WO2025047758A1 - Culture medium for culturing virus-producing cells - Google Patents

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

Culture medium for virus-producing cells

The present invention relates to cell culture technology, in particular to a culture medium for virus-producing cells, a culture method, etc.

To meet the needs for human gene therapy and vaccine production, rapid and stable production technologies for viruses and viral vectors are essential, and in particular, the culture of cells that produce viruses and viral vectors (hereinafter also referred to as "virus-producing cells") is extremely important.
When designing and optimizing a medium for virus-producing cells, the components of the medium are an important issue. The components contained in the medium affect the growth and proliferation of cells. In addition, the medium for virus-producing cells also affects the virus proliferation ability (i.e., virus production ability) in the cells. Therefore, it is necessary to select appropriate components. For example, nutrients, vitamins, minerals, amino acids, sugars, etc. are important for cell proliferation. In addition, the concentration of each component in the medium affects cell growth and virus production, so by appropriately adjusting the concentration of each component, it is possible to improve cell proliferation and virus productivity (Non-Patent Documents 1 to 4). On the other hand, some components may be harmful to cells at high concentrations, so it is necessary to consider the balance.

Liste-Calleja L, J Biosci Bioeng. 2014;117(4):471-7 Petiot E, Vaccine. 2015;33(44):5974-81 Cervera L, BMC Proc. 2011;5 Suppl 8(Suppl 8):P126 Shen CF, Vaccine. 2012;30(2):300-6

The present invention aims 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.

In view of the above problems, the present inventors have focused on medium components that improve virus productivity and have conducted intensive research. As a result, they have found that a medium containing a specific component at a specific concentration can improve the virus productivity of virus-producing cells, and have succeeded in preparing a medium additive (supplement) suitable for producing the medium, thereby completing the present invention.
That is, the present invention is as follows.

[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, the final concentrations of each component being within the following ranges when added to a medium:
(4) L-aspartic acid: 40 to 790 mg/L
(10) Glutathione: 0.5 to 5 mg/L.
[2] The agent according to [1], which contains L-aspartic acid and glutathione, and when added to a medium, the final concentrations of each component 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 medium are within the following ranges:
(1) Potassium: 8.65-86.5 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: 0.618-6.18mg/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: 17.54-175.4 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 medium are within the following ranges:
(1) Potassium: 8.65-86.5 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: 0.618-6.18mg/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: 17.54-175.4 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 to which the medium additive according to any one of [1] to [5] has been added.
[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 concentration of each component in the medium composition being within the following ranges:
(4) L-aspartic acid: 250 to 1,000 mg/L
(10) Glutathione: 1.5 to 6 mg/L.
[8] The medium composition according to [7], which contains L-aspartic acid and glutathione, and the concentrations of each component in the medium composition are within the following ranges:
(4) L-aspartic acid: 250 to 1,000 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 concentration of each component in the medium composition is within the following range:
(1) Potassium: 290-772 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: 3-59.6mg/L
(7) Riboflavin: 0.7 to 4.8 mg/L
(8) Ethanolamine: 10.2 to 102 mg/L
(9) Buffer: 90 to 8,800 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: 290-772 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: 3-59.6mg/L
(7) Riboflavin: 0.7 to 4.8 mg/L
(8) Ethanolamine: 10.2 to 102 mg/L
(9) Buffer: 90 to 8,800 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 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, the method comprising adjusting the concentrations of each component in the medium composition to fall within 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 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 concentration of each component in the medium composition is within the following range:
(1) Potassium: 290-772 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: 3-59.6mg/L
(7) Riboflavin: 0.7 to 4.8 mg/L
(8) Ethanolamine: 10.2 to 102 mg/L
(9) Buffer: 90 to 8,800 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 buffering agent 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 to 45 mg/L
(3) Iron: 17 to 123 mg/L
(5) L-tryptophan: 48 to 480 mg/L
(6) Folic acid: 3-59.6mg/L
(7) Riboflavin: 0.7 to 4.8 mg/L
(8) Ethanolamine: 10.2 to 102 mg/L
(9) Buffer: 90 to 8,800 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 a virus-infected virus-producing cell in the medium composition according to any one of [7] to [11].

[01] A medium additive 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, and a glutamine source, the final concentration of each component being within the following ranges when added to a medium:
(1) Potassium: 8.65-86.5 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: 0.618-6.18mg/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: 17.54-175.4 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, the agent according to [01] above, wherein the final concentrations of each component when added to a medium are within the following ranges:
(1) Potassium: 8.65-86.5 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: 0.618-6.18mg/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: 17.54-175.4 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 to which the medium additive according to any one of [01] to [03] has been added.
[05A] The medium composition according to [04], which contains 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: 290-772 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: 3-59.6mg/L
(7) Riboflavin: 0.7 to 4.8 mg/L
(8) Ethanolamine: 10.2 to 102 mg/L
(9) Buffer: 90 to 8,800 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: 290-772 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: 3-59.6mg/L
(7) Riboflavin: 0.7 to 4.8 mg/L
(8) Ethanolamine: 10.2 to 102 mg/L
(9) Buffer: 90 to 8,800 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, the concentration of each component in the medium composition being within the following ranges:
(1) Potassium: 290-772 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: 3-59.6mg/L
(7) Riboflavin: 0.7 to 4.8 mg/L
(8) Ethanolamine: 10.2 to 102 mg/L
(9) Buffer: 90 to 8,800 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.
[08] The medium composition according to [07], wherein the concentration of each component in the medium composition is within the following range:
(1) Potassium: 290-370 mg/L
(2) Magnesium: 15-25 mg/L
(3) Iron: 18-30 mg/L
(4) L-aspartic acid: 500 to 1,000 mg/L
(5) L-tryptophan: 100 to 200 mg/L
(6) Folic acid: 5-20mg/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, the concentration of each component in the medium composition being within the following ranges:
(1) Potassium: 290-772 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: 3-59.6mg/L
(7) Riboflavin: 0.7 to 4.8 mg/L
(8) Ethanolamine: 10.2 to 102 mg/L
(9) Buffer: 90 to 8,800 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.
[010] The medium composition according to [09], wherein the concentration of each component in the medium composition is within the following range:
(1) Potassium: 290-370 mg/L
(2) Magnesium: 15-25 mg/L
(3) Iron: 18-30 mg/L
(4) L-aspartic acid: 500 to 1,000 mg/L
(5) L-tryptophan: 100 to 200 mg/L
(6) Folic acid: 5-20mg/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 range:
(1) Potassium: 290-772 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: 3-59.6mg/L
(7) Riboflavin: 0.7 to 4.8 mg/L
(8) Ethanolamine: 10.2 to 102 mg/L
(9) Buffer: 90 to 8,800 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.
[012] A method for producing a medium composition for use in culturing virus-producing cells, comprising adjusting the concentrations of potassium, magnesium, iron, L-aspartic acid, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffer, glutathione, a glutamine source, and copper to the following ranges:
(1) Potassium: 290-772 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: 3-59.6mg/L
(7) Riboflavin: 0.7 to 4.8 mg/L
(8) Ethanolamine: 10.2 to 480 mg/L
(9) Buffer: 90 to 8,800 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 a step of 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 a virus-infected virus-producing cell in a medium composition according to any one of [04] to [010].

