Method for preparing influenza virus vaccine by using Vero cells

Abstract

The invention discloses a method for preparing an influenza virus vaccine by using Vero cells, and belongs to the technical field of biology. The method comprises the following steps: firstly, carrying out serum-free culture amplification on Vero cells in a bioreactor containing a microcarrier; inoculating an influenza virus working seed batch, and maintaining culture in a serum-free culture medium; carrying out three-stage clarification and ultrafiltration concentration on the harvested virus liquid; purifying the virus through sucrose density gradient centrifugation and molecular sieve chromatography; and finally cracking the virus, quantifying, and adding polymer microspheres to prepare a monovalent virus stock solution. The serum-free culture medium comprises pea protein hydrolysate, an amino acid mixture and a cell homeostasis regulating agent. The risk of exogenous animal source components is avoided, the virus harvesting titer is remarkably improved, meanwhile, the immunogenicity of hemagglutinin and neuraminidase is completely reserved, technological parameters are controllable, amplification is easy, and the method is suitable for industrial production.

Description

Methods for preparing influenza virus vaccines using Vero cells Technical Field

[0001] This invention relates to a method for preparing influenza virus vaccines using Vero cells, and involves methods for preparing various influenza virus vaccine strains on Vero cells, belonging to the field of biotechnology. Background Technology

[0002] Influenza, as a global respiratory infectious disease, poses a continuous threat to human health. Vaccination is the most effective means of preventing and controlling influenza. Traditional influenza vaccine production mainly relies on chicken embryo culture technology. Although this process is mature, it suffers from problems such as long production cycles, limited scale, susceptibility to the supply of SPF chicken embryos, potential introduction of chicken-derived allergens, and antigenic drift during viral passage in chicken embryos. These limitations make it difficult to meet the urgent needs for vaccine production capacity and speed in response to large-scale or sudden influenza outbreaks. Using Vero cells to prepare influenza vaccines theoretically offers significant advantages such as strong process controllability, ease of scale-up, absence of exogenous animal proteins, and short production cycles. However, Vero cells often face challenges such as weakened adhesion, limited growth and metabolism, and lower viral yield under serum-free culture conditions.

[0003] Existing technologies, such as CN120924503A, improve cell condition and increase viral titer by adding specific functional proteins, such as red algae protein and soybean protein hydrolysate, to serum-free culture media. CN102078605B develops an advanced chromatographic purification process to remove host impurities, but its cell culture stage still largely uses a serum-containing system, failing to completely avoid the risks of animal-derived components. There is an urgent need in this field for an optimized technical solution that integrates the entire process of cell culture, virus amplification, antigen purification, and formulation, enabling efficient and high-titer production of influenza virus in Vero cells under serum-free conditions to meet the industrial demand for large-scale, high-quality influenza vaccine production. Summary of the Invention

[0004] This invention aims to provide a method for the large-scale, efficient, and safe preparation of influenza virus vaccines using Vero cells. By optimizing cell culture, virus proliferation, purification, inactivation, and formulation processes, combined with specific serum-free culture medium additives and a novel microsphere adjuvant system, the method enhances virus yield and immunogenicity, enabling the industrial production of stable, high-quality vaccine stock solutions.

[0005] To achieve the above objectives, the present invention provides a method for preparing an influenza virus vaccine using Vero cells, comprising the following steps: Step S1, passage resuscitating Vero cells in a bioreactor containing microcarriers and performing high-density amplification using serum-free medium; Step S2, inoculating with a working seed batch of influenza virus and maintaining viral culture in serum-free medium supplemented with TPCK-trypsin until the viral fluid is harvested; Step S3, clarifying and concentrating the viral fluid; Step S4, purifying the concentrated viral fluid to obtain a purified viral suspension; Step S5, sequentially inactivating and lysing the purified viral suspension, adding polymer microspheres and buffer, and aseptically filtering to prepare a monovalent vaccine stock solution.

[0006] The serum-free culture medium contains pea protein hydrolysate, an amino acid mixture, and at least one cell homeostasis regulator.

[0007] The amino acid mixture includes, but is not limited to, at least two of glutamine, arginine, aspartic acid, leucine, isoleucine, valine, and their salts.

[0008] Preferably, the amino acid mixture comprises glutamine, arginine, isoleucine, valine, and aspartic acid.

