within mitochondria. The form of iron is not limited as long as it can function in the medium additive and the medium composition obtained by adding the medium additive to a basal medium. Iron is typically included in the medium additive as an iron salt. Examples of iron salts include inorganic salts, organic acid salts, and complexes (complex salts) of iron. Examples of inorganic salts include sulfate, chloride, and nitrate. Examples of organic acid salts include acetate and citrate. There are no particular limitations on the iron complex as long as it can form a complex with iron and transport iron into cells. However, synthetic, non-proteinaceous compounds that are water-soluble and stable at the pH of the medium (nearly neutral), i.e., do not precipitate as iron hydroxide, are selected. Examples of iron salts include, but are not limited to, ferrous citrate, sodium ferrous citrate, ammonium ferrous citrate, ferrous acetate, ferrous oxalate, ferrous succinate, sodium ferrous citrate succinate, ferrous pyrophosphate, ferric pyrophosphate, ferrous lactate, ferrous gluconate, ferrous formate, ferric formate, ammonium ferric potassium oxalate, ferrous sulfate, ferric sulfate, ammonium ferrous sulfate, ferric carbonate, ferrous chloride, and ferric chloride. Sodium ferrous citrate is preferred. Iron salts may be anhydrous or hydrated. Iron is included in the medium additive so that the final concentration when added to the basal medium is in the range of 3.19 to 31.9 mg / L, preferably 5 to 20 mg / L, and particularly preferably 8 to 15 mg / L. The iron concentration in the medium additive is usually 31.9 to 319 mg / L, preferably 50 to 200 mg / L, and particularly preferably 80 to 150 mg / L. If the iron concentration in the medium additive is too high, the final concentration will be too high when added to the basal medium, and if an excess of essential metals is contained in the medium, cell growth will be inhibited, which is undesirable.If the iron concentration in the medium additive is too low, the final concentration will be too low when added to the basal medium, and the essential metals required by cells will not be supplied, and cell growth will be inhibited, which is undesirable.When iron is provided as an iron salt, the above concentration represents the concentration converted into only the functional molecule (i.e., free iron). The iron concentration can be measured by any method known in the art, but is usually measured by ICP MS.

[0014] (1.4 L-Aspartic Acid) L-Aspartic acid is known to be involved in the activation of the electron transport chain (Biotechnol. Prog. 2000, 16, 872-884). L-Aspartic acid functions as an electron donor for enzyme complex 1 (NADH: quinone oxidoreductase) in the electron transport chain, activating energy production and thereby playing an important role in cellular metabolism and proliferation. L-Aspartic acid is also known to be a target of amino acid consumption specific to viruses (Vaccine 33 (2015) 5974-5981). The form of L-aspartic acid is not limited as long as it can exhibit its function in medium additives and medium compositions obtained by adding the medium additives to a basal medium. L-Aspartic acid may be isolated and purified from natural products or their processed products, or may be synthetic. Salts are also possible, and specific examples include salts with inorganic bases, organic bases, inorganic acids, organic acids, and amino acids. Free L-aspartic acid is preferred. L-aspartic acid is contained in the medium additive so that the final concentration when added to a basal medium is 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 mg / L or more, or 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800 mg / L or less. In one embodiment of the present invention, L-aspartic acid is contained in the medium additive so that the final concentration when added to a basal medium is in the range of 171 to 1710 mg / L, preferably 200 to 800 mg / L, and particularly preferably 400 to 600 mg / L. In another embodiment of the present invention, L-aspartic acid is contained in the medium additive so that the final concentration when added to a basal medium is in the range of 40 to 790 mg / L, preferably 60 to 700 mg / L, and particularly preferably 80 to 600 mg / L. The concentration of L-aspartic acid in the medium additive is typically 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, or 1700 mg / L or more, or 18,000, 17,000, 16,000, 15,000, 14,000, 13,000, 12,000, 11,000, 10,000, 9,000, or 8,000 mg / L or less.In one embodiment of the present invention, the concentration of L-aspartic acid in the medium additive is typically 1710 to 17100 mg / L, preferably 2000 to 8000 mg / L, and particularly preferably 4000 to 6000 mg / L. In another embodiment of the present invention, the concentration of L-aspartic acid in the medium additive is typically 400 to 7900 mg / L, preferably 600 to 7000 mg / L, and particularly preferably 800 to 6000 mg / L. If the concentration of L-aspartic acid in the medium additive is too high, the final concentration when added to the basal medium will be too high, lowering the pH of the medium and making it impossible to maintain an appropriate pH for the cells, which is undesirable. If the concentration of L-aspartic acid in the medium additive is too low, the final concentration when added to the basal medium will be too low, preventing the supply of metabolites to the TCA cycle and causing the cells to suffer from energy deficiency, which is undesirable. When L-aspartic acid is provided as a salt, the above concentration represents the concentration converted into only the functional molecule (i.e., free L-aspartic acid). The L-aspartic acid concentration can be measured by any method known in the art, but is usually measured by UPLC.

[0015] (1.5 L-tryptophan) L-tryptophan is one of the amino acids required for intracellular protein synthesis and also plays an important role in cell growth and intracellular signal transduction pathways. The form of L-tryptophan is not limited as long as it can exhibit its function in the medium additive and in the medium composition obtained by adding the medium additive to a basal medium. It may be isolated and purified from natural products or their processed products, or it may be a synthetic product. It can also be in the form of a salt, specifically, salts with inorganic bases, organic bases, inorganic acids, organic acids, and amino acids. Free L-tryptophan is preferred. L-tryptophan is included in the medium additive so that the final concentration when added to the basal medium is in the range of 48 to 480 mg / L, preferably 60 to 300 mg / L, and particularly preferably 100 to 200 mg / L. The concentration of L-tryptophan in the medium additive is typically 480 to 4,800 mg / L, preferably 600 to 3,000 mg / L, and particularly preferably 1,000 to 2,000 mg / L. If the concentration of L-tryptophan in the medium additive is too high, the final concentration when added to the basal medium will be too high, which is undesirable because it is toxic to cells. If the concentration of L-tryptophan in the medium additive is too low, the final concentration when added to the basal medium will be too low, which is undesirable because the essential amino acid tryptophan will not be synthesized intracellularly and the tryptophan required by cells will not be supplied. When L-tryptophan is provided as a salt, the above concentration represents the concentration converted into only the functional molecule (i.e., free L-tryptophan). L-tryptophan concentration can be measured by any method known in the art, but is typically measured by UPLC.

[0016] (1.6 Folic Acid) Folic acid is a component necessary for promoting nucleic acid synthesis (Journal of Biotechnology 233 (2016) 34-41). The form of folic acid is not limited as long as it can exert its function in a medium additive and a medium composition obtained by adding the medium additive to a basal medium. It may be isolated and purified from natural products or their processed products, or it may be a synthetic product. It can also be in the form of a salt, and examples of such salts include 5-methyltetrahydrofolic acid (5MeTHF), tetrahydrofolic acid (THF), and 5-formyltetrahydrofolic acid (5CHOTHF). Folic acid is preferred. Folic acid is included in the medium additive so that the final concentration when added to the basal medium is in the range of 0.618 to 6.18 mg / L, preferably 1 to 5 mg / L, and particularly preferably 1.5 to 3 mg / L. The concentration of folic acid in the medium additive is typically 6.18 to 61.8 mg / L, preferably 10 to 50 mg / L, and particularly preferably 15 to 30 mg / L. If the concentration of folic acid in the medium additive is too high, the final concentration when added to the basal medium will be too high, resulting in low solubility in the medium and precipitation in the medium. Furthermore, because it is highly susceptible to degradation, the possibility of decomposition products adversely affecting certain cells cannot be ruled out, making this undesirable. If the concentration of folic acid in the medium additive is too low, the final concentration when added to the basal medium will be low, resulting in a lack of folic acid necessary for nucleic acid synthesis, which will affect cell growth, making this undesirable. When folic acid is provided as a salt, the above concentration represents the concentration converted to the functional molecule (i.e., free folic acid) only. Folic acid concentration can be measured by any method known in the art, but is typically performed by LCMS.

