Method for producing coxsackie virus by using HEK293 cell

Abstract

The invention belongs to the technical field of biology, and relates to a method for producing CVB1 virus by culturing HEK293 cells in vitro, which comprises the following steps: (1) culturing host cells in a culture medium I which comprises a basic culture medium of L-alanyl-L-glutamine; (2) adding a culture medium II into the cell culture in the step (1), and continuously culturing the host cells; (3) adding a culture medium III into the cell culture in the step (2), inoculating viruses, and continuously culturing the host cells inoculated with the viruses; and (4) obtaining a cell culture containing the target virus. The invention also relates to culture media for the production of CVB1 viruses, and their use for the production of CVB1 viruses.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for producing CVB1 virus by culturing HEK293 cells in vitro. This invention also relates to culture media for producing CVB1 virus, and their use in producing CVB1 virus. Background Technology

[0002] Coxsackieviruses (CVs) are classified into two groups, A and B, based on their genotypes. Group A contains 23 serotypes, while group B contains 6 serotypes. Coxsackievirus B1 (CVB1) is a common human pathogen belonging to the enterovirus family. It is a non-enveloped, single-stranded, positive-sense RNA virus. Infants and young children are the most susceptible populations, with a lower infection rate in adults. The main clinical symptoms are common cold-like symptoms such as fever, headache, and diarrhea. Occasionally, severe illnesses such as aseptic meningitis, myocarditis, hepatitis, pancreatitis, and hand-foot-and-mouth disease may occur. Currently, there are no specific drugs for treating enteroviruses, including CVB1. Therefore, developing a CVB1 vaccine is an important measure to prevent neonatal infection and reduce morbidity. It has been confirmed that natural CVB1 can kill various tumor cell lines. Furthermore, due to its small genome, ability to cross the blood-brain barrier, and mild symptoms in adults, recombinant CVB1 obtained through artificial attenuation can be developed into an oncolytic virus drug.

[0003] Developing CVB1 into a vaccine or therapeutic oncolytic virus drug requires the development of large-scale virus preparation methods. For the production process of viral drugs, it is necessary to consider control strategies for various process parameters in both cell culture and virus culture stages, as well as the selection of culture modalities, to ensure process stability, good reproducibility, and ultimately maximize virus yield to meet the requirements of commercial scale-up production.

[0004] Currently, the vast majority of toxin production processes for enteroviruses are based on adherent cells prepared in serum-containing culture media, with very few employing suspension cell culture and toxin production processes: for example, Chen Guojun et al. [1] Laboratory-grade CVB1 virus was prepared by adhering Vero cells to serum-containing RPMI-1640 medium; Yang Ting et al. [2]KMB17 cells were cultured in a cell factory using serum-containing medium to produce CVA16. CN112641798A reported that Vero cells were first cultured in DMEM containing 10% FBS in culture flasks to adhere to the cells. After reaching 90% confluence, the medium was replaced with serum-free DMEM, followed by inoculation with CVB3 virus for toxin production. While adherence technology provides good support for cell growth, subsequent scale-up is limited and requires high labor costs. Other reports have described the use of microcarriers or sheet carriers for cell culture to produce enteroviruses; however, the success rate of cell transfer between carriers during scale-up becomes a key factor affecting process stability. Therefore, current adherence and microcarrier processes are gradually evolving towards suspension processes, which offer greater scale-up convenience. Furthermore, both adherence and microcarrier culture processes typically require the addition of newborn calf or fetal bovine serum to the cell culture medium to promote cell growth and toxin production. However, introducing serum increases the cost of removing HCP, exogenous factors, mycoplasma, or non-product viruses during subsequent virus purification, and adds additional quality control risks. Therefore, more and more processes are shifting towards serum-free media or chemically defined media with clearly defined components. Currently, several domestic and international companies have developed commercially available serum-free media for various cell types, which can effectively meet the culture needs of some production cell lines for antibody production or virus preparation. Based on the Quality by Design (QbD) principle, using chemically defined media with clearly defined components in cell suspension culture and virus production can significantly improve product quality from the source, enhance batch-to-batch stability, reduce exogenous contamination, alleviate purification pressure, and facilitate large-scale commercialization.

[0005] Because CVB1 has the property of causing cell lysis during production, continuous culture is not very suitable; only basic batch or fed-batch culture modes are suitable. To increase the yield of a single batch of virus, cells are usually inoculated and produced at high densities. However, high cell density rarely results in a correspondingly high viral yield; often, inoculation after increasing cell density leads to a decrease in viral yield, a phenomenon known as the "cell density effect" (CDE). Numerous studies have shown that cell densities exceeding (1-5) × 10⁻⁶ at inoculation have been found to be ineffective. 6 A concentration of 1 cell / ml significantly reduces the level of toxin production per single cell; Kamen et al. [3] In adenovirus production, the density of inoculated cells was found to be 2 × 10⁻⁶. 6 The single-cell toxicity level is only 5 × 10⁶ cells / ml, which is equivalent to a cell density of 5 × 10⁶ cells / ml. 5The cell density effect occurs at 10% of the target cell density per ml. This effect is due to several factors. First, the cell culture environment and available energy resources limit cell growth. High-density cell growth leads to insufficient oxygen supply and the accumulation of metabolic byproducts such as lactic acid and ammonia in the culture medium, significantly impacting cell growth and hindering high-density cell growth or causing a decrease in viral titer. Second, the timing and conditions of viral inoculation also play a role. Therefore, to achieve higher viral titers, it is necessary to maximize cell density within a specific culture system while simultaneously addressing the cell density effect in virus production. This necessitates a highly efficient method for preparing Coxsackievirus CVB1 to overcome these technical challenges. Summary of the Invention

[0006] Through extensive experimental research, the inventors of this application discovered a culture medium for producing CVB1 virus and, based on this culture medium, provided a method for in vitro culture of HEK293 cells to produce CVB1 virus. This method can obtain high-density cells while maintaining high-titer virus production, significantly improving the production efficiency of CVB1 virus. This invention is thus provided.