[0009] In step S1, the process of reviving and passaged Vero cells is as follows: after reviving Vero cells, they are cultured in a medium containing fetal bovine serum at 35-37°C and 5% CO2. When the cells reach 80-90% confluence, they are digested and passaged using trypsin-EDTA solution.

[0010] In step S2, before virus inoculation, the cells are washed 2-3 times with a 0.01 mol / L pH 7.4 phosphate buffer solution preheated to 37°C to remove residual serum.

[0011] The cell homeostasis regulator in the serum-free culture medium in step S1 is at least one of nicotinamide, N-acetylcysteine, and ectoine.

[0012] The polymer microspheres are either poloxamer-PLGA blend microspheres or PLGA / chitosan composite microspheres.

[0013] The preparation method of poloxamer-PLGA blend microspheres is as follows: Add 0.5-1g of polyvinyl alcohol to 50-100mL of water, stir at 100-300rpm for 3-5min in a water bath at 70-80℃, and cool to room temperature to obtain the aqueous phase; refrigerate overnight for later use; add 0.1-1g of polylactic acid-glycolic acid copolymer and 0.01-0.1g of poloxamer to 5-10mL of dichloromethane, and stir at 100-300rpm at room temperature. Stir for 3-5 minutes to obtain the oil phase; mix the prepared oil phase and the aqueous phase at 8000-10000 rpm for 3-5 minutes, then add 20-50 mL of polyvinyl alcohol aqueous solution, stir at 300-500 rpm for 6 hours at room temperature to allow the dichloromethane to fully evaporate and the polymer microspheres to solidify; then centrifuge at 4℃ and 5000-8000 rpm for 10-20 minutes to collect the microsphere precipitate; wash and disperse with water; and freeze-dry under vacuum to obtain the final product.

[0014] The water washing and dispersion treatment is as follows: the precipitate is washed 3-5 times with pre-cooled water at 0-4℃ to remove residual polyvinyl alcohol, solvent and unencapsulated free polymer; the washed microsphere precipitate is redispersed in 1-5mL of mannitol aqueous solution.

[0015] The preparation method of poloxamer-PLGA blend microspheres is as follows: 1-10 mg of chitosan is added to 5-10 mL of acetic acid aqueous solution, stirred at 100-300 rpm for 3-5 min, filtered for sterilization, and stored at 0-4℃; 0.1-1 g of polylactic acid-glycolic acid copolymer and 0.01-0.1 g of poloxamer are added to 5-10 mL of dichloromethane, stirred at 100-300 rpm for 3-5 min at room temperature to obtain the oil phase; the reserved chitosan acetic acid solution is added to the reserved oil phase, homogenized at 5000-6000 rpm for 1-3 min at 0-4℃ to form a water / oil type primary emulsion; then transferred to 100-300 mL of polyvinyl alcohol aqueous solution, stirred at 400-800 rpm for 6-8 h at room temperature to allow the dichloromethane to fully evaporate and the microspheres to solidify; then centrifuged to precipitate; washed with water and resuspended, then freeze-dried under vacuum to obtain the final product.

[0016] The centrifugal sedimentation process involves centrifuging at 0-4℃ and 5000-8000 rpm for 10-20 minutes, and then collecting the sediment.

[0017] The aforementioned water washing and resuspension treatment involves washing the water 3-5 times with pre-cooled water at 0-4℃, and then resuspending it in 3-5 mL of 5wt% trehalose aqueous solution.

[0018] The vacuum freeze-drying process involves drying at a temperature of -60 to -40°C and a vacuum of 80-100 Pa for 6-8 hours.

[0019] Trypsin-EDTA digestion solution is used for the digestion and dissociation of adherent Vero cells. Trypsin is responsible for hydrolyzing cell surface proteins, while EDTA weakens intercellular connections by chelating calcium and magnesium ions. The two work synergistically to achieve gentle and efficient cell digestion.

[0020] The beneficial effects of the present invention are as follows: 1. Compared with the prior art, the present invention adopts the Vero cell-microcarrier serum-free culture system, avoids exogenous animal-derived components, improves production safety and process consistency, is suitable for industrial scale-up production, and has strong adaptability to multiple influenza virus strains such as H3N2 and B.