The present invention provides a medium composition for culturing virus-producing cells with 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. This allows for more efficient virus production. The resulting virus can function as a useful viral vector for gene therapy and vaccine production.

1 is a graph showing the results of investigating the proliferation ability of virus-producing cells in a medium containing L-aspartic acid and glutathione (reduced form). The results are obtained under conditions of high L-aspartic acid concentrations. The average value of n=2 is shown. 2 is a graph showing the results of investigating the proliferation ability of virus-producing cells in a medium containing L-aspartic acid and glutathione (reduced form). The results are obtained under conditions where the L-aspartic acid concentration is low. The average value of n=2 is shown. 3 is a graph showing the results of investigating the proliferation ability of virus-producing cells in a medium containing L-aspartic acid and glutathione (reduced form). The results are from experiments in which different basal media were used. The average value of n=2 is shown. Figure 4 is a graph showing the results of investigating the proliferation ability of virus-producing cells in various media. The results are obtained under conditions of low cell density and low virus concentration. In the graph, "Supplement" refers to AJI Supplement. Figure 5 is a graph showing the results of investigating the virus production ability of virus-producing cells in various media. The results are obtained under conditions of low cell density and low virus concentration. The results are shown as the mean value ± standard deviation (n = 3). In the graph, "Supplement" means AJI Supplement. Figure 6 is a graph showing the results of investigating the proliferation ability of virus-producing cells in various media. The results are obtained under conditions of high cell density and high virus concentration for infection. The results are shown as the mean ± standard deviation (n = 3). In the graph, "Supplement" means AJI Supplement. Figure 7 is a graph showing the results of investigating the virus production ability of virus-producing cells in various media. The results are obtained under conditions of high cell density and high virus concentration for infection. The results are shown as the mean value ± standard deviation (n = 3). In the graph, "Supplement" means AJI Supplement.

The present invention will now be described. Terms used in the present specification 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 "medium additive of the present invention"). The medium additive of the present invention can usually be added to a basal medium (described later). For example, when the medium additive is a solution, the medium additive is added to a basal medium in an amount of 1/20 to 1/5, preferably 1/15 to 1/8, more preferably 1/10. One embodiment of the medium additive 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 additive 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, and a glutamine source. Usually, 2, 3, 4, 5, 6, 7, 8, 9, or 10 members, and preferably all 11 members.
In another embodiment of the present invention, the medium additives include magnesium, folic acid, riboflavin, ethanolamine, a buffering agent, and glutathione.
In another embodiment of the present invention, the medium additive comprises potassium and iron.
The medium additive may be a solution of each desired component in a suitable solvent (e.g., water), or the solution may be freeze-dried or otherwise converted into a solid powder. It may also be a simple mixture of each solid component.
Each component will be described below.

(1.1 Potassium)
Potassium is a nutrient necessary for cell survival and proliferation, and is necessary for maintaining physiological functions of cells, 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 is usually included in the medium additive as a potassium salt, although the form 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 to a basal medium. Examples of potassium salts include, but are not limited to, potassium chloride, potassium hydroxide, potassium sulfate, potassium phosphate, dipotassium hydrogen phosphate, potassium carbonate, potassium hydrogen carbonate, etc. Potassium chloride is preferable. The potassium salt may be anhydrous or hydrated.
Potassium is contained 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 concentration of potassium 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. If the potassium concentration in the medium additive is too high, the final concentration when added to the basal medium will be too high, which is undesirable because the osmotic pressure will increase and cell growth will decrease. If the potassium concentration in the medium additive is too low, the final concentration when added to the basal medium will be low, which is undesirable because the intracellular sodium-potassium pump will not start and the ATP required for cell growth will not be obtained. When potassium is provided as a potassium salt, the above concentration represents the concentration when converted into only the functional molecule (i.e., free potassium).
The potassium concentration can be measured by any method available in the art, but is usually measured by ICP MS.

(1.2 Magnesium)
Magnesium acts as a cofactor for enzyme activity and regulates many enzymatic reactions in cells. Magnesium 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. Magnesium is not limited in its form as long as it can exert its function in the medium additive and in the medium composition obtained by adding the medium additive to a basal medium, and is usually 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, magnesium acetate, etc. Preferred examples include magnesium sulfate. Magnesium salts may be anhydrous or hydrated.
Magnesium is contained 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 concentration of magnesium in the medium additive is usually 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 when added to the basal medium will be too high, and if an excess of essential metals is contained in the medium, cell growth will be inhibited, which is not preferable. If the magnesium concentration in the medium additive is too low, the final concentration when added to the basal medium will be low, and essential metals required by cells will not be supplied, and cell growth will be inhibited, which is not preferable. When magnesium is provided as a magnesium salt, the above concentration represents the concentration when converted into only the functional molecule (i.e., free magnesium).
The magnesium concentration can be measured by any method available in the art, but is usually measured by ICP MS.

(1.3 Iron)
Iron is also involved in the structure of enzymes and proteins in the electron transport system, 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 system in mitochondria. The form of iron 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 to a basal medium, and is usually included in the medium additive as an iron salt. Examples of iron salts include inorganic salts, organic acid salts, complexes (complex salts), and the like of iron. Examples of inorganic salts include sulfates, chlorides, and nitrates. Examples of organic acid salts include acetates and citrates. There is no particular restriction on the iron complex as long as it can form a complex with iron and transport iron into cells, but a synthetic non-proteinaceous one that is easily soluble in water and has a stable complex at the pH (near neutral) of the medium, i.e., does not precipitate as iron hydroxide, is 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, potassium ferric oxalate ammonium, ferrous sulfate, ferric sulfate, ammonium ferrous sulfate, ferric carbonate, ferrous chloride, ferric chloride, etc. Preferably, sodium ferrous citrate is used. The iron salt may be anhydrous or hydrated.
Iron is contained 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 concentration of iron 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 when added to the basal medium will be too high, and if an excess of essential metals is contained in the medium, cell growth will be inhibited, which is not preferable. If the iron concentration in the medium additive is too low, the final concentration when added to the basal medium will be low, and essential metals required by cells will not be supplied, and cell growth will be inhibited, which is not preferable. When iron is provided as an iron salt, the above concentration represents the concentration when converted into only functional molecules (i.e., free iron).
The iron concentration can be measured by any method available in the art, but is usually measured by ICP MS.