[0017] (1.7 Riboflavin) Riboflavin, another name for vitamin B2, plays an important role in the cellular electron transport system (Biotechnol. Prog. 2000, 16, 872-884). The electron transport system is a sequential process consisting of multiple protein complexes present in mitochondrial membranes and is essential for intracellular energy production. Therefore, including riboflavin in cell culture media contributes to proper cell growth and metabolism. Riboflavin may be used in any form as long as it can function in medium additives and medium compositions obtained by adding the medium additives to a basal medium. It may be isolated and purified from natural products or their processed products, or may be synthetic. Derivatives are also possible, including, but not limited to, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), riboflavin butyrate (e.g., riboflavin tetrabutyrate), their salts, and their hydrates. Riboflavin and riboflavin derivatives may also be in the form of salts. Examples of such salts include acid addition salts and salts with bases. Riboflavin is included in the medium additive so that the final concentration when added to the basal medium is in the range of 0.18 to 1.8 mg / L, preferably 0.3 to 1.5 mg / L, and particularly preferably 0.4 to 1.0 mg / L. The concentration of riboflavin in the medium additive is usually 1.8 to 18 mg / L, preferably 3 to 15 mg / L, and particularly preferably 4 to 10 mg / L. If the concentration of riboflavin in the medium additive is too high, the final concentration when added to the basal medium will be too high, resulting in low solubility in the medium and precipitation in the medium. In addition, since riboflavin is highly susceptible to degradation, the possibility of decomposition products adversely affecting certain cells cannot be ruled out, making this undesirable. If the concentration of riboflavin in the medium additive is too low, the final concentration when added to the basal medium will be low, preventing riboflavin metabolic products from being supplied to the electron transport system, thereby reducing its function, making this undesirable. When riboflavin is provided as a salt, the above concentration represents the concentration converted into only the functional molecule (i.e., free riboflavin). The riboflavin concentration can be measured by any method known in the art, but is usually measured by LCMS.

[0018] (1.8 Ethanolamine) Ethanolamine, also known as 2-aminoethanol or monoethanolamine, is involved in the synthesis of phosphatidylethanolamine, a phospholipid that is a major component of cell membranes. Phosphatidylethanolamine plays an important role in maintaining the structure of cell membranes and regulating their fluidity and permeability. Therefore, adding ethanolamine to a culture medium can provide an appropriate lipid environment for cells. Ethanolamine may be used in any form, as long as it can function in a culture medium additive and in a culture medium composition obtained by adding the additive to a basal medium. It may be isolated and purified from natural products or their processed products, or it may be a synthetic product. Derivatives are also possible. Examples of such derivatives include phosphoethanolamine (also known as phosphorylethanolamine), monomethylethanolamine, dimethylethanolamine, N-acylphosphatidylethanolamine, phosphatidylethanolamine, and lysophosphatidylethanolamine. Ethanolamine and ethanolamine derivatives may be in the form of salts. Examples of such salts include salts with inorganic bases, organic bases, inorganic acids, organic acids, and amino acids. Preferred examples 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, trifluoroacetic 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. Hydrochloride salts are particularly preferred. Ethanolamine is included in the medium additive so that the final concentration when added to the basal medium is in the range of 10.2 to 102 mg / L, preferably 15 to 60 mg / L, and particularly preferably 20 to 40 mg / L. The concentration of ethanolamine in the medium additive is typically 102 to 1020 mg / L, preferably 150 to 600 mg / L, and particularly preferably 200 to 400 mg / L. If the concentration of ethanolamine in the medium additive is too high, the final concentration when added to the basal medium will be too high, which is undesirable because it will inhibit cell proliferation.If the concentration of ethanolamine in the medium additive is too low, the final concentration when added to the basal medium will be low, which is undesirable because it will not provide the cell membrane components necessary for cell growth. When ethanolamine is provided as a salt, the above concentration represents the concentration converted into only the functional molecule (i.e., free ethanolamine). The ethanolamine concentration can be measured by any method known in the art, but is usually measured by UPLC.

[0019] (1.9 Buffering Agents) The buffering agent contained in the medium additive of the present invention and in the medium composition obtained by adding the medium additive to a basal medium is not particularly limited as long as it can control the pH of the final medium composition to a predetermined value (e.g., 7.0 to 7.4), and examples thereof include N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid] (HEPES), MOPS, MES, phosphate, bicarbonate, etc. The concentration of the buffering agent used can be adjusted appropriately depending on the type of buffering agent used. When HEPES is used as a buffering agent, HEPES is contained in the medium additive so that the final concentration when added to the basal medium is in the range of 90 to 900 mg / L, preferably 100 to 600 mg / L, and particularly preferably 200 to 400 mg / L. The concentration of HEPSE in the medium additive is typically 900 to 9,000 mg / L, preferably 1,000 to 6,000 mg / L, and particularly preferably 2,000 to 4,000 mg / L. If the concentration of the buffer in the medium additive is too high or too low, the desired buffering capacity will not be obtained, which is undesirable. For example, in the case of HEPES, the buffer concentration can be measured by any method known in the art, but typically by LCMS-MS.

[0020] (1.10 Glutathione) Glutathione has antioxidant properties and functions to protect cells from intracellular oxidative stress (Journal of Biotechnology 233 (2016) 34-41, Appl Microbiol Biotechnol (2015) 99:9935-9949). The form of glutathione is not limited as long as it can exert its function in a medium additive and a medium composition obtained by adding the medium additive to a basal medium. It may be isolated and purified from a natural product or a processed product thereof, or may be a synthetic product. Glutathione may be either oxidized glutathione or reduced glutathione, with reduced glutathione being preferred. It can also be in the form of a salt. Examples of such salts include salts with inorganic bases, organic bases, inorganic acids, organic acids, and amino acids. Preferred examples 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, trifluoroacetic 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. Glutathione is contained in the medium additive so that its final concentration when added to the basal medium is 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg / L or more, and 6, 5, 4, or 3 mg / L or less. In one embodiment of the present invention, glutathione is contained in the medium additive so that its final concentration when added to the basal medium is 0.6 to 6 mg / L, preferably 1 to 5 mg / L, and particularly preferably 1.5 to 3 mg / L. In another embodiment of the present invention, glutathione is contained in the medium additive so that the final concentration when added to a basal medium is in the range of 0.5 to 5 mg / L, preferably 1 to 4 mg / L, and particularly preferably 2 to 3 mg / L. The concentration of glutathione in the medium additive is usually 5, 6, 7, 8, 9, or 10 mg / L or more, and 60, 50, 40, or 30 mg / L or less. In one embodiment of the present invention, the concentration of glutathione in the medium additive is 6 to 60 mg / L, preferably 10 to 50 mg / L, and particularly preferably 15 to 30 mg / L.In another embodiment of the present invention, the concentration of glutathione in the medium additive is 5 to 50 mg / L, preferably 10 to 40 mg / L, and particularly preferably 20 to 30 mg / L. If the concentration of glutathione in the medium additive is too high, the final concentration when added to the basal medium will be too high, and since it is highly susceptible to decomposition, the decomposition products may have adverse effects on some cells, which is undesirable. If the concentration of glutathione in the medium additive is too low, the final concentration when added to the basal medium will be too low, which is undesirable because it disrupts the necessary redox balance within the cells. When glutathione is provided as a salt, the above concentration represents the concentration converted to the functional molecule (i.e., free glutathione) only. Glutathione concentration can be measured by any method known in the art, but a simple method is to use a glutathione quantification kit (e.g., DOJINDO: G257).