[0021] 2. This invention optimizes the serum-free culture medium formulation and synergistically combines cell homeostasis regulators with high-density culture and PLGA / chitosan composite microsphere technology to significantly improve virus titer and hemagglutinin titer, while preserving antigen integrity and enhancing immunogenicity and long-lasting immune response. Detailed Implementation

[0022] The parameters and sources of some substances in the examples are as follows: Vero cells were obtained from the European Animal Cell Collection Center (ECACC), number 03129010; the original Vero cell bank was established by adhering to the culture vessel twice at the Institute of Medical Biology, Chinese Academy of Medical Sciences; the cells used in this experiment were passage 141 to form a working seed bank, which was established and preserved by the Institute of Medical Biology, Chinese Academy of Medical Sciences.

[0023] Influenza A virus A / New York / 32 / 2020 (H3N2), ID: 21 / 324, was derived from the National Institute for Biological Sciences (NIBSC) in the UK and was passaged and deposited by the Institute of Medical Biology, Chinese Academy of Medical Sciences.

[0024] Influenza virus strains: Influenza B virus BY B / Phuket / 3073 / 2013, ID: 14 / 312E3; Influenza B virus BV B / Brisbane / 60 / 2008 / NYMCBX35 / 41950, ID: 15 / 300E13; both were derived from NIBSC in the UK and were passaged and deposited by the Institute of Medical Biology, Chinese Academy of Medical Sciences.

[0025] Trypsin-EDTA digestion solution: a standard digestion solution containing 0.25wt% trypsin and 0.02wt% disodium EDTA, free of phenol red; catalog number: CB1411; source: Beijing Kingclone.

[0026] Sodium dodecyl sarcosinate: CAS No.: 137-16-6; Source: Thermo Fisher; Model No.: 28311.

[0027] Chromatographic column: Sephacryl S-400 HR gel filtration column.

[0028] MEM culture medium: Catalog number: 11095-080; Source: Thermo Fisher; Lot number: 3284175.

[0029] Pea protein hydrolysate: Batch number: BCBZ3060; Source: Sigma-Aldrich; Product number: P1025; Protein content ≥80%.

[0030] Polylactic acid-glycolic acid copolymer: Model: DG5050; Lactic acid:glycolic acid = 50:50; Source: Jinan Daigang; Molecular weight: 30000-60000 Da.

[0031] Polyvinyl alcohol: Batch number: SLCC4907; CAS number: 9002-89-5; Source: Sigma-Aldrich; Product number: P8136; Molecular weight: 35,000-50,000 Da.

