Supplementary material added in virus culture process and use method thereof

Abstract

The invention provides a supplementary material added in a virus culture process and a use method of the supplementary material. The supplementary material comprises the following components: glucose, GluMAX and soybean lecithin. Supplementary materials are added into the culture medium, so that glycometabolism, lipid metabolism, nucleic acid translation and protein synthesis of host cells can be remarkably enhanced; effect pathways of interferon can be influenced, interferon stimulation gene expression is inhibited, and therefore the cell antiviral effect is inhibited. The cell maintenance can be promoted, and the cell apoptosis after virus infection is delayed. In conclusion, the virus yield can be remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of culture medium, and particularly relates to a feed added in the process of virus culture and a use method thereof. BACKGROUND

[0002] In the biomedical field of virology, vaccine development, antiviral drug screening and gene therapy, in vitro culture and efficient amplification of viruses are crucial core technologies. The industry has long been faced with the common challenges of low virus yield, long production cycle and high cost, especially when producing large-scale vaccines of viruses with the risk of pandemic outbreak, such as influenza vaccines. Traditional virus culture media, such as DMEM, MEM, etc., are all general-purpose media designed to meet the basic needs of the growth and maintenance of ordinary mammalian cells. The core of these media is to provide balanced carbon sources (such as glucose), nitrogen sources (such as various amino acids), vitamins, inorganic salts, etc., to support the metabolic pathways of cells in normal physiological conditions, i.e., mainly relying on oxidative phosphorylation (OXPHOS) to efficiently produce energy. However, viruses are strict intracellular parasites, and their life activities completely depend on the metabolic system of host cells. A large number of studies have shown that after viral infection, the host's metabolic network is hijacked, and the host's metabolic mode is changed. The core features of this transformation include: significant enhancement of glycolysis, intensified glutamine consumption, and overall remodeling of lipid metabolism. Therefore, the traditional general-purpose medium designed based on normal cell metabolism cannot provide the best metabolic environment required for virus replication, and instead becomes a key bottleneck that limits virus titer, productivity and stability.

[0003] Lecithin is a natural phospholipid mixture, the main components of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, etc. Its chemical nature is phospholipid, which is the most important constituent material of all biological membrane structures, and is involved in biological processes such as cell membrane synthesis, cell metabolism, virus proliferation and secretion. Commercial lecithin is mainly extracted from soybeans or egg yolks, and soybean lecithin does not contain animal-derived ingredients and has a mature application history in cell culture. In the aspect of virus culture, a large number of documents have proved that enhancing lipid metabolism can significantly promote the proliferation of various viruses, and recently there have also been documents reporting that antiviral drugs developed targeting lipid metabolism targets have good effects. Therefore, lipid metabolism should be one of the focuses of virus medium optimization.

[0004] In the field of virus medium development, existing technologies mainly focus on empirical optimization and basic component supplementation of traditional basal medium, with less attention to lipid metabolism. These improvements have achieved certain results, but have not fundamentally solved the problem of specific metabolic needs of virus replication. The specific limitations are: 1. Simple fortification of nutrients: Current technologies usually indirectly support virus replication by adding high concentrations of glucose, amino acids, serum or serum substitutes to the basal medium. This approach lacks specificity and does not precisely meet the material and energy requirements of virus biosynthesis, especially lipid synthesis.

[0005] 2. Challenges in regulating single metabolic pathways: Some studies have attempted to regulate specific metabolic pathways. For example, some technologies mention adding glycolysis inhibitors (such as 2-DG) or activators to the medium to regulate energy supply. However, these attempts are often point-like regulation, and the complex correlation of metabolic networks often makes the regulation results unstable and even contradictory. In addition, pathway inhibitors are usually chemicals, and safety is a challenge.