(1.4 L-Aspartic Acid)
L-aspartic acid is known to be involved in the activation of the electron transport system (Biotechnol. Prog. 2000, 16, 872-884). L-aspartic acid functions as an electron donor for enzyme complex 1 (NADH: quinone oxidoreductase) of the electron transport system, and plays an important role in cell metabolism and proliferation by activating energy production. 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 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. It can also be made into a salt, and specific examples thereof include salts with inorganic bases, organic bases, inorganic acids, organic acids, and salts with amino acids.
Free L-aspartic acid is preferred.
L-aspartic acid is included in the medium additive so that the final concentration when added to the basal medium is 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 mg/L or more, and 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 included in the medium additive so that the final concentration when added to the basal medium is in the range of 171 to 1710 mg/L, preferably 200 to 800 mg/L, 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, particularly preferably 80 to 600 mg/L. The concentration of L-aspartic acid in the medium additive is usually 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 mg/L or more, and 18000, 17000, 16000, 15000, 14000, 13000, 12000, 11000, 10000, 9000, 8000 mg/L or less. In one embodiment of the present invention, the concentration of L-aspartic acid in the medium additive is usually 1710 to 17100 mg/L, preferably 2000 to 8000 mg/L, 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 usually 400 to 7900 mg/L, preferably 600 to 7000 mg/L, 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 becomes too high, the pH in the medium decreases, and the appropriate pH of the cells cannot be maintained, which is not preferable. If the concentration of L-aspartic acid in the medium additive is too low, the final concentration when added to the basal medium becomes low, metabolites are not supplied to the TCA cycle, and the cells become energy insufficient, which is not preferable. When L-aspartic acid is provided as a salt, the above concentrations represent the concentrations calculated in terms of only the functional molecule (ie, free L-aspartic acid).
The concentration of L-aspartic acid can be measured by any method available in the art, but is usually measured by UPLC.

(1.5 L-tryptophan)
L-tryptophan is one of the amino acids necessary for protein synthesis in cells, 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 exert its function in the medium additive and in the medium composition obtained by adding the medium additive to a basal medium. L-tryptophan may be isolated and purified from natural products or processed products thereof, or may be a synthetic product. It may also be in the form of a salt, and specific examples thereof include salts with inorganic bases, organic bases, inorganic acids, organic acids, and salts with amino acids.
Free L-tryptophan is preferred.
L-tryptophan is contained 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 usually 480 to 4800 mg/L, preferably 600 to 3000 mg/L, and particularly preferably 1000 to 2000 mg/L. If the concentration of L-tryptophan in the medium additive is too high, the final concentration when added to the basal medium becomes 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 becomes low, and tryptophan, an essential amino acid, is not synthesized in the cells, which is undesirable because the tryptophan required for the cells cannot be supplied. When L-tryptophan is provided as a salt, the above concentration represents the concentration when converted into only the functional molecule (i.e., free L-tryptophan).
The L-tryptophan concentration can be measured by any method available in the art, but is usually measured by UPLC.

(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 the medium additive and in the medium composition obtained by adding the medium additive to a basal medium. Folic acid may be isolated and purified from natural products or processed products thereof, or may be a synthetic product. It may 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 usually 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, and the solubility in the medium will be low, resulting in precipitation in the medium. In addition, since it is very easily decomposed, it is not possible to deny that the decomposition products may have an adverse effect on some cells, which is not preferable. 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, and folic acid necessary for nucleic acid synthesis will not be supplied, which will affect cell growth, which is not preferable. When folic acid is provided as a salt, the above concentration represents the concentration when converted into only the functional molecule (i.e., free folic acid).
The concentration of folic acid can be measured by any method available in the art, but is usually measured by LCMS.

(1.7 Riboflavin)
Riboflavin is another name for vitamin B2 and plays an important role in the electron transport system of cells (Biotechnol. Prog. 2000, 16, 872-884). The electron transport system is a sequential process consisting of multiple protein complexes present in the membranes of mitochondria, and is essential for energy production in cells. Therefore, the inclusion of riboflavin in a cell culture medium contributes to proper cell growth and metabolism. Riboflavin may be in any form as long as it can exert 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 processed products thereof, or may be a synthetic product. It may also be a derivative, and examples of such derivatives include, but are not limited to, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), riboflavin butyrate (e.g., riboflavin tetrabutyrate), or salts thereof, hydrates thereof, and the like. Riboflavin and riboflavin derivatives may be in the form of a salt. Examples of such salts include acid addition salts and salts with bases.
Riboflavin is contained 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, and the solubility in the medium will be low, resulting in precipitation in the medium. In addition, since it is very easily decomposed, it is not preferable because the decomposition products may have adverse effects on some cells. If the concentration of riboflavin in the medium additive is too low, the final concentration when added to the basal medium will be low, and the metabolic products of riboflavin will not be supplied to the electron transport system, resulting in a decrease in its function, which is not preferable. When riboflavin is provided as a salt, the above concentration represents the concentration when converted into only the functional molecule (i.e., free riboflavin).
The riboflavin concentration can be measured by any method available in the art, but is usually measured by LCMS.

1.8 Ethanolamine
Ethanolamine, also called 2-aminoethanol or monoethanolamine, is involved in the synthesis of phosphatidylethanolamine, a phospholipid that is a major component of the cell membrane. Phosphatidylethanolamine plays an important role in maintaining the structure of the cell membrane and regulating the fluidity and permeability of the cell membrane. Therefore, by including ethanolamine in the medium, it is possible to provide a suitable lipid environment for cells. The form of ethanolamine 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 to a basal medium. Ethanolamine may be isolated and purified from natural products or their processed products, or may be a synthetic product. It is also possible to make it into a derivative. Examples of such derivatives include phosphoethanolamine (also known as phosphorylethanolamine), monomethylethanolamine, dimethylethanolamine, N-acylphosphatidylethanolamine, phosphatidylethanolamine, and lysophosphatidylethanolamine.
The salt may be in the form of an ethanolamine or ethanolamine derivative salt. Examples of such salts include salts with inorganic bases, organic bases, inorganic acids, and organic acids, and salts with amino acids. Preferred salts 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. Particularly preferred are hydrochlorides.
Ethanolamine is contained 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 usually 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 becomes too high, which is undesirable because it inhibits cell proliferation. If the concentration of ethanolamine in the medium additive is too low, the final concentration when added to the basal medium becomes low, which is undesirable because it does not supply cell membrane components necessary for cell proliferation. When ethanolamine is provided as a salt, the above concentration represents the concentration when converted to only the functional molecule (i.e., free ethanolamine).
The concentration of ethanolamine can be measured by any method available in the art, but is usually measured by UPLC.