[0032] Example 1: Method for preparing influenza virus vaccine using Vero cells, as follows: Step S1, Cell Culture and Expansion S1.1 Cell Resuscitation and Culture: Vero cells were resuscitated, with an initial inoculation density of 2.5 × 10⁻⁶ cells. 5 Cells / mL were seeded in roller bottles containing MEM medium with 10 wt% fetal bovine serum and cultured at 37°C and 5% CO2. S1.2 Cell digestion and passage: When cells reached over 80% confluence, the culture medium was aspirated, and the cells were washed with 0.01 mol / L sterile phosphate buffer solution (pH 7.4). Cells were then passaged at 0.1 mL / cm³. 2 Add trypsin-EDTA digestion solution and digest at 37℃ until most cells become rounded and detach. Add serum-containing complete culture medium to stop digestion, disperse by pipetting to form a single-cell suspension, and passage at a ratio of 1:3-4 for a total of 3 passages to obtain sufficient cell mass for inoculation into the bioreactor; S1.3 Microcarrier culture: Seed the expanded Vero cells into a bioreactor loaded with Cytodex-1 microcarriers at a concentration of 3 g / L, and culture in serum-free medium. Set the rotation speed to 100 rpm, pH to 7.4, and dissolved oxygen >40% to allow cells to attach to the microcarriers and grow to a density of 1.5-2.0 × 10⁶ cells / year. 6cells / mL; serum-free culture medium, model: FreeStyle™ F17, source: Thermo Fisher; Step S2, virus inoculation and culture S2.1 Cell washing: Discard the culture medium in the reactor, wash the cells three times with a preheated 37°C, pH 7.4 0.01 mol / L phosphate buffer solution to remove residual serum; S2.2 Virus inoculation: Inoculate with pre-diluted to 10 4 TCID 50 / mL of working seed batch of influenza virus, using influenza A H3N2 strain A / New York / 32 / 2020, with a multiplicity of infection (MOF) of 0.01; S2.3 Virus maintenance culture: Add serum-free medium from step S1.3, supplement with 2μg / mL TPCK-trypsin to lyse viral hemagglutinin and promote viral replication, and continue culturing at 35℃; S2.4 Process monitoring and harvest: Take samples regularly for monitoring during culture; after 96 hours of culture, when the HA titer reaches its peak, terminate the culture; Step S3, virus clarification and concentration S3.1 Clarification: Use 0.65μm, 0.45μm, and 0.22μm chromatography filters for three-stage depth filtration to remove cell debris and microcarriers, obtaining a clear virus solution; S3.2 Concentration: Use a tangential flow ultrafiltration system with a molecular weight cutoff of 300kDa for concentration. Concentration: Concentrate the clarified virus solution to 1 / 10 of its original volume; Step S4, Virus Purification S4.1 Density Gradient Centrifugation: Prepare a 15-60 wt% continuous sucrose gradient using 0.01 mol / L phosphate buffer solution at pH 7.4. Spread the concentrated virus solution on top of the gradient and pre-centrifuge at 5000 rpm for 15 min at 4°C. The supernatant obtained is then centrifuged at 30000 rpm for 2.5 h. Bands will form in the 20-30% sucrose range; collect the bands rich in virus particles; S4.2 Buffer Replacement: Replace the collected antigen bands using ultrafiltration with a molecular weight cutoff of 100 kDa at 4°C. The buffer solution is 0.01 mol / L pH 7.4. Phosphate buffer solution was used to concentrate the solution to a target protein concentration of 1 mg / mL; S4.3 Purification: A molecular sieve column was used, equilibrated with 0.01 mol / L phosphate buffer solution at pH 7.4, and the sample was loaded to obtain a purified virus suspension; Step S5, Inactivation, Lysis and Preparation S5.1 Inactivation: β-propiolactone was added dropwise to the purified virus suspension at 4℃ and 200 rpm to a final concentration of 0.1 vol%, and stirring was maintained for 36 h. Then, the suspension was allowed to stand in a 37℃ water bath for 2 h to ensure complete loss of infectivity, resulting in an inactivated virus solution; S5.2 Lysis: Sodium dodecyl sarcosinate was added to the inactivated virus solution at 4℃ and 200 rpm to a final concentration of... 0.1 wt%, continuously lyse for 90 min to fully disrupt the viral envelope structure and form viral lysate containing hemagglutinin (HA) and neuraminidase (NA); S5.3 Quantification and dilution: Quantify the above viral lysate by determining the hemagglutinin content using the one-way immunodiffusion method (SRID), and dilute to a final concentration of 30 μg / mL according to the formula of 15 μg hemagglutinin per 0.5 mL dose; S5.4 Preparation of monovalent viral stock solution: Add 0.02 mol / L pH 7.4 phosphate buffer, 1 mg / mL gelatin, and 0.5 mg / mL sodium alginate, filter at 0.2 μm under sterile conditions, and dispense into 0.5 mL pre-filled syringes to obtain the monovalent viral stock solution.

[0033] Example 2 uses Vero cells to prepare an influenza virus vaccine. The method is basically the same as in Example 1, except that the serum-free culture medium in steps S1.3 and S2.3 is based on FreeStyle™ F17 serum-free culture medium and also includes the following components: 200 mg / L pea protein hydrolysate, 0.3 g / L glutamine, 0.2 g / L arginine, 0.2 g / L isoleucine, 0.1 g / L valine, 0.1 g / L aspartic acid, and 0.5 mg / mL nicotinamide.

[0034] Example 3 uses Vero cells to prepare an influenza virus vaccine. The method is basically the same as in Example 1, except that the serum-free culture medium in steps S1.3 and S2.3 is based on the serum-free culture medium FreeStyle™ F17 and also includes the following components: 200 mg / L pea protein hydrolysate, 0.3 g / L glutamine, 0.2 g / L arginine, 0.2 g / L isoleucine, 0.1 g / L valine, 0.1 g / L aspartic acid, and 0.5 mg / mL N-acetylcysteine.