[0006] 3. Lack of attention and limitations on lipid metabolism: Although lipids are essential for the replication of enveloped viruses, current medium technologies for lipid supplementation are passive and indirect. The most common way is to rely on lipoproteins, cholesterol and fatty acids extracted from serum. For example, CN103045533A discloses a serum-free medium for culturing cells to produce viruses, which contains lipid carriers (such as cyclodextrin) and a small amount of lipid mixture. However, this method has obvious defects: it needs to completely change the original medium, which poses a challenge to the compliance of the change; the ingredients are complex and the source is uncertain, with large batch-to-batch differences, which seriously affects the stability and repeatability of the production process.

[0007] 4. Usually develop special medium for specific products, high R&D and production cost, narrow applicability.

[0008] In summary, the defects of the prior art are that they do not better adapt to the metabolic state of host cells induced after viral infection, especially ignoring the important role of "lipid metabolism remodeling" in this process; the metabolic environment they provide is far from the optimal environment required for virus replication, resulting in limited virus productivity; and the economy is poor. SUMMARY

[0009] Therefore, the present application provides a feed added during virus culture and a method of using the same, which can significantly improve virus yield, has clear ingredients, does not contain animal-derived ingredients, is convenient to use, and is low in cost.

[0010] To achieve the above-mentioned purposes, the technical scheme adopted by the present application is as follows: On the one hand, the present application provides a feed added during virus culture, which comprises: glucose, GluMAX and soy lecithin.

[0011] On the other hand, the present application provides a method of using the feed, which comprises adding the feed to the medium.

[0012] Preferably, the culture medium comprises DMEM, MEM, F12, VP-SFM or Opti-MEM.

[0013] Preferably, the culture medium can be added with the feed before or after inoculation of the virus.

[0014] Preferably, the feed is added in an amount of 0.1-1% of the mass of the culture medium.

[0015] Preferably, after the feed is added to the culture medium, the final concentration of glucose in the culture medium is 1-5 g / L, the final concentration of GluMAX is 0.1-5 mmol / L, and the final concentration of soybean lecithin is 0.001-1 mmol / L.

[0016] Preferably, the culture medium is used for culturing hepatitis virus, herpes virus, flavivirus, enterovirus, and influenza virus.

[0017] Compared with the prior art, the present application has the following beneficial effects: (1) By adding the feed to the culture medium, the present application can significantly enhance the sugar metabolism, lipid metabolism, nucleic acid translation and protein synthesis of the host cell; can affect the effect pathway of interferon, inhibit the expression of interferon-stimulated genes, thereby inhibiting the antiviral effect of the cell; can promote cell maintenance and delay cell apoptosis after viral infection. In summary, the present application can significantly improve the viral yield.

[0018] (2) The feed of the present application can be applied to the culture of various viruses, such as influenza virus, hepatitis virus, herpes virus, and flavivirus, and can also be applied to common mammalian cells, such as HEK-293T and MDCK. The applicable culture medium includes DMEM, MEM, F12, VP-SFM or Opti-MEM, and the application range is wide.

[0019] (3) The feed of the present application has clear components and does not contain animal-derived components. It is convenient to use and low in cost. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The hemagglutination titer comparison chart provided by Example 1 and Comparative Example 1 of the present application; Figure 2 The total single-cell viral yield comparison chart provided by Example 1 and Comparative Example 1 of the present application; Figure 3 The single expansion comparison chart of the reactor culture harvest liquid ultrafiltration and concentration provided by Example 2 of the present application. DETAILED DESCRIPTION

[0021] The present application will be further described in detail below with reference to specific examples, so that those skilled in the art can more clearly understand the present application.

[0022] The existing culture medium for culturing viruses has the following defects: the commercial general-purpose culture medium cannot well adapt to the change in the metabolic state of the host cell induced after virus infection, especially ignoring the important role of "lipid metabolism remodeling" in this process; the metabolic environment provided by the culture medium is different from the optimal environment required for virus replication, resulting in limited virus production; and the economic efficiency is poor.

[0023] To solve the above technical problems, the present application provides a feed added in the process of virus culture, and the composition comprises glucose, GluMAX and soybean lecithin.