[0021] (1.11 Glutamine Source) Glutamine is an amino acid that plays an important role in cellular metabolism. The glutamine source contained in the medium additive of the present invention and the medium composition obtained by adding the medium additive to a basal medium is not particularly limited as long as it can provide glutamine to the final medium composition, but examples include L-glutamine, alanyl-L-glutamine (an amino acid in which alanine and glutamine are bonded via a peptide bond), and glutamic acid (which can function as a glutamine precursor). Alanyl-L-glutamine is preferred. Alanyl-L-glutamine is more stable than general glutamine. The form of the glutamine source is not limited as long as it can exert its function in the medium additive and the medium composition obtained by adding the medium additive. It may be isolated and purified from natural products or their processed products, or may be a synthetic product. Salts are also possible; specific examples include salts with inorganic bases, organic bases, inorganic acids, organic acids, and amino acids. Alanyl-L-glutamine is preferred. The glutamine source is included in the medium additive so that the final concentration when added to the basal medium is in the range of 17.54 to 175.4 mg / L, preferably 20 to 100 mg / L, and particularly preferably 40 to 70 mg / L. The concentration of the glutamine source in the medium additive is typically 175.4 to 1754 mg / L, preferably 200 to 1000 mg / L, and particularly preferably 400 to 700 mg / L. Here, the above concentration of the glutamine source represents the concentration when converted to functional molecules (i.e., glutamine) only. The glutamine concentration can be measured by any method known in the art, but is typically measured by UPLC.

[0022] 2. Medium Composition The present invention provides a medium composition for use in culturing virus-producing cells (hereinafter also referred to as the "medium composition of the present invention"). One embodiment of the medium composition of the present invention contains at least one member selected from the group consisting of L-aspartic acid and glutathione, and preferably contains both L-aspartic acid and glutathione. Another embodiment of the medium composition of the present invention contains at least one member selected from the group consisting of potassium, magnesium, iron, L-aspartic acid, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, glutathione, a glutamine source, and copper. Typically, two, three, four, five, six, seven, eight, nine, ten, or eleven members are included, and preferably all twelve are included. The medium composition of the present invention may be provided in a liquid form, or may be prepared in a more concentrated form than the concentration at the time of use, or in a solid form such as a lyophilized powder, which is then diluted with a solvent such as water, or dissolved or dispersed in a solvent such as water, before use. Each component is described below.

[0023] (2.1 Potassium) The potassium contained in the medium composition has the same meaning as (1.1 Potassium) above. The potassium concentration in the medium composition is 290 to 772 mg / L, preferably 290 to 370 mg / L, and particularly preferably 300 to 400 mg / L. When potassium is provided as a potassium salt, the above concentration represents the concentration converted into only the functional molecule (i.e., free potassium).

[0024] (2.2 Magnesium) The magnesium contained in the medium composition has the same meaning as in (1.2 Magnesium) above. The magnesium concentration in the medium composition is 7 to 45 mg / L, preferably 15 to 25 mg / L, and particularly preferably 18 to 23 mg / L. When magnesium is provided as a magnesium salt, the above concentration represents the concentration converted into only the functional molecule (i.e., free magnesium).

[0025] (2.3 Iron) The iron contained in the medium composition has the same meaning as in (1.3 Iron) above. The iron concentration in the medium composition is 17 to 123 mg / L, preferably 18 to 30 mg / L, and particularly preferably 20 to 28 mg / L. When iron is provided as an iron salt, the above concentration represents the concentration converted into only the functional molecule (i.e., free iron).

[0026] (2.4 L-Aspartic Acid) The L-aspartic acid contained in the medium composition has the same meaning as (1.4 L-Aspartic Acid) above. The concentration of L-aspartic acid in the medium composition is 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, or 380 mg / L or more, and 2010, 1910, 1810, 1710, 1610, 1510, 1410, 1310, 1210, 1110, or 1010 mg / L or less. In one embodiment of the present invention, the concentration of L-aspartic acid in the medium composition is 220 to 3894 mg / L, preferably 500 to 1000 mg / L, and particularly preferably 600 to 900 mg / L. In another embodiment of the present invention, the concentration of L-aspartic acid in the medium composition is 250 to 1000 mg / L, preferably 270 to 910 mg / L, and particularly preferably 290 to 810 mg / L. When L-aspartic acid is provided as a salt, the above concentration represents the concentration converted into only the functional molecule (i.e., free L-aspartic acid).

[0027] (2.5 L-tryptophan) The L-tryptophan contained in the medium composition has the same meaning as (1.5 L-tryptophan) above. The concentration of L-tryptophan in the medium composition is 48 to 480 mg / L, preferably 100 to 200 mg / L, and particularly preferably 150 to 200 mg / L. When L-tryptophan is provided as a salt, the above concentration represents the concentration converted into only the functional molecule (i.e., free L-tryptophan).

[0028] (2.6 Folic Acid) The folic acid contained in the medium composition has the same meaning as (1.6 Folic Acid) above. The concentration of folic acid in the medium composition is 3 to 59.6 mg / L, preferably 5 to 20 mg / L, and particularly preferably 8 to 15 mg / L. When folic acid is provided as a salt, the above concentration represents the concentration converted into only the functional molecule (i.e., free folic acid).

[0029] (2.7 Riboflavin) The riboflavin contained in the medium composition has the same meaning as (1.7 Riboflavin) above. The concentration of riboflavin in the medium composition is 0.7 to 4.8 mg / L, preferably 1 to 2 mg / L, and particularly preferably 1.4 to 2 mg / L. When riboflavin is provided as a salt, the above concentration represents the concentration converted into only the functional molecule (i.e., free riboflavin).

[0030] (2.8 Ethanolamine) The ethanolamine contained in the medium composition has the same meaning as (1.8 Ethanolamine) above. The concentration of ethanolamine in the medium composition is 10.2 to 102 mg / L, preferably 30 to 50 mg / L, and particularly preferably 35 to 45 mg / L. When ethanolamine is provided as a salt, the above concentration represents the concentration converted into only the functional molecule (i.e., free ethanolamine).

[0031] (2.9 Buffering Agent) The buffering agent contained in the medium composition has the same meaning as described above in (1.9 Buffering Agent). When the buffering agent is HEPES, the concentration of the buffering agent in the medium composition is 90 to 8800 mg / L, preferably 1000 to 3000 mg / L, and particularly preferably 2000 to 3000 mg / L.