[0007] On one hand, the present invention provides a method for producing a target virus by culturing cells in vitro, comprising:

[0008] (1) Culture host cells in culture medium I, wherein culture medium I is a basal culture medium containing L-alanyl-L-glutamine;

[0009] (2) Add culture medium II to the cell culture from step (1) and continue culturing the host cells, wherein the culture medium II contains the following components:

[0010] Arginine, cysteine, histidine, isoleucine, leucine, lysine, valine, tyrosine, methionine, phenylalanine, threonine, tryptophan, choline chloride, folic acid, methionine, inositol, nicotinamide, D-calcium pantothenate, pyridoxal, vitamin B2, vitamin B1, thiamine, vitamin B12, L-alanyl-L-glutamine, Cell Boost™ 5, sodium pyruvate, sodium dichloroacetate, NaCl, KCl, KH2PO4 and Na2HPO4;

[0011] (3) Add culture medium III to the cell culture from step (2), inoculate with the virus, and continue culturing the virus-inoculated host cells, wherein the culture medium III contains the following components:

[0012] Arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, tyrosine, choline chloride, folic acid, inositol, nicotinamide, D-calcium pantothenate, pyridoxal, vitamin B2, thiamine, vitamin B12, L-alanyl-L-glutamine, Cell Boost™ 5, sodium pyruvate, sodium dichloroacetate, NaCl, KCl, KH2PO4 and Na2HPO4;

[0013] (4) Obtain cell cultures containing the target virus.

[0014] In some exemplary implementations, Cell Boost™ 5 is selected from any one or more of the following:

[0015] HyClone Cell Boost 5 liquid supplement with HyClone product numbers SH31117.01, SH31117.02, SH31117.03, SH31117.04, SH31117.05, SH31117.06, SH31117.07, SH31117.08, and SH31117.09, and HyClone Cell Boost 5 dry powder supplement with HyClone product numbers SH30865.01, SH30865.02, SH30865.03, and SH30865.04.

[0016] In some implementations, step (1) includes:

[0017] The host cells were cultured in culture medium I until the host cell density reached (1-2) × 10⁻⁶. 6 Cells / ml

[0018] In some embodiments, the concentration of L-alanyl-L-glutamine in the culture medium I is 1.8-2.2 mM.

[0019] In some embodiments, the basal culture medium is selected from HEK293 CD medium, Optipro® 293CD medium, Bepanthen® S001 medium, and any combination thereof.

[0020] In some embodiments, the culture medium I is obtained by the following method:

[0021] L-alanyl-L-glutamine was added to the basal medium at a final concentration of 1.8-2.2 mM. The basal medium was selected from HEK293 CD medium, Optipro® 293CD medium, Bepanthen® S001 medium and any combination thereof.

[0022] In some embodiments, the culture medium I is HEK293 CD medium containing L-alanyl-L-glutamine, wherein the concentration of L-alanyl-L-glutamine in the culture medium I is 1.8-2.2 mM (e.g., 2 mM).

[0023] In some embodiments, the culture is carried out at a culture temperature of 37.0℃ ± 1.0℃ and a pH of 6.8-7.2 in culture medium I.

[0024] In some embodiments, the dissolved oxygen content of the culture medium I is 30-70% air saturation.

[0025] As used in this article, the term "% air saturation" refers to the ratio of the dissolved oxygen value in a liquid to the maximum theoretical dissolved oxygen value (100% saturation) of that liquid under the same temperature, pressure, and salinity conditions.

[0026] In some implementations, the aeration rate is 120-180 ccm during the culture process.

[0027] In some implementations, step (2) includes:

[0028] In step (1), add culture medium II to the cell culture and continue culturing the host cells until the host cell density reaches (2-4) × 10⁻⁶. 6 Cells / ml

[0029] In some embodiments, the culture medium II comprises the following components:

[0030] Arginine, cysteine, histidine, isoleucine, leucine, lysine, valine, tyrosine, phenylalanine, threonine, tryptophan, choline chloride, folic acid, methionine, KCl, inositol, nicotinamide, D-calcium pantothenate, pyridoxal, vitamin B2, vitamin B1, KH2PO4, thiamine, L-alanyl-L-glutamine, Cell Boost™ 5, sodium pyruvate, sodium dichloroacetate, NaCl, Na2HPO4, and water.

[0031] In some embodiments, the concentrations of arginine, cysteine, histidine, isoleucine, leucine, lysine, valine, tyrosine, methionine, phenylalanine, threonine, and nicotinamide in culture medium II are 2-4 g / L, 0.5-1 g / L, 0.75-1.5 g / L, 0.25-0.5 g / L, 0.6-1 g / L, 0.9-1.5 g / L, 0.2-0.3 g / L, choline chloride, folic acid, inositol, and nicotinamide, respectively. The concentrations of D-calcium pantothenate (15-30 mg / L), pyridoxal (15-30 mg / L), vitamin B2 (1-3 mg / L), vitamin B1 (15-40 mg / L), thiamine (15-30 mg / L), L-alanyl-L-glutamine (150-250 mM), CellBoost™ 5 (15-35 g / L), sodium pyruvate (5-15 g / L), sodium dichloroacetate (50-100 g / L), NaCl (1-5 g / L), KCl (25-75 mg / L), KH2PO4 (25-75 mg / L), and Na2HPO4 (250-350 mg / L) are as follows: mg / L.

[0032] In some embodiments, the culture medium II comprises the following components:

[0033] Arginine at a concentration of 2-4 g / L, cysteine ​​at a concentration of 0.5-1 g / L, histidine at a concentration of 0.75-1.5 g / L, isoleucine at a concentration of 1-2 g / L, leucine at a concentration of 1-2 g / L, lysine at a concentration of 1-2.5 g / L, valine at a concentration of 1-2 g / L, tyrosine at a concentration of 0.7-1.2 g / L, methionine at a concentration of 0.25-0.5 g / L, phenylalanine at a concentration of 0.6-1 g / L, threonine at a concentration of 0.9-1.5 g / L, tryptophan at a concentration of 0.2-0.3 g / L, choline chloride at a concentration of 15-30 mg / L, folic acid at a concentration of 15-30 mg / L, inositol at a concentration of 25-75 mg / L, nicotinamide at a concentration of 15-30 mg / L, and other amino acids at a concentration of 15-30 mg / L. The following substances are present: D-calcium pantothenate (mg / L), pyridoxal (15-30 mg / L), vitamin B2 (1-3 mg / L), vitamin B1 (15-40 mg / L), thiamine (15-30 mg / L), L-alanyl-L-glutamine (150-250 mM), Cell Boost™ 5 (15-35 g / L), sodium pyruvate (5-15 g / L), sodium dichloroacetate (50-100 g / L), NaCl (1-5 g / L), KCl (25-75 mg / L), KH2PO4 (25-75 mg / L), Na2HPO4 (250-350 mg / L), and the balance being water.