[0024] In another aspect, the present application provides a method for using the feed, which comprises adding the feed to the culture medium.

[0025] Further, the culture medium comprises DMEM, MEM, F12, VP-SFM or Opti-MEM.

[0026] Further, the feed can be added to the culture medium before or after inoculation of the virus.

[0027] Further, the amount of the feed added to the culture medium is 0.1-1% of the mass of the culture medium.

[0028] Further, after the feed is added to the culture medium, the final concentration of glucose in the culture medium is 1-g / L, the final concentration of GluMAX is 0.1-5mmol / L, and the final concentration of soybean lecithin is 0.001-1mmol / L.

[0029] Further, the feed is suitable for various viruses, such as influenza virus, hepatitis virus, herpes virus, flavivirus and enterovirus, and is suitable for common mammalian cells, such as HEK-293T and MDCK.

[0030] Firstly, the present application can significantly enhance the sugar metabolism, lipid metabolism, nucleic acid translation and protein synthesis of the host cell by adding the feed to the culture medium; can affect the effect pathway of interferon, inhibit the expression of interferon-stimulated genes, thereby inhibiting the antiviral effect of the cell; can promote cell maintenance and delay cell apoptosis after virus infection; and can significantly improve the virus yield.

[0031] Secondly, the feed of the present application is suitable for various viruses, such as influenza virus, hepatitis virus, herpes virus, flavivirus and enterovirus, and is suitable for common mammalian cells, such as HEK-293T and MDCK. The culture medium suitable for use includes DMEM, MEM, F12, VP-SFM or Opti-MEM, and the application range is wide.

[0032] Finally, the feed component of the present application is clear and does not contain animal-derived components. It is convenient to use and low in cost.

[0033] Example 1 This example adds a feed to a DMEM serum-free medium for culturing H3N2 influenza virus. The specific steps are as follows: using a T75 culture flask, DMEM serum-free medium mixed with 1% feed is added to the T75 culture flask, wherein after the feed is added to the DMEM serum-free medium, the final concentration of glucose in the medium is 2 g / L, the final concentration of GluMAX is 1 mmol / L, and the final concentration of soybean lecithin is 0.01 mmol / L. Inoculate the T75 culture flask with influenza virus (H1N1, H3N2, H5N1, BV, BY), the MOI of the influenza virus inoculation is 0.001, and the trypsin concentration is 4 μg / mL. The virus culture conditions are 34°C, and the virus culture is completed after 72 h, with a total single-cell virus yield of 9987 virus / cell. The yield of influenza virus cultured in the T75 culture flask is increased by 400%. Table 1 shows the hemagglutination titer, hemagglutinin concentration, and total single-cell virus yield data of the virus after culture in the medium of Example 1.

[0034] Table 1

[0035] Comparative Example 1 This comparative example provides a DMEM serum-free medium for culturing H3N2 influenza virus, and the culture method is the same as that of Example 1, except that no feed is added. The results of Example 1 and Comparative Example 1 are compared, and the results are shown in Table 1. Figures 1-2 . Figure 1 The change trend of the hemagglutination titer of Example 1 and Comparative Example 1 is shown. The virus titer of Example 1 is always higher than that of Comparative Example 1, and the highest hemagglutination titer of Example 1 is 4 times that of Comparative Example 1. Figure 2 The change trend of the total single-cell virus yield of Example 1 and Comparative Example 1 is shown. The total single-cell virus yield of Example 1 is always higher than that of Comparative Example 1, and the total single-cell virus yield of Example 1 is 9987 virus / cell, while that of Comparative Example 1 is 1863 virus / cell. The total single-cell virus yield of Example 1 is 5.36 times that of Comparative Example 1. Figure 2 , wherein A represents Comparative Example 1 and B represents Example 1. Table 2 shows the hemagglutination titer, hemagglutinin concentration, and total single-cell virus yield data of the virus after culture in the medium of Comparative Example 1.