[0032] (2.10 Glutathione) The glutathione contained in the medium composition has the same meaning as (1.10 Glutathione) above. The concentration of glutathione in the medium composition is 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg / L or more, and 6, 5, 4, or 3 mg / L or less. In one embodiment of the present invention, the concentration of glutathione in the medium composition is 2 to 6 mg / L, preferably 2.5 to 3.5 mg / L, and particularly preferably 2.8 to 3.2 mg / L. In another embodiment of the present invention, the concentration of glutathione in the medium composition is 1.5 to 6 mg / L, preferably 2 to 5 mg / L, and particularly preferably 3 to 4 mg / L. When glutathione is provided as a salt, the above concentration represents the concentration converted into only the functional molecule (i.e., free glutathione).

[0033] (2.11 Glutamine Source) The glutamine source contained in the medium composition has the same meaning as described above in (1.11 Glutamine Source). The concentration of the glutamine source in the medium composition is 17.54 to 1169 mg / L, preferably 145 to 1030 mg / L, and particularly preferably 290 to 880 mg / L. Here, the above concentration of the glutamine source represents the concentration converted into the functional molecule (i.e., glutamine) only.

[0034] (2.12 Copper) Copper is a trace element involved in many enzymatic reactions within cells. For example, it functions as a coenzyme for enzymes involved in cellular metabolism and antioxidant enzymes. These enzymes catalyze important intracellular reactions and are necessary for maintaining normal cellular function, making copper an important component for cell proliferation. Copper may be present in any form within the medium composition as long as it can perform its function. Copper salts are typically included in the medium composition as copper salts. Examples of copper salts include inorganic salts, organic acid salts, and complexes (complex salts). Examples of inorganic salts include sulfates, chlorides, and nitrates. Examples of organic acid salts include acetates and citrates. Copper complexes are not particularly limited as long as they form complexes with copper and transport copper into cells. Examples of copper salts include, but are not limited to, copper nitrate, copper acetate, and copper sulfate. Copper sulfate is preferred. Copper salts may be anhydrous or hydrated. The copper concentration in the medium composition is 0.002 to 0.024 mg / L, preferably 0.004 to 0.015 mg / L, and particularly preferably 0.006 to 0.01 mg / L. When copper is provided as a copper salt, the above concentration represents the concentration converted into only the functional molecule (i.e., free copper). The copper concentration is usually measured by ICP MS.

[0035] The medium composition of the present invention can be prepared, for example, by adding the medium additive of the present invention (described above in "1. Medium Additive") to a basal medium, and this embodiment is preferred because of its simplicity. For example, when the medium additive of the present invention is in the form of a solution, the medium composition of the present invention can be obtained by adding 1 / 20 to 1 / 5, preferably 1 / 15 to 1 / 8, and more preferably 1 / 10, of the medium additive to a basal medium.

[0036] As used herein, the term "basal medium" refers to a medium containing carbon sources, nitrogen sources, inorganic salts, and the like that are essential for cell culture. The basal medium that can be used as a component of the medium of the present invention is not particularly limited and may be selected appropriately depending on the cells to be cultured. The basal medium may be prepared by a method known per se, or a commercially available product may be used.

[0037] Usable basal media include, for example, 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 basal 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 mixtures of these media. The basal medium may be commercially available or may be one currently under development.

[0038] In addition, examples of the basal medium used to prepare the medium composition of the present invention for culturing virus-producing cells (described later) include CDM4HEK293 (Cytiva), SFM4HEK293 (Cytiva), and HyCell ＴＭ TransFx-H (Cytiva), Pro293a (LONZA), Pro293s (LONZA), BalanCD HEK293 (FUJIFILM), EX-CELL® 293 (SAFC), EX-CELL® CD293 Viral Vector Medium (SAFC), 293 SFMII (Thermo Fisher Scientific), CD293 (Thermo Fisher Scientific), FreeStyle293 ＴＭ Expression medium (Thermo Fisher Scientific), Expi293 ＴＭExpression medium (Thermo Fisher Scientific), LV MAX ＴＭ Production medium (Thermo Fisher Scientific), Viral Production Medium (Thermo Fisher Scientific), etc. are preferred. As basal media under development, B10-06, PBH-05, etc. are preferred.

[0039] In one embodiment of the present invention, the medium composition of the present invention is prepared by adding the medium additive of the present invention to a basal medium. In this case, if the basal medium already contains the components (1) to (11) essential for the medium composition, the concentration of the component in the medium composition is the sum of the concentration in the medium additive and the concentration in the basal medium. Component (12), copper, is not contained in the medium additive, so its concentration corresponds to that in the basal medium.

[0040] Furthermore, in addition to the medium additives and basal medium, the medium composition of the present invention can also contain components favorable for cell growth and virus production. Examples of such components include sugars such as glucose, fructose, sucrose, and maltose; amino acids; 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; and N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid). buffers such as N-[tris(hydroxymethyl)methyl]glycine (BES)); antibiotics such as amphotericin B, kanamycin, gentamicin, streptomycin, and penicillin; Type I collagen, Type II collagen, and the like; Examples of such 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.

[0041] The medium composition 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 is preferably derived from a mammal (e.g., fetal bovine serum, human serum, etc.), and more preferably human serum. The concentration of the serum may be within a known concentration range. The medium composition of the present invention is preferably serum-free.

[0042] 3. Virus-producing cells and methods for culturing virus-producing cells The medium composition of the present invention, preferably a medium composition obtained by adding the medium additive of the present invention to a basal medium, can be used to produce any virus capable of replicating in the medium composition. Viruses of interest in the present invention include, for example, viruses belonging to the following families: orthomyxoviruses, paramyxoviruses, reoviruses, picornaviruses, flaviviruses, arenaviruses, herpesviruses, poxviruses, and adenoviruses. These may also be recombinant viruses. In a preferred embodiment of the present invention, the virus of interest is a virus that can form part of a vaccine, particularly a human vaccine, and the medium composition of the present invention can be used to cultivate cells producing these viruses. The virus may be, in particular, poliovirus, rabies virus, Japanese encephalitis virus, yellow fever virus, rubella virus, mumps virus, dengue virus, or measles virus, viruses causing various forms of hepatitis, AIDS virus, chickenpox virus, herpesvirus, viruses causing respiratory syncytial virus (RSV), cytomegalovirus, EBV, rotavirus, or influenza virus. In another preferred embodiment of the present invention, the target viruses are adenoviruses and adeno-associated viruses (AAVs), and the medium composition of the present invention can be used to culture cells that produce these viruses. Because adenoviruses and AAVs have the ability to carry genes, they can be used as viral vectors in gene therapy, gene transfer research, and the like. Therefore, adenovirus and AAV-producing cells can be used to mass-produce adenovirus vectors carrying a desired gene and then introduce that gene into target cells. Furthermore, adenoviruses and AAVs incorporating a desired gene can be mass-produced using virus-producing cells and used as vaccines.

[0043] The virus-producing cells are not particularly limited as long as they are capable of propagating the desired virus, and mammalian cells such as human, monkey, and rodent cells are used. Specific examples include HEK293 cells, Vero cells, CV-1 cells, LLC-MK2 cells, MDCK cells, MDBK cells, WI-38 cells, MRC5 cells (human fibroblasts), and BHK21 cells, all of which are commercially available. HEK293 cells or Vero cells are preferably used when the target virus is adenovirus or AAV, and MDCK cells are preferably used when the target virus is influenza virus.

[0044] The present invention provides a method for culturing virus-producing cells (hereinafter also referred to as the "culturing method of the present invention"). The culturing method of the present invention comprises a step of culturing virus-producing cells in the medium composition of the present invention. The culture of virus-producing cells can be carried out under the culture conditions used for culturing ordinary animal cells. For example, the culture conditions can be 95% humidity, CO ２ Culture at a concentration of 5 to 10% (v / v) is exemplified, but the present invention is not limited to such conditions. Culture can be carried out at, for example, 30 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 are achieved. The culture period is not particularly limited, and is, for example, 12 to 150 hours, preferably 48 to 120 hours.