[0034] In some exemplary embodiments, the culture medium II comprises the following components:

[0035] The concentrations of the following compounds are listed: arginine (3.16 g / L), cysteine ​​(0.8 g / L), histidine (1.05 g / L), isoleucine (1.31 g / L), leucine (1.31 g / L), lysine (1.81 g / L), valine (1.17 g / L), tyrosine (0.9 g / L), methionine (377 mg / L), phenylalanine (0.83 g / L), threonine (1.2 g / L), tryptophan (250 mg / L), choline chloride (25 mg / L), folic acid (25 mg / L), inositol (50 mg / L), nicotinamide (25 mg / L), D-calcium pantothenate (25 mg / L), pyridoxal (25 mg / L), and 2.5 mg / L... Vitamin B2 at a concentration of 25 mg / L, Vitamin B1 at a concentration of 25 mg / L, Thiamine at a concentration of 200 mM, L-alanyl-L-glutamine at a concentration of 20 g / L, Cell Boost™ 5 at a concentration of 20 g / L, Sodium pyruvate at a concentration of 11 g / L, Sodium dichloroacetate at a concentration of 75 g / L, NaCl at a concentration of 2 g / L, KCl at a concentration of 50 mg / L, KH2PO4 at a concentration of 50 mg / L, Na2HPO4 at a concentration of 287 mg / L, and the balance being water.

[0036] In some embodiments, the volume ratio of the culture medium II to the cell culture of step (1) is 1:80 to 1:200.

[0037] In some embodiments, the culture is carried out at a culture temperature of 37.0℃ ± 1.0℃.

[0038] In some implementations, the dissolved oxygen level of the cell culture is 30-70% air saturation during the culture process.

[0039] In some implementations, the aeration rate is 120-180 ccm during the culture process.

[0040] In some implementations, step (2) further includes: when the cell density of the host cells reaches (2-4) × 10 6 After reaching a cell density of 100 cells / ml, the cells were perfused using medium I for 4 days. The perfusion rates for days 1-4 were 0.4-0.6 V / day, 0.65-0.85 V / day, 1.15-1.35 V / day, and 1.9-2.1 V / day, respectively, where V represents the total volume of the cell culture.

[0041] In some embodiments, the perfusion culture is carried out at a culture temperature of 37.0℃ ± 1.0℃.

[0042] In some implementations, the dissolved oxygen level of the cell culture is 30-70% air saturation during perfusion culture.

[0043] In some implementations, the aeration rate is 120-180 ccm during perfusion culture.

[0044] In some implementations, step (3) includes:

[0045] Add culture medium III to the cell culture in step (2), inoculate with the virus at an MOI of 0.01-0.05, and continue to culture the host cells after inoculation with the virus.

[0046] In some embodiments, the culture medium III comprises the following components:

[0047] Arginine, cysteine, histidine, isoleucine, lysine, phenylalanine, threonine, tryptophan, leucine, inositol, nicotinamide, valine, tyrosine, thiamine, L-alanyl-L-glutamine, Cell Boost™ 5, methionine, and water.

[0048] In some embodiments, in culture medium III, the concentrations of arginine (2-4 g / L), cysteine ​​(0.5-1 g / L), histidine (0.75-1.5 g / L), isoleucine (1-2 g / L), leucine (1-2 g / L), lysine (1-2.5 g / L), methionine (0.25-0.5 g / L), phenylalanine (0.6-1 g / L), threonine (0.9-1.5 g / L), tryptophan (0.2-0.3 g / L), inositol (25-75 mg / L), nicotinamide (15-30 mg / L), valine (1-2 g / L), tyrosine (0.7-1.2 g / L), and thiamine (15-30 mg / L) are respectively. The concentration of L-alanyl-L-glutamine is 150-250 mM, and the concentration of Cell Boost™ 5 is 15-35 g / L.

[0049] In some embodiments, the culture medium III comprises the following components:

[0050] Arginine at a concentration of 2-4 g / L, cysteine ​​at a concentration of 0.5-1 g / L, histidine at a concentration of 0.75-1.5 g / L, isoleucine at a concentration of 1-2 g / L, leucine at a concentration of 1-2 g / L, lysine at a concentration of 1-2.5 g / L, methionine at a concentration of 0.25-0.5 g / L, phenylalanine at a concentration of 0.6-1 g / L, threonine at a concentration of 0.9-1.5 g / L, tryptophan at a concentration of 0.2-0.3 g / L, inositol at a concentration of 25-75 mg / L, nicotinamide at a concentration of 15-30 mg / L, valine at a concentration of 1-2 g / L, tyrosine at a concentration of 0.7-1.2 g / L, and lysine at a concentration of 15-30 mg / L. Thiamine at a concentration of 150-250 mM, L-alanyl-L-glutamine at a concentration of 15-35 g / L, and the balance being water.

[0051] In some exemplary embodiments, the culture medium III comprises the following components:

[0052] Arginine at a concentration of 3.16 g / L, cysteine ​​at a concentration of 0.8 g / L, histidine at a concentration of 1.05 g / L, isoleucine at a concentration of 1.31 g / L, leucine at a concentration of 1.31 g / L, lysine at a concentration of 1.81 g / L, methionine at a concentration of 377 mg / L, phenylalanine at a concentration of 0.83 g / L, threonine at a concentration of 1.2 g / L, tryptophan at a concentration of 250 mg / L, inositol at a concentration of 50 mg / L, nicotinamide at a concentration of 25 mg / L, valine at a concentration of 1.17 g / L, tyrosine at a concentration of 0.9 g / L, thiamine at a concentration of 25 mg / L, L-alanyl-L-glutamine at a concentration of 200 mM, Cell Boost™ 5 at a concentration of 20 g / L, and the balance being water.

[0053] In some embodiments, the volume ratio of culture medium III to the cell culture of step (2) is 1:80 to 1:200.

[0054] In some embodiments, the inoculation temperature is 35.0℃±1.0℃.

[0055] In some implementations, in step (3), the culture temperature is 35.0℃±1.0℃ and the culture time is 24-48h.

[0056] In some implementations, the dissolved oxygen level of the cell culture is 30-70% air saturation during the culture process.

[0057] In some implementations, the aeration rate is 120-180 ccm during the culture process.

[0058] In some implementations, step (4) further includes: separating the cell culture supernatant from the cell culture containing the target virus, and purifying the target virus from the cell culture supernatant.

[0059] In some implementations, the host cell is derived from a mammal.

[0060] In some implementations, the host cell is a HEK293 cell.

[0061] In some implementations, the host cell is a cell that has been acclimated to suspension.

[0062] In some exemplary embodiments, the suspension acclimatization includes the following steps:

[0063] (i) The cells were separated into single cells and cultured as single-cell clones;

[0064] (ii) Select single-cell clones with the highest proliferation rate in the top 30% and perform continuous passage culture;

[0065] (iii) The culture medium used for the first subculture is a basal medium containing 10%-20% serum. During the subculture process, the basal medium containing serum is used in a gradient decreasing manner as the number of subcultures increases, until a basal medium without serum is used.