[0036] Table 2

[0037] Comparative Example 2 This comparative example provides a DMEM serum-free medium for culturing H3N2 influenza virus, and the culture method is the same as that of Example 1, except that no soybean lecithin is added. Table 3 shows the hemagglutination titer, hemagglutinin concentration, and total single-cell virus yield data of the virus after culture in the medium of Comparative Example 2.

[0038] Table 3

[0039] Comparative Example 3 This comparative example provides a DMEM serum-free medium for culturing H3N2 influenza virus, and the culture method is the same as that of Example 1, except that no glucose and GluMAX are added. Table 4 shows the hemagglutination titer, hemagglutinin concentration, and total single-cell virus yield data of the virus after culture in the medium of Comparative Example 3.

[0040] Table 4

[0041] Comparative Example 4 This comparative example provides a DMEM serum-free medium for culturing H3N2 influenza virus, and the culture method is the same as that of Example 1, except that the final concentration of GluMAX is 0.1 mmol / L and the final concentration of soybean lecithin is 0.001 mmol / L. Table 5 shows the hemagglutination titer, hemagglutinin concentration, and total single-cell virus yield data of the virus after culture in the medium of Comparative Example 4.

[0042] Table 5

[0043] Example 2 This example uses a 1L four-way reactor to culture H3N2 influenza virus, and #3 and #4 are the culture tanks with added feed, with the addition ratio being 0.5% and 1%, respectively. #1 and #2 have no addition. The virus culture steps are to inject 1L of DMEM serum-free medium mixed with the feed into the reactor, inoculate the influenza virus at an MOI of 0.001, and add trypsin at a concentration of 4 μg / mL. The virus culture conditions are 34°C, pH setting at 7.2, and the virus culture is completed after 72 h. As shown in Table 6, the single dilution values of #3 and #4 after ultrafiltration and concentration (20 times) are 203 and 237 μg / mL, respectively, and the influenza virus yield in the 1L reactor is increased by 179.5%. Figure 3

[0044] Table 6

[0045] Example 3 ​The comparative example 2 provides a VP medium for culturing H1N1 influenza virus, and the culturing method is the same as that of example 1. Table 8 is the data of hemagglutination titer, hemagglutinin concentration and total single-cell virus yield of the virus after culturing with the medium of example 4.

[0046] Table 7

[0047] Example 4 The comparative example 2 provides a VP medium for culturing H1N1 influenza virus, and the culturing method is the same as that of example 1. Table 8 is the data of hemagglutination titer, hemagglutinin concentration and total single-cell virus yield of the virus after culturing with the medium of example 4.

[0048] Table 8

[0049] The specific raw materials in the present application are all existing substances, which can be directly purchased from the market.

[0050] The above only is the preferred embodiment of the present application, and is not used to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A feed additive for use in virus culture, characterized in that, include: Glucose, GluMAX, and soy lecithin.

2. The method of using the replenishing agent according to claim 1, characterized in that, Add feed to the culture medium.

3. The method of using the replenishing agent according to claim 2, characterized in that, The culture medium includes: DMEM, MEM, F12, VP-SFM or Opti-MEM.

4. The method of using the replenishing agent according to claim 2, characterized in that, The culture medium can be replenished either after or before virus inoculation.

5. The method of using the replenishing agent according to claim 2, characterized in that, The amount of feed added is 0.1-1% of the mass of the culture medium.

6. The method of using the replenishing agent according to claim 2, characterized in that, After the feed is added to the culture medium, the final concentration of glucose in the culture medium is 1-5 g / L, the final concentration of GluMAX is 0.1-5 mmol / L, and the final concentration of soybean lecithin is 0.001-1 mmol / L.

7. The method of using the feeding material according to claim 2, characterized in that, The feed is suitable for culturing influenza virus, hepatitis virus, herpes virus, flavivirus, and enterovirus.

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