[0045] The incubator used for cell culture is not particularly limited as long as it is capable of culturing the target cells, and examples thereof 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, and roller bottles.

[0046] 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.

[0047] 4. Virus Production Method The present invention provides a virus production method (hereinafter also referred to as the "production method of the present invention"). The virus production method of the present invention is characterized by culturing virus-infected virus-producing cells (hereinafter also referred to as "infected cells") in the medium composition of the present invention. The culture may be either adherent culture or suspension culture, but suspension culture is preferred. As used herein, "adherent culture" refers to culturing cells attached to a culture substrate. Specifically, it refers to a method in which adherent cells grow while adhering to the surface of the culture substrate and while adhering to each other. Examples of culture substrates include, but are not limited to, multiwell plates, culture dishes, petri dishes, culture flasks, microcarriers, hollow fibers, etc. 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. The virus is produced by collecting a supernatant from the culture medium obtained by culturing infected cells. The virus-producing cells in which virus production has been achieved are then collected and disrupted, and the resulting cell lysate containing virus particles is subjected to appropriate processes such as filter filtration, ultracentrifugation, chromatography, or ultrafiltration to purify the virus particles and produce the final product. In the present invention, the term "virus-infected cells" encompasses not only cells in which live virus has been added to virus-producing cells and the virus has grown therein, but also cells in which nucleic acids encoding proteins necessary for viral infection have been introduced into virus-producing cells and the virus has been produced and grown therein. The virus (particles) and vectors obtained using the production method of the present invention can be used as active ingredients in pharmaceutical compositions. The pharmaceutical compositions can be used ex vivo in patient-derived cells or administered directly to patients.

[0048] The present invention will be described in detail below using examples, but the present invention is not limited in any way. Unless otherwise specified, the reagents and materials used are commercially available or can be prepared according to known literature. Furthermore, those skilled in the art will understand that any substance having the same effect or action can be substituted.

[0049] <Preparation of the medium composition of the present invention 1> L-aspartic acid and glutathione were dissolved in distilled water to prepare a medium additive. The medium additive was added to a basal medium to prepare the medium composition of the present invention. The concentrations of each component in the medium composition were as follows (each concentration is shown as the concentration of the functional molecule only): (4) L-aspartic acid: 250-1000 mg / L (10) Glutathione: 1.5-6 mg / L

[0050] <Preparation of Medium Composition of the Present Invention 2> A medium additive was prepared by dissolving potassium, magnesium, iron, L-aspartic acid, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, glutathione, and a glutamine source in distilled water. The medium additive was added to a basal medium to prepare a medium composition of the present invention (hereinafter also referred to as "AJI Supplement"). The concentrations of each component in the medium composition are as follows (each concentration is the concentration of the functional molecule only): (1) Potassium: 290-772 mg / L (2) Magnesium: 7-45 mg / L (3) Iron: 17-123 mg / L (4) L-Aspartic Acid: 250-1000 mg / L (5) L-Tryptophan: 48-408 mg / L (6) Folic Acid: 3-59.6 mg / L (7) Riboflavin: 0.7-4.8 mg / L (8) Ethanolamine: 10.2-102 mg / L (9) Buffer: 90-8800 mg / L (10) Glutathione: 1.5-6 mg / L (11) Glutamine Source: 17.54-1169 mg / L. The concentration of (12) Copper in the medium composition was 0.002-0.024 mg / L, carried over from the basal medium.

[0051] Similarly, a medium composition was prepared with the following concentrations of each component (each concentration is the concentration of the functional molecule only): (1) potassium: 290-370 mg / L, (2) magnesium: 15-25 mg / L, (3) iron: 18-30 mg / L, (4) L-aspartic acid: 290-810 mg / L, (5) L-tryptophan: 100-200 mg / L, (6) folic acid: 5-20 mg / L, (7) riboflavin: 1-2 mg / L, (8) ethanolamine: 30-50 mg / L, (9) buffer: 1000-3000 mg / L, (10) glutathione: 3-4 mg / L, (11) glutamine source: 145-1030 mg / L, and (12) copper: 0.004-0.015 mg / L.

[0052] Potassium chloride was used as potassium, anhydrous magnesium sulfate was used as magnesium, sodium ferric citrate was used as iron, ethanolamine was ethanolamine hydrochloride was used as ethanolamine, HEPES was used as buffer, reduced glutathione was used as glutathione, and alanyl L-glutamine was used as glutamine source.

[0053] <Serum-free adaptation and suspension of HEK293 cells> 1: Reagents used: (1) Fetal Bovine Serum (FBS, Cytiva) (2) DMEM / F12 (Thermo Fisher Scientific Inc.) (3) 293 SFM II (Thermo Fisher Scientific Inc.) (4) L-Glutamine (200 mM) (Thermo Fisher Scientific Inc.)

[0054] 2: Cells used Cell line: HEK293 2. sus(ATCC:CRL-1573.3)

[0055] 3: Serum-free adaptation and suspension method Serum-free adaptation and suspension were performed according to the following procedure. (1): HEK293 2.sus cells were induced to sleep in 10% FBS / DMEM F12 medium and statically cultured (seeding density: 2 × 10 ５ cells / mL T75 Flask x 1 Culture period: 3 days CO ２ (2): Before reaching 100% confluence, the cells were passaged and statically cultured in 5% FBS / DMEM F12 medium (seeding density: 2 × 10５ cells / mL T75 Flask x 1 cultured for 3 days CO ２ (3): Before reaching 100% confluence, the cells were passaged and statically cultured in 2.5% FBS / DMEM F12 medium (seeding density: 2 × 10 ５ cells / mL T75 Flask x 2 Culture days 3 days CO ２ (4): Before reaching 100% confluence, the cells were subcultured and cultured with shaking in 2.5% FBS / DMEM F12 medium (seeding density: 3 × 10 ５ cells / mL, 2 petri dishes, 3-4 days of culture, CO ２ (5) The cells were cultured for 4 days with shaking for two passages in a medium containing a 1:1 mixture of 2.5% FBS / DMEM F12 medium and 293SFM II + 4 mM L-Gln (FBS final concentration: 1.25%) (seeding density: 3 × 10 ５ cells / mL, 2 petri dishes, 4 days of culture, CO ２ (6) The cells were cultured for two passages in a 1:3 mixture of 2.5% FBS / DMEM F12 medium and 293SFM II + 4 mM L-Gln (FBS final concentration: 0.625%) (seeding density: 3 × 10 ５ cells / mL, 2 petri dishes, 4 days of culture, CO ２ (7) The cells were cultured for two passages in a 1:7 mixture of 2.5% FBS / DMEM F12 medium and 293SFM II + 4 mM L-Gln with shaking (FBS final concentration: 0.313%) (seeding density: 3 × 10 ５ cells / mL, 2 petri dishes, 4 days of culture, CO ２ (8) The cells were cultured for two passages in a 1:15 mixture of 2.5% FBS / DMEM F12 medium and 293SFM II + 4 mM L-Gln (FBS final concentration: 0.156%) (seeding density: 3 × 10 ５ cells / mL, 2 petri dishes, 4 days of culture, CO ２ (9): Shaking culture was performed for three passages in 293SFM II + 4 mM L-Gln (FBS final concentration: 0%) (seeding density: 3 × 10５ cells / mL, 2 petri dishes, 4 days of culture, CO ２ 5% 90 rpm). (10): A cell bank was created.