[0066] (iv) Select single-cell clones with a cell viability greater than 90% in a serum-free basal culture medium.

[0067] In some exemplary embodiments, step (iii) includes sequentially passage the single-cell clone obtained in step (ii) using a basal medium containing serum concentrations of 15%, 10%, 5%, and 0%.

[0068] In some exemplary embodiments, step (iv) further includes inoculating the obtained single-cell clones with a cell viability greater than 90% with the virus, continuing to culture the cells for 24 to 48 hours until the virus is harvested, and selecting the single-cell clones with the highest virus yield.

[0069] In some embodiments, the basal culture medium is selected from QuaMono Plus CHO monoclonal medium, DMEM, OptiPRO SFM, HEK293 CD medium, or any combination thereof.

[0070] In some implementations, the virus is Coxsackievirus.

[0071] In some implementations, the virus is Coxsackievirus B1 (CVB1).

[0072] On the other hand, the present invention provides a culture medium, which is culture medium II or culture medium III as defined above.

[0073] On the other hand, the present invention provides a kit comprising any two or all three of culture media I, culture media II, and culture media III, wherein culture media I, culture media II, and culture media III are as defined above.

[0074] On the other hand, the present invention provides the use of the culture medium or kit of the present invention for preparing viruses in host cells.

[0075] In some implementations, the host cell is derived from a mammal.

[0076] In some implementations, the host cell is a HEK293 cell.

[0077] In some implementations, the virus is Coxsackievirus.

[0078] In some implementations, the virus is Coxsackievirus B1 (CVB1).

[0079] Beneficial effects of the invention

[0080] This application provides a culture medium for producing CVB1 virus, and based on the culture medium, a method for in vitro culture of HEK293 cells to produce CVB1 virus. This method involves adding feed media with different nutrient compositions at different stages of bioreactor culture, modifying the fermentation culture method, culturing cells at the optimal growth temperature and providing optimal nutrient supply in the early stages of cell culture, perfusion culture while maintaining good cell conditions to obtain high cell density, and slowing cell growth by cooling during inoculation, adding nutrients more conducive to cell toxin production, allowing the virus to gradually package and accumulate, thereby achieving stable high-density, high-titer toxin production, significantly improving the production efficiency of CVB1 virus, and showing great promise for industrial application. Attached Figure Description

[0081] Figure 1 Cell growth curves for Examples 1-7. The date of inoculation of the cell seed culture into the bioreactor is denoted as D0, the date of start of perfusion culture is denoted as D3, and the date of virus inoculation is denoted as D7.

[0082] Figure 2Cell growth curves for Comparative Examples 1-4. The date of inoculation of the cell seed culture into the bioreactor is recorded as D0; the date of virus inoculation in Comparative Examples 1-2 and 4 is recorded as D3; the date of start of perfusion culture in Comparative Example 3 is recorded as D3; and the date of virus inoculation is recorded as D7. Detailed Implementation

[0083] The invention will now be described in the following non-limiting embodiments.

[0084] Those skilled in the art will understand that the embodiments are described by way of example only and are not intended to limit the scope of protection claimed in this application. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0085] The HEK293 cells used in the following examples were HEK293-S / 1F4 monoclonal cells, purchased from Zhongshan Kangtianshenghe Biotechnology Co., Ltd. as Wayne 293. TM Cells were printed and plated using a single-cell isolation system (CYTENA, f.sight). Cell division was continuously monitored using a single-clone validation system (MD, Clone Select Image). Subsequently, multiple clones with faster proliferation rates were selected for amplification and passage. During passage, the single-clone strains were acclimatized to suspension using a stepwise serum reduction strategy with an initial concentration of 20% or 10% serum (FBS). Finally, the FBS was reduced to 0, and single-clone strains with good cell proliferation and high toxin production levels were selected as candidate suspension cell lines.

[0086] The Coxsackievirus CVB1 virus seed used in the following examples is the oncolytic backbone strain CVB1 with low toxicity and high oncolytic activity, developed by the National Engineering Research Center for Diagnostic Reagents and Vaccines for Infectious Diseases at Xiamen University, a collaborating unit (patent publication number: WO2019201192A1), based on its own intellectual property rights.

[0087] Example 1: Cell Culture and Virus Production Scheme 1

[0088] 1. Preparation of cell seed solution

[0089] Seed culture medium: HEK293 CD Medium (liquid medium A21001 or dry powder medium A22001 purchased from QuaCell) with a final concentration of 2mM GlutaMAX (purchased from Thermo Fisher Scientific, active ingredient L-alanyl-L-glutamine).

[0090] Cell culture:

[0091] (1) Cell resuscitation: HEK293 cells were seeded into 125 mL shake flasks containing 20 mL of seed culture medium and cultured in a CO2 shaker at a temperature of 37.0℃±1.0℃, a CO2 concentration of 5.0%, and a rotation speed of 120 rpm until the cell density reached (1.5-3)×10⁻⁶ cells / mL. 6 Cells / ml were used to obtain the revived cell seed solution;

[0092] (2) Primary cell seed culture: The revived cell seed culture was inoculated into 125 mL shake flasks containing 30 mL of seed culture medium at a passage ratio of 1:5. The cells were cultured in a CO2 shaker at a temperature of 37.0℃±1.0℃, a CO2 concentration of 5.0%, and a rotation speed of 120 rpm until the cell density reached (4-6)×10⁶ cells / year. 6 One cell / ml was used to obtain the primary cell seed solution;

[0093] (3) Secondary cell seed culture: The primary cell seed culture was inoculated into two 1L shake flasks containing 200mL seed culture medium at a passage ratio of 1:10. The cells were cultured in a CO2 shaker at a temperature of 37.0℃±1.0℃, a CO2 concentration of 5.0%, and a rotation speed of 120 rpm until the cell density reached (4-6)×106 cells / ml to obtain the secondary cell seed culture, which was used for subsequent experiments.

[0094] 2. Culture medium preparation:

[0095] Fermentation basal medium and perfusion medium: HEK293 CD Medium with GlutaMAX at a final concentration of 2mM and Poloxamer 188 at a final concentration of 1g / L added. 2L of fermentation basal medium and 9L of perfusion medium, for a total of 11L.

[0096] Feed culture medium: Prepare the feed culture medium according to the nutrient ratios in Tables 1 and 2 below. After stirring and dissolving, filter aseptically through a 0.22μm microporous membrane to obtain feed culture medium A and feed culture medium B.

[0097] Table 1: Components of Feed A Medium

[0098] Table 2: Components of Feed B Medium

[0099] The Cell Boost™ 5 was purchased from Hyclone.