1.9 Buffers
The buffer 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 finally prepared 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, phosphates, bicarbonates, etc.
The concentration of the buffer used can be appropriately adjusted depending on the type of buffer used. When HEPES is used as a buffer, HEPES is included 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 usually 900 to 9000 mg/L, preferably 1000 to 6000 mg/L, and particularly preferably 2000 to 4000 mg/L. If the concentration of the buffer in the medium additive is too high or too low, the desired buffering capacity cannot be obtained, which is not preferable.
The concentration of the buffer, for example, in the case of HEPES, can be measured by any method available in the art, but can usually be measured by LCMS-MS.

(1.10 Glutathione)
Glutathione has an antioxidant effect and has the function of protecting cells from oxidative stress within the cells (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 the medium additive and the medium composition obtained by adding the medium additive to a basal medium. It may be isolated and purified from natural products or processed products thereof, or it may be a synthetic product. Glutathione may be either oxidized glutathione or reduced glutathione, but reduced glutathione is preferable. It can also be made into a salt. Examples of such salts include salts with inorganic bases, organic bases, inorganic acids, organic acids, and salts with amino acids. Preferred examples of the salts 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 the final concentration when added to the basal medium is 0.5, 0.6, 0.7, 0.8, 0.9, 1 mg/L or more and 6, 5, 4, 3 mg/L or less. In one embodiment of the present invention, glutathione is contained in the medium additive so that the final concentration when added to the basal medium is in the range of 0.6 to 6 mg/L, preferably 1 to 5 mg/L, 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 the basal medium is in the range of 0.5 to 5 mg/L, preferably 1 to 4 mg/L, particularly preferably 2 to 3 mg/L. The concentration of glutathione in the medium additive is usually 5, 6, 7, 8, 9, 10 mg/L or more and 60, 50, 40, 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, 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, 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 becomes too high, and since it is very easily decomposed, the possibility that the decomposition products may have an adverse effect on some cells cannot be denied, which is not preferable. If the concentration of glutathione in the medium additive is too low, the final concentration when added to the basal medium becomes low, which is not preferable, since it disrupts the balance of oxidation and reduction required within the cells. When glutathione is provided as a salt, the above concentration represents the concentration when converted into only the functional molecule (i.e., free glutathione).
The glutathione concentration can be measured by any method available in the art, but is conveniently measured using a glutathione quantification kit (eg, DOJINDO: G257).

1.11 Glutamine Source
Glutamine is an amino acid and plays an important role in cell 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 prepared, and examples thereof include L-glutamine, alanyl-L-glutamine (an amino acid in which alanine and glutamine are bonded by a peptide bond), and glutamic acid (which can function as a precursor of glutamine). Alanyl-L-glutamine is preferable. 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 to which the medium additive is added. It may be isolated and purified from natural products or processed products thereof, or may be a synthetic product. It may also be a salt, and specific examples thereof include salts with inorganic bases, organic bases, inorganic acids, organic acids, and salts with 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 usually 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 into only the functional molecule (i.e., glutamine).
The glutamine concentration can be measured by any method available in the art, but is usually measured by UPLC.

2. Medium Composition The present invention provides a medium composition for use in culturing virus-producing cells (hereinafter, also referred to as "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. Usually, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 members are contained, and preferably all 12 members are contained.
The medium composition of the present invention may be provided in a liquid state, or may be prepared in a more concentrated state than the concentration at the time of use, or in a solid state such as a freeze-dried powder, and then diluted with a solvent such as water, or dissolved or dispersed in a solvent such as water, at the time of use.
Each component will be described below.

(2.1 Potassium)
The potassium contained in the medium composition has the same meaning as described above in (1.1 Potassium).
The concentration of potassium 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 calculated in terms of only the functional molecule (i.e., free potassium).

(2.2 Magnesium)
The magnesium contained in the medium composition has the same meaning as described above in (1.2 Magnesium).
The concentration of magnesium 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 calculated in terms of only the functional molecule (i.e., free magnesium).

(2.3 Iron)
The iron contained in the medium composition has the same meaning as defined above in (1.3 Iron).
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 calculated in terms of only the functional molecule (i.e., free iron).

2.4 L-Aspartic Acid
The L-aspartic acid contained in the medium composition has the same meaning as defined above in (1.4 L-aspartic acid).
The concentration of L-aspartic acid in the medium composition is 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380 mg/L or more, and 2010, 1910, 1810, 1710, 1610, 1510, 1410, 1310, 1210, 1110, 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, 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, particularly preferably 290 to 810 mg/L. When L-aspartic acid is provided as a salt, the above concentrations represent the concentrations calculated in terms of only the functional molecule (ie, free L-aspartic acid).

(2.5 L-tryptophan)
The L-tryptophan contained in the medium composition has the same meaning as above (1.5 L-tryptophan).
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).

(2.6 Folic Acid)
The folic acid contained in the medium composition has the same meaning as defined above in (1.6 Folic acid).
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 calculated in terms of only the functional molecule (i.e., free folic acid).

(2.7 Riboflavin)
The riboflavin contained in the medium composition has the same meaning as defined above (1.7 Riboflavin).
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).

2.8 Ethanolamine
The ethanolamine contained in the medium composition has the same meaning as described above (1.8 Ethanolamine).
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 calculated in terms of only the functional molecule (i.e., free ethanolamine).

2.9 Buffers
The buffer contained in the medium composition has the same meaning as described above in (1.9 Buffer).
When the buffer is HEPES, the concentration of the buffer in the medium composition is 90 to 8800 mg/L, preferably 1000 to 3000 mg/L, and particularly preferably 2000 to 3000 mg/L.

(2.10 Glutathione)
The glutathione contained in the medium composition has the same meaning as defined above in (1.10 Glutathione).
The concentration of glutathione in the medium composition is 0.5, 0.6, 0.7, 0.8, 0.9, 1 mg/L or more and 6, 5, 4, 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, 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, particularly preferably 3 to 4 mg/L. When glutathione is provided as a salt, the above concentration represents the concentration when converted into only the functional molecule (i.e., free glutathione).

2.11 Glutamine Source
The glutamine source contained in the medium composition is the same as that 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 only the functional molecule (i.e., glutamine).

(2.12 Copper)
Copper is a type of trace element and is involved in many enzyme reactions in cells. For example, it acts as a coenzyme for enzymes involved in cell metabolism and antioxidant enzymes. These enzymes catalyze important reactions in cells and are necessary to maintain normal cell function, so copper is an important component for cell growth. Copper is not limited in its form as long as it can exert its function in the medium composition, and is usually included in the medium composition as a copper salt. Examples of copper salts include inorganic salts, organic acid salts, complexes (complex salts), and the like of copper. Examples of inorganic salts include sulfates, chlorides, and nitrates. Examples of organic acid salts include acetates and citrates. There is no particular restriction on the copper complex as long as it can form a complex with copper and transport copper into cells. Examples of copper salts include, but are not limited to, copper nitrate, copper acetate, copper sulfate, and the like. Preferably, copper sulfate is used. The copper salt 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.

The medium composition of the present invention can be prepared, for example, by adding the medium additive of the present invention (see "1. Medium additive" above) 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.