[0056] <Virus Preparation Method> 1: Reagents used (1) Fetal bovine serum (FBS, Fetal Bovine Serum, Cytiva) Minimum Essential Medium Eagle (hereinafter MEM, Sigma-Aldrich) Minimum Essential Medium (autoclavable) (Thermo Fisher Scientific Co., Ltd.) Sodium bicarbonate (Fujifilm Wako Pure Chemical Industries, Ltd.) Neutral red (Fujifilm Wako Pure Chemical Industries, Ltd.) Agarose (Becton, Dickinson and Company) Antibacterial agent (penicillin-streptomycin solution) (Fujifilm Wako Pure Chemical Industries, Ltd.) PBS (-) (Fujifilm Wako Pure Chemical Industries, Ltd.) 0.05% trypsin (Thermo Fisher Scientific Co., Ltd.)

[0057] 2: Virus strains and host cells used Cell line: HEK-293 (ATCC 1573 ＴＭ ) Virus strain: Human adenovirus 32 (ATCC VR-625 ＴＭ )

[0058] 3: Implementation Method 3.1 Passage Method (1) After thawing the frozen host cells in a thermostatic bath set at approximately 37°C, 10 mL of 10% FBS-containing MEM is placed in a 15 mL centrifuge tube, and 1 mL of the thawed cell solution is added and suspended. (2) Centrifuge (1000 rpm, 190 x g, 24°C) for 5 minutes using a centrifuge (AX-310, Tomy Seiko Co., Ltd.) and remove the supernatant. (3) Add 1 mL of 10% FBS-containing MEM to a 15 mL centrifuge tube and resuspend. Add 5 mL of 10% FBS-containing MEM to a 25 mL centrifuge tube. ２ Add 0.5 mL of the cell suspension to the culture flask and incubate in CO ２ Incubator (Set temperature: 37°C, CO ２ (4) The cells were cultured at a concentration of 5% for several days. ２Confirm that a monolayer sheet has formed at the bottom of the culture flask. Remove the medium, wash the cells twice with PBS, add 1 mL of 0.05% trypsin to detach the cells, and transfer the recovery liquid to a 15 mL centrifuge tube. (5) Mix the recovery liquid with an equal volume of MEM containing 10% FBS, centrifuge for 5 minutes (1000 rpm, 190 x g, 24 °C), and remove the supernatant. (6) Add 1 mL of MEM containing 10% FBS to the 15 mL centrifuge tube and resuspend. Add 5 mL of MEM containing 10% FBS to a 25 cm ２ 0.1 mL of the suspension was added to the culture flask, and CO ２ The cells are cultured in an incubator for several days. (7) Once the cells have formed into a monolayer sheet, repeat steps (4) to (6) to subculture the cells.

[0059] 3.2 75cm ２ Culture flask preparation method (1) Subculture and 25 cm ２ The medium of the host cells that had formed a monolayer sheet on the bottom of the culture flask was removed, and the cells were washed twice with PBS. Then, 25 cm of 0.05% trypsin was added to the cells. ２ Add 1 mL to each culture flask to detach the cells, and transfer the recovery solution to a 15 mL centrifuge tube. (2) Mix the recovery solution with an equal volume of 10% FBS-containing MEM, centrifuge for 5 minutes (1000 rpm, 190 x g, 24°C), and remove the supernatant. (3) Add 1 mL of 10% FBS-containing MEM to the 15 mL centrifuge tube and resuspend. Place the entire resuspended solution in a sterilized medium bottle, add 10% FBS-containing MEM, and add 75 cm ２ 15 mL of each was inoculated into a culture flask and CO ２ (4) The host cells are cultured in an incubator until they reach 75 cm ２ Confirm that a single layer sheet is formed on the bottom of the culture flask, and use it to purify the inoculated virus solution.

[0060] 3.3 How to prepare a 6-well plate (1) Subculture and ２ The medium of the host cells that had formed a monolayer sheet on the bottom of the culture flask was removed, and the cells were washed twice with PBS. Then, 25 cm of 0.05% trypsin was added to the cells. ２Add 1 mL to each culture flask to detach the cells, and transfer the recovery solution to a 15 mL centrifuge tube. (2) Mix the recovery solution with an equal volume of MEM containing 10% FBS, and centrifuge for 5 minutes (1000 rpm, 190 × g, 24 °C) to remove the supernatant. (3) Add 1 mL of MEM containing 10% FBS to the 15 mL centrifuge tube and resuspend. The cell count was 0.15 × 10 ６ The culture medium was added so that the concentration of cells / mL was 1000. 2 mL of the medium was seeded in a 6-well plate. ２ (4) After confirming that the host cells have formed a monolayer sheet on the bottom of the plate, use it to measure the concentration of the inoculated virus solution and the amount of virus.

[0061] 3.4 Purification method of inoculated virus solution Frozen stored virus was thawed, appropriately diluted with MEM, and added to 75 cm ２ Add CO to the culture flask ２ The virus is allowed to adsorb to the cells in an incubator for 1 hour. After that, the virus solution is removed, MEM is added, and the cells are incubated in CO ２ The cells are cultured in an incubator until cytopathic effect (CPE) appears, and the supernatant containing the virus solution and the cells are collected. After collection, the cells are frozen and thawed three times (resuspended at least five times) and centrifuged for five minutes (2000 rpm, 780 x g, 4°C). The supernatant is used as the virus inoculum solution. The virus inoculum solution is dispensed into 1 mL aliquots in cryogenic tubes and stored frozen (-80°C) in an ultra-low temperature freezer until use.

[0062] 3.5 Measurement of concentration of inoculated virus solution The inoculated virus solution was serially diluted with PBS (10 to 10 ６ To a 6-well plate containing cultured HEK-293 cells, 0.2 mL of each virus dilution was added to each well, and the plate was shaken every 15 minutes to allow the virus to adsorb to the cells for 1 hour. After 1 hour, equal amounts of medium A and medium B were mixed and quickly added to the Petri dishes in 2 mL portions. The mixture was left at room temperature until the agarose solidified, and then heated in CO ２ After culturing in an incubator for several days, 2 mL of medium containing neutral red (medium A + medium B) was overlaid and incubated overnight in CO ２After culturing in an incubator, the number of plaques is counted using a handy colony counter (AS ONE Corporation). The number of samples for each concentration is 3. Medium A: 9.5 mL of 10xMEM, 3 mL of 7.5% sodium bicarbonate, 5 mL of FBS, 0.5 mL of antibiotic, 32 mL of sterile distilled water. Medium B: 0.8 g of agarose, 50 mL of distilled water.

[0063] 4. Virus Production Experiments in Media Containing L-Aspartic Acid and Glutathione (Reduced Form) 4.1 Conditions with Low Concentrations of L-Aspartic Acid and Glutathione (Reduced Form) 4.1.1 Reagents Used: (1) 293 SFM II (Thermo Fisher Scientific Co., Ltd.) (2) B10-06 (Ajinomoto Co., Inc., developed product) (3) L-Aspartic Acid (4) Glutathione (Reduced Form) *L-Glutamine was added to 293 SFM II to a final concentration of 4 mM to prepare glutamine-containing 293 SFM II. *Alanyl-Glutamine was added to B10-06 to a final concentration of 4 mM.