[0100] 3. Cell Culture and Virus Production:

[0101] (1) The secondary cell seed liquid obtained in step 1 was inoculated into the bioreactor at a volume ratio of 5-10% of the fermentation basal medium. The pH was adjusted to 7.0 by introducing CO2 or adding NaHCO3 solution. The fermentation temperature was controlled at 37.0℃±1.0℃. Air was introduced at a flow rate of 150ccm. The fermentation process was carried out by cascading pure oxygen control at 0-150ccm to maintain dissolved oxygen at 30-70%.

[0102] (2) During the fermentation process, the cell density should reach (1-2)×10⁻⁶. 6 20 mL of feed A medium was rapidly fed in a single batch at a rate of 10 cells / mL.

[0103] (3) The cell density reaches (2-4)×10 6 When the cell count / ml reaches a certain level, ATF perfusion culture is initiated using perfusion medium at concentrations of 0.5 V / day (1 L / day), 0.75 V / day (1.5 L / day), 1.25 V / day (2.5 L / day), and 2.0 V / day (4 L / day).

[0104] (4) After the temperature drops to 35℃, stop the ATF perfusion and change the medium, and quickly add 20mL of feed culture medium at once, and inoculate the Coxsackievirus CVB1 seed at MOI=0.03;

[0105] (5) After the Coxsackievirus CVB1 inoculation is completed, continue fermentation culture for 24-48 hours. When the lesions are complete (cell viability is less than 80%), fermentation is completed, and the Coxsackievirus CVB1 titer is measured by taking the fermentation broth.

[0106] The cell proliferation rate is stable during virus production. Figure 1 The peak density was 1.26E+07 cells / ml, and the Coxsackievirus CVB1 titer (TCID1) was measured after culture. 50 The value ( / ml) is 1.12E+10.

[0107] Example 2: Cell Culture and Virus Production Scheme 2

[0108] Cell seed culture, fermentation basal medium, perfusion medium, and fed medium were prepared according to the method in Example 1.

[0109] Cell Culture and Virus Production:

[0110] (1) Inoculate the cell seed liquid into the bioreactor at a volume ratio of 5-10% of the fermentation basal medium. Adjust the pH to 6.8 using NaHCO3 solution, control the fermentation temperature at 35.0℃±1.0℃, introduce air at a flow rate of 150ccm, and use a cascade control system to maintain dissolved oxygen at 30-70% during fermentation.

[0111] Steps (2), (3), (4), and (5) are completely consistent with those in Example 1.

[0112] Compared to Example 1, the method in Example 2 involved a lower culture pH and a lower culture temperature before cell inoculation with the virus. The cell proliferation rate remained stable during virus production. Figure 1 The peak density was 9.50E+06 cells / ml, and the Coxsackievirus CVB1 titer (TCID1) was measured after culture. 50 The value ( / ml) is 5.62E+09.

[0113] Example 3: Cell Culture and Virus Production Scheme 3

[0114] Cell seed culture, fermentation basal medium, perfusion medium, and fed medium were prepared according to the method in Example 1.

[0115] Cell Culture and Virus Production:

[0116] (1) Inoculate the cell seed liquid into the bioreactor at a volume ratio of 5-10% of the fermentation basal medium. Adjust the pH to 7.0 by introducing CO2 or adding NaHCO3 solution. Control the fermentation temperature at 37.0℃±1.0℃. Introduce air at a flow rate of 150ccm. During the fermentation process, use a cascade control system to maintain dissolved oxygen at 30-70% by using pure oxygen at a flow rate of 0-150ccm.

[0117] (2) During the fermentation process, the cell density should reach (1-2)×10⁻⁶. 6 24 mL of feed A medium was rapidly fed in a single batch at a rate of 10 cells / ml.

[0118] (3) The cell density reaches (2-4)×10 6 When the cell / ml ratio reaches a certain level, ATF perfusion culture is initiated using perfusion medium at concentrations of 0.5 V / Day, 0.75 V / Day, 1.25 V / Day, and 2.0 V / Day.

[0119] (4) After the temperature drops to 35℃, stop the ATF perfusion and change the medium, and quickly add 24mL of feed culture medium at once, and inoculate the Coxsackievirus CVB1 seed at MOI=0.03;

[0120] (5) After the Coxsackievirus CVB1 inoculation is completed, continue fermentation culture for 24-48 hours. When the lesions are complete (cell viability is less than 80%), fermentation is completed, and the Coxsackievirus CVB1 titer is measured by taking the fermentation broth.

[0121] Compared to Example 1, the method in Example 3 involved increasing the amounts of Feed A and Feed B in the supplemental culture media. Cell proliferation remained stable during virus production. Figure 1 The peak density was 1.34E+07 cells / ml, and the Coxsackievirus CVB1 titer (TCID1) was measured after culture. 50 The value ( / ml) is 7.50E+09.

[0122] Example 4: Cell Culture and Virus Production Scheme 4

[0123] Cell seed culture, fermentation basal medium, perfusion medium, and fed medium were prepared according to the method in Example 1.

[0124] Cell Culture and Virus Production:

[0125] (1) Inoculate the cell seed liquid into the bioreactor at a volume ratio of 5-10% of the fermentation basal medium. Adjust the pH to 7.2 by introducing CO2 or adding NaHCO3 solution. Control the fermentation temperature at 37.0℃±1.0℃. Introduce air at a flow rate of 150ccm. During the fermentation process, use a cascade control system to maintain dissolved oxygen at 30-70% by using pure oxygen at a flow rate of 0-150ccm.

[0126] Steps (2), (3), (4), and (5) are completely consistent with those in Example 2.

[0127] Compared to Example 1, the method in Example 4 involved increasing the culture pH before virus inoculation. Cell proliferation rate remained stable during virus production. Figure 1 The peak density was 1.25E+07 cells / ml, and the Coxsackievirus CVB1 titer (TCID1) was measured after culture. 50 The value ( / ml) is 1.00E+10.

[0128] Example 5: Cell Culture and Virus Production Scheme 5

[0129] Cell seed culture, fermentation basal medium, perfusion medium, and fed medium were prepared according to the method in Example 1.

[0130] Cell Culture and Virus Production:

[0131] (1) Inoculate the cell seed liquid into the bioreactor at a volume ratio of 5-10% of the fermentation basal medium. Adjust the pH to 7.0 by introducing CO2 or adding NaHCO3 solution. Control the fermentation temperature at 37.0℃±1.0℃. Introduce air at a flow rate of 150ccm. During the fermentation process, use a cascade control system to maintain dissolved oxygen at 30-70% by using pure oxygen at a flow rate of 0-150ccm.