In this specification, the term "basal medium" refers to a medium containing carbon sources, nitrogen sources, inorganic salts, etc., which are essential for cell culture. There are no particular limitations on the basal medium that can be used as a component of the medium of the present invention, and it may be appropriately selected 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.

Usable basal media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM/F12, McCoy's 5A medium, Minimum Essential Medium (MEM), Eagle's Minimum Essential Medium (EMEM), alpha modified Eagle's Minimum Essential Medium (alpha modified Eagle's Medium), and alpha modified Eagle's Medium (alpha modified Eagle's Medium). Examples of the basal medium include α-MEM, α-MEM, 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.

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

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 components (1) to (11) essential to the medium composition are already contained in the basal medium, 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. As for component (12), copper, it is not contained in the medium additive, so its concentration corresponds to that in the basal medium.

In addition to the medium additives and the basal medium, the medium composition of the present invention can also contain components that are favorable for cell growth and virus production. Such components include, for example, 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; N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES)), N-[tris(hydroxymethyl)methyl]glycine (N-[tris (hydroxymethyl)methyl]glycine (Tricine) and other buffers; antibiotics such as amphotericin B, kanamycin, gentamicin, streptomycin, and penicillin; cell adhesion factors and extracellular matrix components such as type I collagen, type II 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; hormones such as dexamethasone, hydrocortisone, estradiol, progesterone, glucagon, and insulin, and the appropriate components can be selected and used depending on the type of cells to be cultured.

The medium composition may contain serum or may be serum-free. There are no particular limitations on the serum, so long as it is derived from an animal and does not inhibit cell growth, but it is preferably mammalian serum (e.g., fetal bovine serum, human serum, etc.), and more preferably human serum. The concentration of serum may be within a known concentration range. The medium composition of the present invention is preferably serum-free.

3. Virus-producing cells and a method 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 for producing any virus capable of replicating in the medium composition. Viruses that are the subject of the present invention include, for example, viruses belonging to the following families: orthomyxoviruses, paramyxoviruses, reoviruses, picornaviruses, flaviviruses, arenaviruses, herpesviruses, poxviruses, and adenoviruses. These may be recombinant viruses.
In a preferred embodiment of the present invention, the viruses of interest are those that may form part of a vaccine, in particular a human vaccine, and the medium composition of the present invention may be used for the cultivation of cells producing these viruses. The viruses may in particular be poliovirus, rabies virus, Japanese encephalitis virus, yellow fever virus, rubella virus, mumps virus, dengue virus or measles virus, viruses of various forms of hepatitis, AIDS virus or chickenpox virus, herpes virus, viruses of the disease caused by respiratory syncytial virus, cytomegalovirus, EBV, rotavirus or influenza virus.
In another preferred embodiment of the present invention, the target virus is adenovirus and adeno-associated virus (AAV), and the medium composition of the present invention can be used for culturing cells that produce these viruses. Since adenovirus and AAV have the ability to carry genes, they can be used as virus vectors in gene therapy and gene transfer research. Therefore, it is possible to mass-produce adenovirus vectors carrying specific genes using adenovirus and AAV-producing cells, and to introduce the genes into target cells. Furthermore, adenovirus and AAV with specific genes can be mass-produced using virus-producing cells to be used as vaccines.

There are no particular limitations on the virus-producing cells as long as they are capable of propagating the desired virus, and mammalian cells such as human, monkey, and rodent cells can be used. Specific examples include HEK293 cells, Vero cells, CV-1 cells, LLC-MK2 cells, MDCK cells, MDBK cells, WI-38 cells, MRC5 cells (human fibroblast cells), 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.

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 virus-producing cells can be cultured under the same culture conditions as those for culturing normal animal cells. For example, the culture can be performed at 95% humidity and a _CO2 concentration of 5-10% (v/v), but the present invention is not limited to such conditions. The culture can be performed at, for example, 30-37°C, but may be performed at a temperature outside the above range as long as the desired cell growth and virus production can be achieved. The culture period is not particularly limited, and is, for example, 12-150 hours, preferably 48-120 hours.

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

The culture vessel may be either 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 the surface of the culture vessel to cells. The cell-supporting substrate may be any substance intended for cell adhesion.

4. Method for Producing Virus The present invention provides a method for producing a virus (hereinafter, also referred to as the "production method of the present invention"). The method for producing a virus of the present invention is characterized in that virus-producing cells infected with a virus (hereinafter, also referred to as the "infected cells") are cultured in the medium composition of the present invention. The culture may be an adhesion culture or a suspension culture, but is preferably a suspension culture. In this specification, "adhesion culture" means culturing by adhering to a culture substrate, and specifically means a method in which adherent cells are grown 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, multi-well plates, culture dishes, petri dishes, culture flasks, microcarriers, hollow fibers, and the like. The culture may be a stationary culture on a substrate. In this specification, "suspension culture" refers to a cell culture method performed in a state in which cells are not adhered to a 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 the infected cells. The virus-producing cells in which virus production has been achieved are then collected and disrupted, and the resulting cell disruption liquid containing the virus particles is appropriately subjected to a process such as filter filtration, ultracentrifugation, chromatography, or ultrafiltration to purify the virus particles into the final product.
In the present invention, the term "cells infected with a virus" refers not only to cells in which a live virus has been added to a virus-producing cell and the virus has proliferated within the cell, but also to cells in which a nucleic acid encoding a protein necessary for viral infection has been introduced into a virus-producing cell and the virus has been produced and proliferated within the cell.
The virus (particle) or vector obtained by the production method of the present invention can be used as an active ingredient in a pharmaceutical composition, which can be used ex vivo in cells derived from a patient or can be administered directly to a patient.

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, etc. Furthermore, those skilled in the art will understand that they can be substituted with other substances that have similar effects and actions.

<Production of 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 are as follows (each concentration is shown as the concentration of the functional molecule only).
(4) L-aspartic acid: 250 to 1,000 mg/L
(10) Glutathione: 1.5 to 6 mg/L

<Production 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 shown as the concentration of the functional molecule only).
(1) Potassium: 290-772 mg/L
(2) Magnesium: 7 to 45 mg/L
(3) Iron: 17 to 123 mg/L
(4) L-aspartic acid: 250 to 1,000 mg/L
(5) L-tryptophan: 48 to 408 mg/L
(6) Folic acid: 3-59.6mg/L
(7) Riboflavin: 0.7 to 4.8 mg/L
(8) Ethanolamine: 10.2 to 102 mg/L
(9) Buffer: 90 to 8,800 mg/L
(10) Glutathione: 1.5 to 6 mg/L
(11) Glutamine source: 17.54 to 1169 mg/L
In addition, the concentration of (12) copper in the medium composition was 0.002 to 0.024 mg/L, which was carried over from the basal medium.