[0064] 4.1.2. Virus strains and host cells used Cell strain: 1. Floating HEK293 prepared in 2. sus (ATCC: CRL-1573.3) Virus strain: 2. Human adenovirus 32 (ATCC VR-625) prepared in 2. ＴＭ )

[0065] 4.1.3 Experimental Method (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 in 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 incubated in 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 the culture medium so that the cell suspension reaches 500 cells / mL. Add 5 mL of the adjusted cell suspension to the 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 ２ After 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 culture medium (see Table 2) for each group to the 15 mL centrifuge tube and resuspend. Count the cell number using a hemocytometer and obtain 3 × 10 ５ Add fresh culture medium so that the cell suspension reaches 500 cells / mL. 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, the number of cells is counted and 5 x 10 ５ The culture medium for each group is adjusted to give a concentration of 10 cells / mL, and 5 mL is added to each petri dish. The frozen virus solution for inoculation is thawed, and the virus solution is added to the petri dish to give an MOI of 10 (see Table 1).

[0066]

[0067] The concentrations of each component in the culture medium were adjusted to be as shown in Table 2.

[0068]

[0069] (13) CO ２The culture is incubated with shaking in an incubator for two days. (14) After incubation, the culture medium is recovered from the dish, freeze-thawed three times (resuspended at least five times), centrifuged (2000 rpm, 190 x g, 4°C, 10 minutes), and the supernatant is collected. (15) 1 mL of the supernatant is added to a cryostat tube and stored frozen (-80°C) until use. (16) The virus concentration of the frozen supernatant is measured at a later date using the method in 3.5. The results are shown in Figure 1.

[0070] 4.2 Conditions with low L-aspartic acid concentration 4.2.1. Reagents used: Prepared in the same manner as in 4.1.1. 4.2.2. Virus strain and host cells used: Prepared in the same manner as in 4.1.2. 4.2.3. Experimental method: Implemented in the same manner as in 4.1.3 (1) to (11). (12) After culturing, the number of cells was counted, and 5 x 10 ５ The culture medium for each group is adjusted to 10 cells / mL, and 5 mL is added to each petri dish. The frozen virus solution for inoculation is thawed, and the virus solution is added to the petri dish at an MOI of 10 (see Table 3).

[0071]

[0072] The concentrations of each component were adjusted to the concentrations shown in Table 4.

[0073]

[0074] The procedure was carried out in the same manner as in 4.1.3 (13) to (15). (16) The virus concentration of the frozen supernatant was measured later using the method in 3.5. The results are shown in Figure 2.

[0075] 4.3 Conditions in which L-aspartic acid and glutathione (reduced) were added to other media 4.3.1. Reagents used (1) 293 SFM II (Thermo Fisher Scientific Inc.) (2) L-aspartic acid (3) Glutathione (reduced) *L-Glutamine was added to 293 SFM II to a final concentration of 4 mM. 4.3.2. Virus strain and host cells used Prepared in the same manner as in 4.1.2. 4.3.3. Experimental method Performed in the same manner as in 4.1.3 (1) to (11). (12) After culturing, the number of cells was counted, and 5 x 10 ５The culture medium for each group is adjusted to 10 cells / mL, and 5 mL is added to each petri dish. The frozen virus solution for inoculation is thawed and added to the petri dish at an MOI of 10 (see Table 5).

[0076]

[0077] The procedure was carried out in the same manner as in 4.1.3 (13) to (15). (16) The virus concentration of the frozen supernatant was measured later using the method in 3.5. The results are shown in Figure 3.

[0078] 5. Virus Production Experiments Using Culture Medium and AJI Supplement 5.1 Conditions of Low Cell Density and Virus Concentration 5.1.1. Reagents Used (1) 293 SFM II (Thermo Fisher Scientific Co., Ltd.) (2) L-Glutamine (200 mM) (Thermo Fisher Scientific Co., Ltd.) (3) CDM4HEK293 (Cytiva) (4) B10-06 (Product Developed by Ajinomoto Co., Inc.) (5) PBH-05 (Product Developed by Ajinomoto Co., Inc.) (6) Supplement V0 (Product Developed by Ajinomoto Co., Inc.) was used as the AJI Supplement. *L-Glutamine was added to 293 SFM II to a final concentration of 4 mM to prepare glutamine-containing 293 SFM II. *Alanyl-Glutamine was added to CDM4HEK293, B10-06, and PBH-05 to a final concentration of 4 mM. 5.1.2. Virus strains and host cells used: Prepared as in 4.1.2. 5.1.3. Experimental method: Performed as in 4.1.3 (1) to (9). (10) Add 1 mL of culture medium (see Table 6) from each group to a 15 mL centrifuge tube and resuspend. Count the number of cells using a hemocytometer and obtain 3 x 10 ５ Add fresh culture medium so that the cell suspension reaches 500 cells / mL. Add 5 mL of the adjusted cell suspension to two dishes and incubate in CO ２ The cells were cultured in an incubator with shaking for 3 to 4 days. (11) After culturing, steps (8) to (10) were repeated three times. (12) After culturing for 3 days, the number of cells was counted (Figure 4) and 1 x 10 ５ The culture medium for each group was adjusted to 1 x 10 cells / mL, and 5 mL was added to each petri dish. ５The virus solution was added to the petri dish so that the MOI was 0.1 for cells / mL (see Table 6). The concentrations of each component in each group are as shown in Tables 7 and 8.

[0079]

[0080] A commercially available medium was used for Groups 1 and 2, and a commercially available medium supplemented with Supplement V0 was used for Group 3. For Groups 4 to 7, the concentrations of each component were adjusted to the concentrations shown in Table 7.

[0081]

[0082] Table 8 shows the concentration of each component in Table 7 converted into functional molecules only.

[0083]

[0084] (13) CO ２ Incubate with shaking in an incubator for 6 days at an MOI of 0.1. (14) After culturing, recover the culture medium from the dish, freeze and thaw three times (resuspend at least five times), and then centrifuge (2000 rpm, 190 x g, 4°C, 10 minutes) to collect the supernatant. (15) Add 1 mL of the supernatant to a cryostat tube and store frozen (-80°C) until use. (16) The virus concentration of the frozen supernatant will be measured at a later date using the method in 3.5. The results are shown in Figure 5.

[0085] 5.2 Conditions for high cell density and virus concentration 5.2.1. Reagents used: Prepared in the same manner as in 5.1.1. 5.2.2. Virus strains and host cells used: Prepared in the same manner as in 4.1.2 5.2.3. Experimental method: Performed in the same manner as in 4.1.3 (1) to (9). (10) Add 1 mL of culture medium (see Table 9) for each group to a 15 mL centrifuge tube and resuspend. Count the number of cells using a hemocytometer and obtain 3 x 10 ５ Add the culture medium for each group so that the cell suspension reaches 500 cells / mL. Add 5 mL of the adjusted cell suspension to two dishes and incubate in CO ２ The cells were cultured in an incubator with shaking for 3 to 4 days. (11) After culturing, steps (8) to (10) were repeated three times. (12) After culturing, the number of cells was counted (Figure 6) and 5 x 10 ５The culture medium for each group was adjusted to 5 × 10 cells / mL, and 5 mL was added to each petri dish. ５ The virus solution was added to the petri dish so that the MOI was 10 for cells / mL (see Table 9). The concentrations of each component in each group are as shown in Tables 7 and 8.