[0132] (2) During the fermentation process, the cell density should reach (1-2)×10⁻⁶. 6 18 mL of feed A medium was rapidly fed in a single batch at a rate of 10 cells / mL.

[0133] (3) The cell density reaches (2-4)×10 6 When the cell / ml ratio reaches 1,000 cells / ml, ATF perfusion culture is initiated at 0.5V / Day, 0.75V / Day, 1.25V / Day, and 2.0V / Day.

[0134] (4) After the temperature drops to 35℃, stop the ATF perfusion and change the medium, and quickly add 18mL of feed B medium at one time, and inoculate the Coxsackievirus CVB1 seed at MOI=0.03;

[0135] (5) After the Coxsackievirus CVB1 inoculation is completed, continue fermentation culture for 24-48 hours. When the lesions are complete (cell viability is less than 80%), fermentation is completed, and the Coxsackievirus CVB1 titer is measured by taking the fermentation broth.

[0136] Compared to Example 1, the method in Example 5 involved a reduction in the amount of feed A and feed B culture media added. Cell proliferation rate remained stable during virus production. Figure 1 The peak cell density was 9.64E+06 cells / ml. The Coxsackievirus CVB1 titer (TCID1) was measured after culture. 50 The value ( / ml) is 7.50E+09.

[0137] Example 6: Cell Culture and Virus Production Scheme 6

[0138] Cell seed culture, fermentation basal culture medium, perfusion culture medium, and fed culture medium were prepared according to the method in Example 2.

[0139] Cell Culture and Virus Production:

[0140] (1) Inoculate the cell seed liquid into the bioreactor at a volume ratio of 5-10% of the fermentation basal medium. Adjust the pH to 7.0 by introducing CO2 or adding NaHCO3 solution. Control the fermentation temperature at 37.0℃±1.0℃. Introduce air at a flow rate of 150ccm. During the fermentation process, use a cascade control system to maintain dissolved oxygen at 30-70% by using pure oxygen at a flow rate of 0-150ccm.

[0141] (2) During the fermentation process, the cell density should reach (1-2)×10⁻⁶. 6 20 mL of feed A medium was rapidly fed in a single batch at a rate of 10 cells / mL.

[0142] (3) The cell density reaches (2-4)×10 6 When the cell / ml ratio reaches a certain level, ATF perfusion culture is initiated at 0.5V / Day, 0.75V / Day, 1.25V / Day, and 2.0V / Day.

[0143] (4) After the temperature drops to 35℃, stop the ATF perfusion and change the medium, quickly add 20mL of feed culture medium, and inoculate the Coxsackievirus CVB1 seed at MOI=0.05.

[0144] (5) After the Coxsackievirus CVB1 inoculation is completed, continue fermentation culture for 24-48 hours. When the lesions are complete (cell viability is less than 80%), fermentation is completed, and the Coxsackievirus CVB1 titer is measured by taking the fermentation broth.

[0145] Compared to Example 1, the method in Example 6 increased the viral inoculation amount. The cell proliferation rate remained stable during virus production. Figure 1 The peak density was 9.34E+06 cells / ml, and the Coxsackievirus CVB1 titer (TCID1) was measured after culture. 50 The value ( / ml) is 1.00E+10.

[0146] Example 7: Cell Culture and Virus Production Scheme 7

[0147] Cell seed culture, fermentation basal medium, perfusion medium, and fed medium were prepared according to the method in Example 1.

[0148] Cell Culture and Virus Production:

[0149] (1) Inoculate the cell seed liquid into the bioreactor at a volume ratio of 5-10% of the fermentation basal medium. Adjust the pH to 7.0 by introducing CO2 or adding NaHCO3 solution. Control the fermentation temperature at 37.0℃±1.0℃. Introduce air at a flow rate of 150ccm. During the fermentation process, use a cascade control system to maintain dissolved oxygen at 30-70% by using pure oxygen at a flow rate of 0-150ccm.

[0150] (2) During the fermentation process, the cell density should reach (1-2)×10⁻⁶. 6 10 mL of feed A medium was rapidly fed in a single batch at a rate of 10 cells / ml.

[0151] (3) The cell density reaches (2-4)×10 6 When the cell / ml ratio reaches a certain level, ATF perfusion culture is initiated at 0.5V / Day, 0.75V / Day, 1.25V / Day, and 2.0V / Day.

[0152] (4) After the temperature drops to 35℃, stop the ATF perfusion and change the medium, quickly add 10ml of feed B medium at once, and inoculate the Coxsackievirus CVB1 seed at MOI=0.03;

[0153] (5) After the Coxsackievirus CVB1 inoculation is completed, continue fermentation culture for 24-48 hours. When the lesions are complete (cell viability is less than 80%), fermentation is completed, and the Coxsackievirus CVB1 titer is measured by taking the fermentation broth.

[0154] Compared to Example 1, the method in Example 7 significantly reduced the amount of feed A and feed B added to the culture medium. Cell proliferation rate remained stable during virus production. Figure 2 The peak density was 9.80E+06 cells / ml, and the Coxsackievirus CVB1 titer (TCID1) was measured after culture. 50 The value ( / ml) is 4.22E+09.

[0155] Comparative Example 1: Comparison of Cell Culture and Virus Production - Scheme 1

[0156] The cell seed culture and fermentation basal culture medium were prepared according to the method in Example 1.

[0157] Cell Culture and Virus Production:

[0158] (1) Inoculate the cell seed liquid into the bioreactor at a volume ratio of 5-10% of the fermentation basal medium. Adjust the pH to 7.0 by introducing CO2 or adding NaHCO3 solution. Control the fermentation temperature at 37.0℃±1.0℃. Introduce air at a flow rate of 150ccm. During the fermentation process, use a cascade control system to maintain dissolved oxygen at 30-70% by using pure oxygen at a flow rate of 0-150ccm.

[0159] (2) The cell density reached (2-4)×10 6 When the cell / ml ratio reaches 100 cells / ml, begin cooling;

[0160] (3) After the temperature drops to 35℃, inoculate the Coxsackievirus CVB1 seed at MOI=0.03;

[0161] (4) After the Coxsackievirus CVB1 inoculation is completed, continue fermentation culture for 24-48 hours. When the lesions are complete (cell viability is less than 80%), fermentation is completed, and the Coxsackievirus CVB1 titer is measured by taking the fermentation broth.

[0162] Compared to Example 1, Comparative Example 1 did not involve perfusion culture, nor did it include the addition of feed A and feed B during the culture process. This resulted in insufficient cell proliferation during virus production. Figure 2 The peak density was only 3.24E+06 cells / ml, and the Coxsackievirus CVB1 titer (TCID) was measured after culture. 50 The concentration ( / ml) was 4.22E+08, which was significantly lower than that in Examples 1-7.