Similarly, a medium composition was prepared having the following concentrations of each component (each concentration is shown as the concentration of the functional molecule only):
(1) Potassium: 290-370 mg/L
(2) Magnesium: 15 to 25 mg/L
(3) Iron: 18-30 mg/L
(4) L-aspartic acid: 290 to 810 mg/L
(5) L-tryptophan: 100 to 200 mg/L
(6) Folic acid: 5-20mg/L
(7) Riboflavin: 1 to 2 mg/L
(8) Ethanolamine: 30 to 50 mg/L
(9) Buffer: 1000 to 3000 mg/L
(10) Glutathione: 3 to 4 mg/L
(11) Glutamine source: 145 to 1030 mg/L
(12) Copper: 0.004-0.015mg/L

The potassium used was potassium chloride, the magnesium used was anhydrous magnesium sulfate, the iron used was sodium ferric citrate, the ethanolamine used was ethanolamine hydrochloride, the buffer used was HEPES, the glutathione used was reduced glutathione, and the glutamine source used was alanyl L-glutamine.

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

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

3: Serum-free acclimation and suspension method Serum-free acclimation and suspension were carried out according to the following procedure.
(1): HEK293 2. sus cells were put to sleep in 10% FBS/DMEM F12 medium and statically cultured (seeding density: 2×10 ⁵ cells/mL, T75 flask×1, culture period: 3 days, CO ₂ 5%).
(2): Before reaching 100% confluence, the cells were passaged and statically cultured in 5% FBS/DMEM F12 medium (seeding density: 2×10 ⁵ cells/mL, T75 flask×1, culture period: 3 days, CO ₂ 5%).
(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×2, culture period: 3 days, CO ₂ 5%).
(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, culture period: 3-4 days, 5% CO ₂ , 90 rpm).
(5): The cells were cultured for 2 passages in a 1:1 mixture of 2.5% FBS/DMEM F12 medium and 293SFM II + 4 mM L-Gln for 4 days with shaking (final FBS concentration: 1.25%) (seeding density: 3 x ¹⁰ cells/mL, 2 petri dishes, 4 days of culture, 5% _CO, 90 rpm).
(6): Shaking culture was performed for two passages in a medium prepared by mixing 2.5% FBS/DMEM F12 medium and 293SFM II + 4 mM L-Gln at a ratio of 1:3 (final FBS concentration: 0.625%) (seeding density: 3 x ¹⁰⁵ cells/mL, 2 petri dishes, culture period: 4 days, _CO2 5%, 90 rpm).
(7): Shaking culture was performed for two passages in a medium prepared by mixing 2.5% FBS/DMEM F12 medium and 293SFM II + 4 mM L-Gln at a ratio of 1:7 (final FBS concentration: 0.313%) (seeding density: 3 x ¹⁰⁵ cells/mL, 2 petri dishes, culture period: 4 days, _CO2 5%, 90 rpm).
(8): Shaking culture was performed for two passages in a medium prepared by mixing 2.5% FBS/DMEM F12 medium and 293SFM II + 4 mM L-Gln at a ratio of 1:15 (final FBS concentration: 0.156%) (seeding density: 3 x ¹⁰⁵ cells/mL, 2 petri dishes, culture period: 4 days, _CO2 5%, 90 rpm).
(9): Shaking culture was performed for 3 passages in 293SFM II + 4 mM L-Gln (final FBS concentration: 0%) (seeding density: 3 x 10 ⁵ cells/mL, 2 petri dishes, culture period: 4 days, CO ₂ 5%, 90 rpm).
(10): A cell bank was prepared.

<Virus preparation method>
1: Reagents used (1) Fetal Bovine Serum (FBS, Cytiva)
Minimum Essential Medium Eagle (hereinafter referred to as MEM, Sigma-Aldrich)
Minimum Essential Medium (autoclavable) (Thermo Fisher Scientific Co., Ltd.)
Sodium bicarbonate (FUJIFILM Wako Pure Chemical Corporation)
Neutral Red (FUJIFILM Wako Pure Chemical Corporation)
Agarose (Becton, Dickinson and Company)
Antibacterial agent (penicillin-streptomycin solution) (FUJIFILM Wako Pure Chemical Corporation)
PBS(-) (FUJIFILM Wako Pure Chemical Corporation)
0.05% trypsin (Thermo Fisher Scientific Co., Ltd.)

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

3. Implementation method
3.1 Passaging method (1) The host cells in the frozen storage are thawed in an incubator set at about 37° C., and then 10 mL of MEM containing 10% FBS is placed in a 15 mL centrifuge tube, and 1 mL of the thawed cell liquid is added and suspended.
(2) The mixture is centrifuged (1000 rpm, 190×g, 24° C.) for 5 minutes using a centrifuge (AX-310, Tommy Seiko Co., Ltd.) to remove the supernatant.
(3) Add 1 mL of 10% FBS-containing MEM to a 15 mL centrifuge tube and resuspend the cells. Add 0.5 mL of the cell suspension to a 25 ^cm2 culture flask containing 5 mL of 10% FBS-containing MEM, and culture for several days in a _CO2 incubator (temperature setting: 37°C, _CO2 concentration: 5%, model: MCO-170AICUV-PJ, Panasonic Healthcare Co., Ltd.).
(4) Confirm that the cells are in the form of a monolayer sheet on the bottom of a 25 ^cm2 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 collected solution to a 15 mL centrifuge tube.
(5) The recovery liquid is mixed with an equal volume of MEM containing 10% FBS, and centrifuged for 5 minutes (1000 rpm, 190×g, 24° C.) to remove the supernatant.
(6) Add 1 mL of 10% FBS-containing MEM to a 15 mL centrifuge tube and resuspend. Add 0.1 mL of the suspension to a 25 ^cm2 culture flask containing 5 mL of 10% FBS-containing MEM, and culture in a _CO2 incubator for several days.
(7) Once the cells have formed into a monolayer sheet, steps (4) to (6) are repeated to subculture the cells.

3.2 Method for preparing a 75 cm2 culture flask ⁽ 1) After subculture, remove the medium from the host cells that have formed a monolayer sheet at the bottom of the 25 ^cm2 culture flask. Wash the cells twice with PBS, then add 1 mL of 0.05% trypsin per 25 ^cm2 culture flask to detach the cells, and transfer the recovered solution to a 15 mL centrifuge tube.
(2) The recovery liquid is mixed with an equal volume of MEM containing 10% FBS, and centrifuged for 5 minutes (1000 rpm, 190×g, 24° C.) to remove the supernatant.
(3) Add 1 mL of 10% FBS-containing MEM to a 15 mL centrifuge tube and resuspend. Place the entire resuspended volume in a sterilized medium bottle, add 10% FBS-containing MEM, and inoculate 15 mL of each into 75 ^cm2 culture flasks and culture in a _CO2 incubator.
(4) Confirm that the host cells are in the form of a monolayer sheet on the bottom of a 75 ^cm2 culture flask, and use this for purifying the inoculated virus solution.

