KR-20260066112-A - Culture medium composition

Abstract

The present invention provides a culture medium composition for promoting the production of a virus carrying a target gene introduced into a mammalian cell that produces a virus containing β-alanine, a culture medium composition for producing a virus or virus-like particles containing β-alanine, and the like.

Inventors

• 가타야마, 다카히로
• 히구치, 다쿠야
• 노구치, 게이코

Assignees

• 아지노모토 가부시키가이샤

Dates

Publication Date

20260512

Application Date

20240904

Priority Date

20230905

Claims (20)

1. A culture medium composition for promoting the production of a virus loaded with a target gene, in the culture of a mammalian cell that produces a virus into which a target gene, comprising β-alanine, has been introduced.
2. In paragraph 1, A culture medium composition in which the mammalian cells are cells selected from the group consisting of HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells and HeLa cells.
3. In paragraph 1, A culture medium composition in which the mammalian cells are HEK293 cells.
4. In paragraph 1, A culture medium composition in which the virus-producing mammalian cell is an adeno-associated virus-producing mammalian cell.
5. In any one of paragraphs 1 through 4, A culture medium composition in which the concentration of β-alanine in the culture medium composition is 0.1 μM to 800 μM.
6. In any one of paragraphs 1 through 4, A culture medium composition comprising additionally polyamine or its salt.
7. In paragraph 6, A culture medium composition in which a polyamine or its salt is spermidine or its salt.
8. In Paragraph 7, A culture medium composition in which the concentration of spermidine or its salt in the culture medium composition is 0.1 μM to 700 μM.
9. A culture medium additive for promoting the production of a virus carrying a target gene, in the culture of mammalian cells producing a virus into which a target gene has been introduced, comprising β-alanine.
10. Use of β-alanine in the preparation of a medium composition for promoting the production of a virus loaded with a target gene, in the culture of a mammalian cell producing a virus into which a target gene has been introduced.
11. A method for promoting the production of a virus carrying said target gene, comprising the process of culturing a mammalian cell that produces a virus into which a target gene has been introduced in a medium containing β-alanine.
12. A culture medium composition for improving the ratio of viruses carrying said target gene among the total viruses produced during the culture of mammalian cells producing viruses into which a target gene has been introduced, comprising β-alanine.
13. In Paragraph 12, A culture medium composition in which the mammalian cells are cells selected from the group consisting of HEK293 cells, MDCK cells, Vero cells, A549 cells, PerC6 cells and HeLa cells.
14. In Paragraph 12, A culture medium composition in which the mammalian cells are HEK293 cells.
15. In Paragraph 12, A culture medium composition in which the virus-producing mammalian cell is an adeno-associated virus-producing mammalian cell.
16. In any one of paragraphs 12 through 15, A culture medium composition in which the concentration of β-alanine in the culture medium composition is 0.1 μM to 800 μM.
17. In any one of paragraphs 12 through 15, A culture medium composition comprising additionally polyamine or its salt.
18. In Paragraph 17, A culture medium composition in which a polyamine or its salt is spermidine or its salt.
19. In Paragraph 18, A culture medium composition in which the concentration of spermidine or its salt in the culture medium composition is 0.1 μM to 140 μM.
20. A culture medium additive for improving the ratio of viruses carrying the target gene among the total viruses produced during the culture of mammalian cells producing viruses into which the target gene has been introduced, comprising β-alanine.

Description

Culture medium composition The present invention relates to a culture medium composition, etc., for promoting the production of a virus carrying the target gene or increasing the proportion of the virus carrying the target gene among the total viruses produced, in the culture of a cell producing a virus having a target gene introduced, which includes β-alanine, and also for culturing a cell producing a virus or virus-like particle containing β-alanine. To meet the demands for human gene therapy and vaccine manufacturing, rapid and stable production technology for viruses and similar substances is indispensable. In particular, the cultivation of cells that produce viruses (hereinafter also referred to as virus-producing cells) is of extreme importance. When designing and optimizing culture media for virus-producing cells, the composition of the medium becomes a critical issue. The components included in the medium affect cell growth and proliferation. Additionally, in the case of a culture medium for virus-producing cells, the components included in the medium also affect the virus-producing ability within the cell. Furthermore, for example, when producing viruses using cells into which a gene that produces a protein that suppresses cancer has been introduced, the components included in the medium also affect the proportion of viruses carrying the said gene among the total viruses produced. However, satisfactory results are not necessarily obtained with the components known so far, and for example, when producing adeno-associated viruses using cells into which a gene has been introduced, it is stated that the proportion of adeno-associated viruses carrying the said gene among the said adeno-associated viruses is about 5% to 30% (Non-patent Literature 1). Figure 1 shows the results of verifying the effect of β-alanine on the AAV8 genome titer in Example 1. The vertical axis represents the relative AAV8 genome titer when the AAV8 genome titer obtained when AAV8 is produced using a medium without the addition of β-alanine is set to 100%. The horizontal axis, "Viral Production Medium," represents the case where AAV8 is produced using a medium without the addition of β-alanine, and "Viral Production Medium + β-alanine" represents the case where AAV8 is produced using a medium with β-alanine added to a final concentration of 100 μM. Figure 2 shows the results of verifying the effect of β-alanine on the full/empty capsid ratio (full capsid ratio) in Example 1. The vertical axis represents the relative full capsid ratio when the full capsid ratio obtained when AAV8 is produced using a medium without the addition of β-alanine is set to 100%. The horizontal axis, "virus production medium," represents the case where AAV8 is produced using a medium without the addition of β-alanine, and "virus production medium + β-alanine" represents the case where AAV8 is produced using a medium with β-alanine added to a final concentration of 100 μM. Figure 3 shows the results of verifying the effect of β-alanine on the AAV8 genome titer in Example 2. The axis represents the relative AAV8 genome titer when the AAV8 genome titer obtained when AAV8 is produced using a medium without the addition of β-alanine is set to 100%. The horizontal axis “virus production medium” represents the case where AAV8 is produced using a medium without the addition of β-alanine, and “+100μM β-alanine”, “+300μM β-alanine”, “+1mM β-alanine”, “+1.5mM β-alanine”, and “+2mM β-alanine” represent the case where AAV8 is produced using a medium with β-alanine added to final concentrations of 100μM, 300μM, 1mM, 1.5mM, and 2mM, respectively. Figure 4 shows the results of verifying the effect of β-alanine on the number of capsids (number of viruses) in Example 3. The vertical axis represents the relative number of capsids when the number of capsids obtained when AAV8 is produced using a medium without the addition of β-alanine is set to 100%. The horizontal axis “virus production medium” represents the case where AAV8 is produced using a medium without the addition of β-alanine, and “+300μM β-alanine” represents the case where AAV8 is produced using a medium with β-alanine added to a final concentration of 300μM. Figure 5 shows the results of verifying the effect of β-alanine on the full capsid ratio in Example 3. The vertical axis represents the relative full capsid ratio when the full capsid ratio obtained when AAV8 is produced using a medium without the addition of β-alanine is set to 100%. The horizontal axis “virus production medium” represents the case where AAV8 is produced using a medium without the addition of β-alanine, and “+300μM β-alanine” represents the case where AAV8 is produced using a medium with β-alanine added to a final concentration of 300μM. Figure 6 shows the results of verifying the effect of spermidine trihydrochloride on the AAV8 genome titer in Example 4. The vertical axis represents the relative AAV8 genome titer when the AAV8 genome tit