[0163] Comparative Example 2: Cell Culture and Virus Production Comparison Scheme 2

[0164] 1. The cell seed solution was prepared using the method described in Example 1.

[0165] 2. Culture medium preparation:

[0166] Fermentation basal medium: HEK293 CD Medium with GlutaMAX added to a final concentration of 2mM, totaling 4L.

[0167] 3. Cell Culture and Virus Production:

[0168] (1) Inoculate the cell seed liquid into the bioreactor at a volume ratio of 5-10% of the fermentation basal medium. Adjust the pH to 7.0 by introducing CO2 or adding NaHCO3 solution. Control the fermentation temperature to 37.0℃±1.0℃. Introduce air at a flow rate of 150ccm and adjust the rotation speed to achieve dissolved oxygen of 20-40% for fermentation culture.

[0169] (2) The cell density reached (2-4)×10 6 When the cell count reaches 100 cells / ml, start centrifugation and change the medium to fresh fermentation basal medium (2L).

[0170] (3) After the temperature drops to 35℃, inoculate the Coxsackievirus CVB1 seed at MOI=0.03;

[0171] (4) After the Coxsackievirus CVB1 inoculation is completed, continue fermentation culture for 24-48 hours. When the lesions are complete (cell viability is less than 80%), fermentation is completed, and the Coxsackievirus CVB1 titer is measured by taking the fermentation broth.

[0172] Compared to Example 1, Comparative Example 2 showed reduced dissolved oxygen levels during cell culture before virus inoculation, no perfusion culture was performed, and neither Feed A nor Feed B was added during culture; only the basal fermentation medium was replaced with fresh medium before virus inoculation. Cell proliferation was insufficient during virus production. Figure 2 The peak density was only 3.37E+06 cells / ml, and the Coxsackievirus CVB1 titer (TCID1) was measured after culture. 50 The value ( / ml) was 1.33E+09, which was significantly lower than that in Examples 1-7.

[0173] Comparative Example 3: Cell Culture and Virus Production Comparison Scheme 3

[0174] Cell seed culture, fermentation basal culture medium, and perfusion culture medium were prepared according to the method in Example 1.

[0175] Cell Culture and Virus Production:

[0176] (1) Inoculate the cell seed liquid into the bioreactor at a volume ratio of 5-10% of the fermentation basal medium. Adjust the pH to 7.0 by introducing CO2 or adding NaHCO3 solution. Control the fermentation temperature at 37.0℃±1.0℃. Introduce air at a flow rate of 150ccm. During the fermentation process, use a cascade control system to maintain dissolved oxygen at 30-70% by using pure oxygen at a flow rate of 0-150ccm.

[0177] (2) The cell density reached (2-4)×10 6 When the cell / ml ratio reaches a certain level, ATF perfusion culture is initiated using perfusion medium at concentrations of 0.5 V / Day, 0.75 V / Day, 1.25 V / Day, and 2.0 V / Day.

[0178] (3) After the temperature drops to 35℃, stop the ATF perfusion and change the medium, and inoculate the Coxsackievirus CVB1 seed at MOI=0.03;

[0179] (4) After the Coxsackievirus CVB1 inoculation is completed, continue fermentation culture for 24-48 hours. When the lesions are complete (cell viability is less than 80%), fermentation is completed, and the Coxsackievirus CVB1 titer is measured by taking the fermentation broth.

[0180] Compared to Example 1, Comparative Example 3 did not include the addition of feed media A and B during the culture process. Cell proliferation rate remained stable during virus production. Figure 2 The peak density was 9.00E+06 cells / ml, and the Coxsackievirus CVB1 titer (TCID1) was measured after culture. 50 The value ( / ml) was 2.37E+09, lower than in Examples 1-7.

[0181] Comparative Example 4: Comparison of Cell Culture and Virus Production - Scheme 4

[0182] The cell seed culture and fermentation basal culture medium were prepared according to the method in Example 1.

[0183] Cell Culture and Virus Production:

[0184] (1) Inoculate the cell seed liquid into the bioreactor at a volume ratio of 5-10% of the fermentation basal medium. Adjust the pH to 7.0 by introducing CO2 or adding NaHCO3 solution. Control the fermentation temperature at 37.0℃±1.0℃. Introduce air at a flow rate of 150ccm. During the fermentation process, use a cascade control system to maintain dissolved oxygen at 30-70% by using pure oxygen at a flow rate of 0-150ccm.

[0185] (2) During the fermentation process, the cell density should reach (1-2)×10⁻⁶. 6 20 mL of feed A medium was rapidly fed in a single batch at a rate of 10 cells / ml.

[0186] (3) After the temperature drops to 35℃, add 20mL of feed B medium at once and inoculate the Coxsackievirus CVB1 seed at MOI=0.03;

[0187] (4) After the Coxsackievirus CVB1 inoculation is completed, continue fermentation culture for 24-48 hours. When the lesions are complete (cell viability is less than 80%), fermentation is completed, and the Coxsackievirus CVB1 titer is measured by taking the fermentation broth.

[0188] Comparative Example 4, compared to Example 1, did not undergo perfusion culture, but fed culture media (Feed A and Feed B) were added during the culture process. The cell proliferation capacity was insufficient during virus production. Figure 2 The peak density was 3.28E+06 cells / ml, and the Coxsackievirus CVB1 titer (TCID1) was measured after culture. 50 The viral load ( / ml) was 3.16E+09, significantly lower than in Examples 1-7. Meanwhile, compared to Comparative Example 1, the addition of feed A and Feed B media significantly increased viral yield, even without perfusion.

[0189] Peak cell culture density and final harvested Coxsackievirus CVB1 titer (TCID) in Examples 1-7 and Comparative Examples 1-4 50 The cell growth curves ( / ml) are summarized in Table 3 below, and the cell growth curves are shown in Figure 3. Figures 1-2 As shown.

[0190] Table 3: Summary of Results

[0191] As shown in Table 3, compared with Examples 1-7 which had added feed medium, the final virus titers of Comparative Examples 1-3 which did not have added feed medium were significantly reduced. Furthermore, compared with Comparative Example 1, Comparative Example 4 also showed that the addition of feed medium significantly increased virus yield without perfusion. The above experimental results demonstrate that the addition of the feed medium of this application can significantly increase virus yield in cell culture and virus production.

[0192] References

[0193] [1] Chen Guojun et al., Methodological improvements for the purification of CVB1 and the inactivation of CVB1 [M], 1999.

[0194] [2] Yang Ting et al., Comparative study on different purification methods of Coxsackievirus A16 [M], 2015.

[0195] [3] Kamen A et al., Development and optimization of an adenovirus production process, J Gene Med. 2004;6 (suppl 1):184–192.