3.3 Method for preparing a 6-well plate (1) After subculture, the medium of the host cells that have formed a monolayer sheet at the bottom of a 25 ^cm2 culture flask is removed, and the cells are washed twice with PBS. 1 mL of 0.05% trypsin is added to each 25 ^cm2 culture flask to detach the cells, and the collected solution is transferred to a 15 mL centrifuge tube.
(2) The recovery liquid is mixed with an equal volume of MEM containing 10% FBS, and centrifuged for 5 minutes (1000 rpm, 190×g, 24° C.) to remove the supernatant.
(3) Add 1 mL of 10% FBS-containing MEM to a 15 mL centrifuge tube and resuspend the cells. Add culture medium so that the cell count is 0.15 x 10 ⁶ cells/mL, and seed 2 mL of the mixture into a 6-well plate and culture in a CO ₂ incubator.
(4) Confirm that the host cells have formed a monolayer sheet on the bottom of the plate, and use it to measure the concentration of the inoculated virus solution and the amount of virus.

3.4 Purification method of inoculated virus solution Frozen virus is thawed, appropriately diluted with MEM, and added to a 75 ^cm2 culture flask in which HEK-293 cells have been cultured, and the virus is allowed to adsorb to the cells for 1 hour in a _CO2 incubator.
Thereafter, the virus solution is removed, MEM is added, and the cells are cultured in a CO ₂ incubator. The culture is continued until the appearance of cytopathic effect (CPE), and the supernatant containing the virus solution and the cells are harvested.
After collection, freeze-thaw three times (suspension times: 5 or more) and centrifuge for 5 minutes (2000 rpm, 780 x g, 4°C), and the supernatant is used as the inoculum virus solution. The inoculum virus solution is dispensed into 1 mL portions into ceramic tubes and frozen (-80°C) in an ultra-low temperature freezer until use.

3.5 Measurement of inoculated virus concentration: Serial dilutions (10 to 10 ^6- fold dilutions) of the inoculated virus solution were prepared with PBS. 0.2 mL of each virus dilution was added to each well of a 6-well plate in which HEK-293 cells were cultured, and the 6-well plate was shaken every 15 minutes to allow the virus to adsorb to the cells for 1 hour.
After 1 hour, mix equal amounts of medium A and medium B and quickly add 2 mL each to the petri dish where the adsorption was completed. Leave at room temperature until the agarose solidifies, culture in a _CO2 incubator for several days, overlay 2 mL of medium (medium A + medium B) containing neutral red, culture overnight in a _CO2 incubator, and then count the number of plaques using a handy colony counter (As One Corporation). The number of samples for each concentration is 3.
Medium A: 10xMEM 9.5 mL, 7.5% sodium bicarbonate 3 mL, FBS 5 mL, antibiotic 0.5 mL, sterile distilled water 32 mL
Medium B: 0.8 g agarose, 50 mL distilled water

4. Virus production experiment using medium 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 (developed by Ajinomoto Co., Inc.)
(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.

4.1.2. Virus strains and host cells used Cell line: 1. Suspension-type HEK293 prepared in 2. sus (ATCC: CRL-1573.3)
Virus strain: Human adenovirus 32 (ATCC VR-625 ^™ ) prepared in 2.

4.1.3. Experimental method (1) Cryopreserved cells were thawed in a thermostatic bath set at approximately 37° C., and then 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 mixture is centrifuged (1000 rpm, 190×g, 24° C.) for 5 minutes using a centrifuge (AX-310, Tommy Seiko Co., Ltd.) to remove the supernatant.
(3) Add 1 mL of subculture medium to a 15 mL centrifuge tube and resuspend the cells, then transfer the entire amount to a 50 mL tube and add 29 mL of glutamine-containing 293 SFM II to suspend the cells. Place 5 mL of the cell suspension in a petri dish and culture with shaking (50 rpm) in a _CO2 incubator (set temperature: 37°C, _CO2 concentration: 5%, model: MCO-170AICUV-PJ, Panasonic Healthcare Co., Ltd.) for 2 to 3 days.
(4) After cultivation, the culture medium is collected and transferred 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 a 15 mL centrifuge tube and resuspend the cells. Count the cell count with a hemocytometer and add culture medium to make the cell count 3 x 10 ⁵ cells/mL. Add 5 mL of the adjusted cell suspension to a petri dish and culture with shaking (50 rpm) in a CO ₂ incubator for 3 to 4 days.
(7) After incubation, (4) to (6) are repeated once.
(8) After culturing in a _CO2 incubator for 3 to 4 days, the culture medium is collected and transferred 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 a 15 mL centrifuge tube and resuspend the cells. Count the number of cells using a hemocytometer and add new culture medium to make the cell count 3 x 10 ⁵ cells/mL. Add 5 mL of the adjusted cell suspension to two petri dishes and culture with shaking in a CO ₂ incubator for 3 to 4 days.
(11) After incubation, (8) to (10) are repeated three times.
(12) After culturing, count the number of cells, adjust the culture medium for each group to 5 x ¹⁰ cells/mL, and add 5 mL per petri dish. Thaw the frozen inoculation virus liquid, and add the virus liquid to the petri dish at an MOI of 10 (see Table 1).

The concentrations of each component in the culture medium were adjusted as shown in Table 2.

(13) Culture with shaking in a _CO2 incubator for 2 days.
(14) After culturing, the culture medium is recovered from the petri dish, freeze-thawed three times (number of suspensions: 5 or more times), centrifuged (2000 rpm, 190 x g, 4°C, 10 minutes), and the supernatant is collected.
(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 later using the method described in 3.5. The results are shown in Figure 1.

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 strains and host cells used were prepared in the same manner as in 4.1.2.
4.2.3. Experimental method The experiment was carried out in the same manner as in 4.1.3 (1) to (11).
(12) After culturing, count the number of cells, adjust the culture medium for each group to 5 x ¹⁰ cells/mL, and add 5 mL per petri dish. Thaw the frozen inoculation virus solution, and add the virus solution to the petri dish at an MOI of 10 (see Table 3).

The concentrations of each component were adjusted as shown in Table 4.

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

4.3 Conditions in which L-aspartic acid and glutathione (reduced form) were added to other media
4.3.1. Reagents used (1) 293 SFM II (Thermo Fisher Scientific Co., Ltd.)
(2) L-aspartic acid (3) glutathione (reduced form)
*293 SFM II was supplemented with L-Glutamine to a final concentration of 4 mM.
4.3.2. Virus strains and host cells used were prepared in the same manner as in 4.1.2.
4.3.3. Experimental method The experiment was carried out in the same manner as in 4.1.3 (1) to (11).
(12) After culturing, count the number of cells, adjust the culture medium for each group to 5 x ¹⁰ cells/mL, and add 5 mL per petri dish. Thaw the frozen inoculation virus solution, and add the virus solution to the petri dish at an MOI of 10 (see Table 5).