[0086]

[0087] (13) CO ２ In the case of an MOI of 10, the culture is cultured with shaking in an incubator for two days. (14) After culture, the culture medium is recovered from the petri dish, freeze-thawed three times (resuspended at least five times), centrifuged (2000 rpm, 190 x g, 4 C, 10 minutes), and the supernatant is collected. (15) 1 mL of the supernatant is added to a cryogenic vial and stored frozen (-80°C) until use. (16) The virus concentration of the frozen supernatant is measured at a later date using the method in 3.5. The results are shown in Figure 7.

[0088] According to the present invention, it is possible to provide a medium composition for culturing virus-producing cells with excellent virus productivity and a medium additive suitable for producing the medium. Virus-producing cells cultured in the medium composition of the present invention exhibit a significant increase in the amount of virus produced per cell. Therefore, more efficient virus production becomes possible. The resulting virus can function as a viral vector useful for gene therapy and vaccine production. This application is based on Patent Application No. 2023-138419 filed in Japan (filing date: August 28, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A medium additive for use in culturing virus-producing cells, comprising at least one member selected from the group consisting of L-aspartic acid and glutathione, the final concentrations of each component being within the following ranges when added to a medium: (4) L-aspartic acid: 40-790 mg / L; (10) glutathione: 0.5-5 mg / L.

2. The agent according to claim 1, which contains L-aspartic acid and glutathione, and when added to a culture medium, the final concentrations of each component are in the following ranges: (4) L-aspartic acid: 40 to 790 mg / L; (10) glutathione: 0.5 to 5 mg / L.

3. The agent according to claim 1, further comprising at least one selected from the group consisting of potassium, magnesium, iron, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer and a glutamine source, and the final concentrations of each component when added to a culture medium are in the following ranges: (1) potassium: 8.65 to 86.5 mg / L (2) magnesium: 0.56 to 5.6 mg / L (3) iron: 3.19 to 31.9 mg / L (5) L-tryptophan: 48 to 480 mg / L (6) folic acid: 0.618 to 6.18 mg / L (7) riboflavin: 0.18 to 1.8 mg / L (8) ethanolamine: 10.2 to 102 mg / L (9) buffer: 90 to 900 mg / L (11) glutamine source: 17.54 to 175.4 mg / L.

4. The agent according to claim 1, further comprising potassium, magnesium, iron, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer and a glutamine source, and the final concentrations of each component when added to a culture medium are in the following ranges: (1) potassium: 8.65 to 86.5 mg / L; (2) magnesium: 0.56 to 5.6 mg / L; (3) iron: 3.19 to 31.9 mg / L; (5) L-tryptophan: 48 to 480 mg / L; (6) folic acid: 0.618 to 6.18 mg / L; (7) riboflavin: 0.18 to 1.8 mg / L; (8) ethanolamine: 10.2 to 102 mg / L; (9) buffer: 90 to 900 mg / L; (11) glutamine source: 17.54 to 175.4 mg / L.

5. The agent according to claim 3, wherein the glutamine source is at least one selected from the group consisting of L-glutamine, alanylglutamine and glycylglutamine.

6. A medium composition comprising a basal medium to which is added the medium additive according to any one of claims 1 to 5.

7. A medium composition for use in culturing virus-producing cells, comprising at least one component selected from the group consisting of L-aspartic acid and glutathione, the concentrations of each component in the medium composition being within the following ranges: (4) L-aspartic acid: 250-1000 mg / L; (10) glutathione: 1.5-6 mg / L.

8. The medium composition according to claim 7, comprising L-aspartic acid and glutathione, the concentrations of each component in the medium composition being within the following ranges: (4) L-aspartic acid: 250-1000 mg / L; (10) glutathione: 1.5-6 mg / L.

9. The medium composition according to claim 7, further comprising at least one selected from the group consisting of potassium, magnesium, iron, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer and a glutamine source, the concentrations of each component in the medium composition being within the following ranges: (1) potassium: 290-772 mg / L; (2) magnesium: 7-45 mg / L; (3) iron: 17-123 mg / L; (5) L-tryptophan: 48-480 mg / L; (6) folic acid: 3-59.6 mg / L; (7) riboflavin: 0.7-4.8 mg / L; (8) ethanolamine: 10.2-102 mg / L; (9) buffer: 90-8800 mg / L; (11) glutamine source: 17.54-1169 mg / L; and (12) copper: 0.002-0.024 mg / L.

10. The medium composition according to claim 7, further comprising potassium, magnesium, iron, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer and a glutamine source, the concentrations of each component in the medium composition being within the following ranges: (1) potassium: 290-772 mg / L; (2) magnesium: 7-45 mg / L; (3) iron: 17-123 mg / L; (5) L-tryptophan: 48-480 mg / L; (6) folic acid: 3-59.6 mg / L; (7) riboflavin: 0.7-4.8 mg / L; (8) ethanolamine: 10.2-102 mg / L; (9) buffer: 90-8800 mg / L; (11) glutamine source: 17.54-1169 mg / L; and (12) copper: 0.002-0.024 mg / L.

11. The medium composition according to claim 9, wherein the glutamine source is at least one selected from the group consisting of L-glutamine, alanylglutamine and glycylglutamine.

12. A method for producing a medium composition for use in culturing virus-producing cells, comprising at least one component selected from the group consisting of L-aspartic acid and glutathione, the method comprising adjusting the concentrations of each component in the medium composition to be within the following concentration ranges: (4) L-aspartic acid: 250 to 1,000 mg / L (10) glutathione: 1.5 to 6 mg / L.

13. A method for producing a medium composition for use in culturing virus-producing cells, comprising L-aspartic acid and glutathione, comprising adjusting the concentrations of each component in the medium composition to the following concentration ranges: (4) L-aspartic acid: 250 to 1,000 mg / L; (10) glutathione: 1.5 to 6 mg / L.

14. The method of claim 12, wherein the medium composition further comprises at least one selected from the group consisting of potassium, magnesium, iron, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, and a glutamine source, and the concentrations of each component in the medium composition are within the following ranges: (1) potassium: 290-772 mg / L; (2) magnesium: 7-45 mg / L; (3) iron: 17-123 mg / L; (5) L-tryptophan: 48-480 mg / L; (6) folic acid: 3-59.6 mg / L; (7) riboflavin: 0.7-4.8 mg / L; (8) ethanolamine: 10.2-102 mg / L; (9) buffer: 90-8800 mg / L; (11) glutamine source: 17.54-1169 mg / L; and (12) copper: 0.002-0.024 mg / L.

15. The method of claim 12, wherein the medium composition further comprises potassium, magnesium, iron, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer and a glutamine source, and the concentrations of each component in the medium composition are in the following ranges: (1) potassium: 290-772 mg / L; (2) magnesium: 7-45 mg / L; (3) iron: 17-123 mg / L; (5) L-tryptophan: 48-480 mg / L; (6) folic acid: 3-59.6 mg / L; (7) riboflavin: 0.7-4.8 mg / L; (8) ethanolamine: 10.2-102 mg / L; (9) buffer: 90-8800 mg / L; (11) glutamine source: 17.54-1169 mg / L; and (12) copper: 0.002-0.024 mg / L.

16. The method of claim 14, wherein the glutamine source is at least one selected from the group consisting of L-glutamine, alanylglutamine and glycylglutamine.

17. A method for culturing virus-producing cells, comprising the step of culturing the virus-producing cells in a medium composition according to any one of claims 7 to 11.

18. A method for producing a virus, comprising culturing a virus-infected virus-producing cell in a medium composition according to any one of claims 7 to 11.

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