[0196] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for producing a target virus by culturing cells in vitro, comprising: (1) Culture host cells in culture medium I, wherein culture medium I is a basal culture medium containing L-alanyl-L-glutamine; (2) Add culture medium II to the cell culture from step (1) and continue culturing the host cells, wherein the culture medium II contains the following components: Arginine, cysteine, histidine, isoleucine, leucine, lysine, valine, tyrosine, methionine, phenylalanine, threonine, tryptophan, choline chloride, folic acid, methionine, inositol, nicotinamide, D-calcium pantothenate, pyridoxal, vitamin B2, vitamin B1, thiamine, vitamin B12, L-alanyl-L-glutamine, Cell Boost™ 5, sodium pyruvate, sodium dichloroacetate, NaCl, KCl, KH2PO4 and Na2HPO4; (3) Add culture medium III to the cell culture from step (2), inoculate with the virus, and continue culturing the virus-inoculated host cells, wherein the culture medium III contains the following components: Arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, tyrosine, choline chloride, folic acid, inositol, nicotinamide, D-calcium pantothenate, pyridoxal, vitamin B2, thiamine, vitamin B12, L-alanyl-L-glutamine, Cell Boost™ 5, sodium pyruvate, sodium dichloroacetate, NaCl, KCl, KH2PO4 and Na2HPO4; (4) Obtain cell cultures containing the target virus.

2. The method of claim 1, wherein, Step (1) includes: The host cells were cultured in culture medium I until the host cell density reached (1-2) × 10⁻⁶. 6 cells / ml; Preferably, the concentration of L-alanyl-L-glutamine in culture medium I is 1.8-2.2 mM; Preferably, the basal culture medium is selected from HEK293 CD medium, Optipro® 293CD medium, Bepanthen® S001 medium, and any combination thereof; Preferably, the culture medium I is HEK293 CD medium containing L-alanyl-L-glutamine, and the concentration of L-alanyl-L-glutamine in the culture medium I is 1.8-2.2 mM (e.g., 2 mM). Preferably, the culture is carried out at a culture temperature of 37.0℃±1.0℃ and a pH of 6.8-7.2 in culture medium I; Preferably, the dissolved oxygen content of the culture medium I is 30-70% air saturation.

3. The method of claim 1 or 2, wherein, Step (2) includes: In step (1), add culture medium II to the cell culture and continue culturing the host cells until the host cell density reaches (2-4) × 10⁻⁶. 6 cells / ml; Preferably, in the culture medium II, the concentrations of arginine (2-4 g / L), cysteine ​​(0.5-1 g / L), histidine (0.75-1.5 g / L), isoleucine (1-2 g / L), leucine (1-2 g / L), lysine (1-2.5 g / L), valine (1-2 g / L), tyrosine (0.7-1.2 g / L), methionine (0.25-0.5 g / L), phenylalanine (0.6-1 g / L), threonine (0.9-1.5 g / L), tryptophan (0.2-0.3 g / L), choline chloride (15-30 mg / L), folic acid (15-30 mg / L), inositol (25-75 mg / L), and nicotinamide (15-30 mg / L) are... The concentrations of D-calcium pantothenate (15-30 mg / L), pyridoxal (15-30 mg / L), vitamin B2 (1-3 mg / L), vitamin B1 (15-40 mg / L), thiamine (15-30 mg / L), L-alanyl-L-glutamine (150-250 mM), Cell Boost™ 5 (15-35 g / L), sodium pyruvate (5-15 g / L), sodium dichloroacetate (50-100 g / L), NaCl (1-5 g / L), KCl (25-75 mg / L), KH2PO4 (25-75 mg / L), and Na2HPO4 (250-350 mg / L) are as follows: Preferably, the volume ratio of the culture medium II to the cell culture in step (1) is 1:80 to 1:200; Preferably, the culture is carried out at a culture temperature of 37.0℃±1.0℃.

4. The method of claim 3, wherein, Step (2) further includes: when the cell density of the host cells reaches (2-4) × 10 6 After reaching a cell density of 100 cells / ml, the cells were perfused using medium I for 4 days. The perfusion rates for days 1-4 were 0.4-0.6 V / day, 0.65-0.85 V / day, 1.15-1.35 V / day, and 1.9-2.1 V / day, respectively, where V represents the total volume of the cell culture. Preferably, the perfusion culture is carried out at a culture temperature of 37.0℃±1.0℃.

5. The method according to any one of claims 1-4, wherein, Step (3) includes: Add culture medium III to the cell culture in step (2), inoculate with the virus at an MOI of 0.01-0.05, and continue to culture the host cells after inoculation with the virus; Preferably, in the culture medium III, the concentrations of arginine (2-4 g / L), cysteine ​​(0.5-1 g / L), histidine (0.75-1.5 g / L), isoleucine (1-2 g / L), leucine (1-2 g / L), lysine (1-2.5 g / L), methionine (0.25-0.5 g / L), phenylalanine (0.6-1 g / L), threonine (0.9-1.5 g / L), tryptophan (0.2-0.3 g / L), inositol (25-75 mg / L), nicotinamide (15-30 mg / L), valine (1-2 g / L), tyrosine (0.7-1.2 g / L), and thiamine (15-30 mg / L) are as follows: mg / L, L-alanyl-L-glutamine concentration is 150-250 mM, Cell Boost™ 5 concentration is 15-35 g / L; Preferably, the volume ratio of the culture medium III to the cell culture in step (2) is 1:80 to 1:200; Preferably, the inoculation temperature is 35.0℃±1.0℃.

6. The method according to any one of claims 1-5, wherein, In step (3), the culture temperature is 35.0℃±1.0℃ and the culture time is 24-48h.

7. The method according to any one of claims 1-6, wherein, The host cell is a mammalian cell; Preferably, the host cell is a HEK293 cell.

8. The method according to any one of claims 1-7, wherein, The virus in question is Coxsackievirus. Preferably, the virus is Coxsackievirus B1 (CVB1).

9. A culture medium, which is culture medium II as defined in claim 1 or 3 or culture medium III as defined in claim 1 or 5.

10. A kit comprising any two or all three of culture media I, II, and III, wherein... The culture medium I is defined as in claim 1 or 2, the culture medium II is defined as in claim 1 or 3, and the culture medium III is defined as in claim 1 or 4.

11. Use of the culture medium of claim 8 or the kit of claim 9 for preparing a virus in a host cell; Preferably, the host cell is a mammalian cell; Preferably, the host cell is a HEK293 cell; Preferably, the virus is Coxsackievirus. Preferably, the virus is Coxsackievirus B1 (CVB1).

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