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

5. Virus production experiment using culture medium and AJI Supplement
5.1 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 (developed by Ajinomoto Co., Inc.)
(5) PBH-05 (developed by Ajinomoto Co., Inc.)
(6) As the AJI Supplement, Supplement V0 (developed by Ajinomoto Co., Inc.) was used.
*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 were prepared in the same manner as in 4.1.2.
5.1.3. Experimental method The experiment was carried out in the same manner as in 4.1.3 (1) to (9).
(10) Add 1 mL of culture medium (see Table 6) for each group to a 15 mL centrifuge tube and resuspend. Count the cell count with a hemocytometer and add new culture medium to make the cell count 3 x 10 ⁵ cells/mL. Add 5 mL of the adjusted cell suspension to two petri dishes and culture with shaking in a CO ₂ incubator for 3 to 4 days.
(11) After incubation, (8) to (10) are repeated three times.
(12) After 3 days of culture, the cell number is counted (Figure 4), and the culture medium for each group is adjusted to 1 x ¹⁰⁵ cells/mL, and 5 mL is added per petri dish. The frozen virus inoculation solution is thawed, and for 1 x ¹⁰⁵ cells/mL, the virus solution is added to the petri dish so that the MOI is 0.1 (see Table 6). The concentrations of each component in each group are as shown in Tables 7 and 8.

For groups 1 and 2, a commercially available medium was used, and for group 3, a commercially available medium supplemented with Supplement V0 was used. For groups 4 to 7, the concentrations of each component were adjusted to the concentrations listed in Table 7.

Table 8 shows the concentration of each component in Table 7 converted to functional molecules only.

(13) Culture with shaking in a _CO2 incubator for 6 days at MOI 0.1.
(14) After culturing, the culture medium is recovered from the petri dish, freeze-thawed three times (number of suspensions: 5 or more times), centrifuged (2000 rpm, 190 x g, 4°C, 10 minutes), and the supernatant is collected.
(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 later by the method described in 3.5. The results are shown in Figure 5.

5.2 High cell density and virus concentration conditions
5.2.1. Reagents used: Prepared in the same manner as in 5.1.1.
5.2.2. Virus strains and host cells used were prepared in the same manner as in 4.1.2.
5.2.3. Experimental method The experiment was carried out 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 add culture medium for each group so that the cell count is 3 x 10 ⁵ cells/mL. Add 5 mL of the adjusted cell suspension to two petri dishes and culture with shaking in a CO ₂ incubator for 3 to 4 days.
(11) After incubation, (8) to (10) are repeated three times.
(12) After culturing, the number of cells is counted (FIG. 6), and the culture medium for each group is adjusted to 5×10 ⁵ cells/mL, and 5 mL is added per petri dish. The frozen virus inoculation solution is thawed, and for 5×10 ⁵ cells/mL, the virus solution is added to the petri dish so that the MOI is 10 (see Table 9). The concentrations of each component in each group are as shown in Tables 7 and 8.

(13) Culture with shaking in a _CO2 incubator for 2 days at MOI 10.
(14) After culturing, the culture medium is recovered from the petri dish, freeze-thawed three times (number of suspensions: 5 or more times), centrifuged (2000 rpm, 190 x g, 4 C, 10 minutes), and the supernatant is collected.
(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 later by the method described in 3.5. The results are shown in Figure 7.

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

Claims (18)

A medium additive for use in culturing virus-producing cells, comprising at least one component 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 to 790 mg/L
(10) Glutathione: 0.5 to 5 mg/L. The agent according to claim 1, which contains L-aspartic acid and glutathione, and when added to a 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. 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 medium are within the following ranges:
(1) Potassium: 8.65-86.5 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: 0.618-6.18mg/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: 17.54-175.4 mg/L. 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 medium are within the following ranges:
(1) Potassium: 8.65-86.5 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: 0.618-6.18mg/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: 17.54-175.4 mg/L. 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. A medium composition comprising a basal medium to which the medium additive according to any one of claims 1 to 5 has been added. 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 concentration of each component in the medium composition being within the following ranges:
(4) L-aspartic acid: 250 to 1,000 mg/L
(10) Glutathione: 1.5 to 6 mg/L. The medium composition according to claim 7, which contains L-aspartic acid and glutathione, and the concentrations of each component in the medium composition are within the following ranges:
(4) L-aspartic acid: 250 to 1,000 mg/L
(10) Glutathione: 1.5 to 6 mg/L. 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, and the concentration of each component in the medium composition is within the following range:
(1) Potassium: 290-772 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: 3-59.6mg/L
(7) Riboflavin: 0.7 to 4.8 mg/L
(8) Ethanolamine: 10.2 to 102 mg/L
(9) Buffer: 90 to 8,800 mg/L
(11) Glutamine source: 17.54 to 1169 mg/L
(12) Copper: 0.002 to 0.024 mg/L. 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 to 45 mg/L
(3) Iron: 17 to 123 mg/L
(5) L-tryptophan: 48 to 480 mg/L
(6) Folic acid: 3-59.6mg/L
(7) Riboflavin: 0.7 to 4.8 mg/L
(8) Ethanolamine: 10.2 to 102 mg/L
(9) Buffer: 90 to 8,800 mg/L
(11) Glutamine source: 17.54 to 1169 mg/L
(12) Copper: 0.002 to 0.024 mg/L. 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. 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. The method according to 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 buffering agent, and a glutamine source, and the concentration of each component in the medium composition is within the following ranges:
(1) Potassium: 290-772 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: 3-59.6mg/L
(7) Riboflavin: 0.7 to 4.8 mg/L
(8) Ethanolamine: 10.2 to 102 mg/L
(9) Buffer: 90 to 8,800 mg/L
(11) Glutamine source: 17.54 to 1169 mg/L
(12) Copper: 0.002 to 0.024 mg/L. 13. The method of claim 12, wherein the medium composition further comprises potassium, magnesium, iron, L-tryptophan, folic acid, riboflavin, ethanolamine, a buffering agent, and a glutamine source, the concentrations of each component in the medium composition being in the following ranges:
(1) Potassium: 290-772 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: 3-59.6mg/L
(7) Riboflavin: 0.7 to 4.8 mg/L
(8) Ethanolamine: 10.2 to 102 mg/L
(9) Buffer: 90 to 8,800 mg/L
(11) Glutamine source: 17.54 to 1169 mg/L
(12) Copper: 0.002 to 0.024 mg/L. The method according to claim 14, wherein the glutamine source is at least one selected from the group consisting of L-glutamine, alanylglutamine, and glycylglutamine. A method for culturing virus-producing cells, comprising a step of culturing the virus-producing cells in a medium composition according to any one of claims 7 to 11. 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.