[0035] Example 4 uses Vero cells to prepare an influenza virus vaccine. The method is basically the same as in Example 1, except that the serum-free culture medium in steps S1.3 and S2.3 is based on FreeStyle™ F17 serum-free culture medium and also includes the following components: 200 mg / L pea protein hydrolysate, 0.3 g / L glutamine, 0.2 g / L arginine, 0.2 g / L isoleucine, 0.1 g / L valine, 0.1 g / L aspartic acid, and 0.5 mg / mL ectoine.

[0036] Example 5 Step S1, Cell Culture and Expansion S1.1 Cell Resuscitation and Culture: Vero cells were resuscitated, with an initial seeding density of 2.5 × 10⁻⁶ cells. 5 Cells / mL were seeded in roller bottles containing MEM medium with 10 wt% fetal bovine serum and cultured at 37°C and 5% CO2. S1.2 Cell digestion and passage: When cells reached over 80% confluence, the culture medium was aspirated, and the cells were washed with 0.01 mol / L sterile phosphate buffer solution (pH 7.4). Cells were then passaged at 0.1 mL / cm³. 2Add trypsin-EDTA digestion solution and digest at 37℃ until most cells become rounded and detach. Add serum-containing complete culture medium to stop digestion, disperse by pipetting to form a single-cell suspension, and passage at a ratio of 1:3-4 for a total of 3 passages to obtain sufficient cell mass for inoculation into the bioreactor; S1.3 Microcarrier culture: Seed the expanded Vero cells into a bioreactor loaded with Cytodex-1 microcarriers at a concentration of 3 g / L, and culture in serum-free medium. Set the rotation speed to 100 rpm, pH to 7.4, and dissolved oxygen >40% to allow cells to attach to the microcarriers and grow to a density of 1.5-2.0 × 10⁶ cells / year. 6 cells / mL.

[0037] Step S2, Virus Inoculation and Culture S2.1 Cell Washing: Discard the culture medium in the reactor and wash the cells three times with a preheated (37°C) pH 7.4 0.01 mol / L phosphate buffer solution to remove residual serum; S2.2 Virus Inoculation: Inoculate with pre-diluted to 10... 4 TCID 50 / mL of working seed batch of influenza virus, using influenza A H3N2 strain A / New York / 32 / 2020, with a multiplicity of infection (MOI) of 0.01; S2.3 Virus maintenance culture: serum-free medium was added, and 2μg / mL of TPCK-trypsin was added to lyse viral hemagglutinin and promote viral replication. Culture was continued at 35℃; S2.4 Process monitoring and harvest: samples were taken periodically for monitoring during culture; culture was terminated after 96 hours when the HA titer reached its peak; Step S3, virus clarification and concentration: S3.1 Clarification: three-stage depth filtration was performed using 0.65μm, 0.45μm, and 0.22μm chromatography filters to remove cell debris and microcarriers, obtaining a clarified virus solution; S3.2 Concentration: concentration was performed using a tangential flow ultrafiltration system with a molecular weight cutoff of 300kDa, and the above... The clarified virus solution was concentrated to 1 / 10 of its original volume; Step S4, Virus Purification S4.1 Density Gradient Centrifugation: A 15-60 wt% continuous sucrose gradient was prepared using 0.01 mol / L phosphate buffer solution at pH 7.4. The concentrated virus solution was placed on top of the gradient and pre-centrifuged at 5000 rpm for 15 min at 4°C. The supernatant was then centrifuged at 30000 rpm for 2.5 h. Bands formed in the 20-30% sucrose range, and the bands rich in virus particles were collected; S4.2 Buffer Replacement: The collected antigen bands were replaced by ultrafiltration at 4°C using a molecular weight cutoff of 100 kDa. The buffer solution was 0.01 mol / L phosphate buffer solution at pH 7.4. S4.3 Purification: Using a molecular sieve column, the solution was equilibrated with a 0.01 mol / L phosphate buffer solution at pH 7.4 to obtain a purified virus suspension; Step S5, Inactivation, Lysis and Preparation: S5.1 Inactivation: β-propiolactone was added dropwise to the purified virus suspension at 4℃ and 200 rpm to a final concentration of 0.1 vol%, and stirring was maintained for 36 h. Then, the suspension was allowed to stand in a 37℃ water bath for 2 h to ensure complete loss of infectivity, resulting in an inactivated virus solution; S5.2 Lysis: Sodium dodecyl sarcosinate was added to the inactivated virus solution at 4℃ and 200 rpm to a final concentration of... 0.1 wt%, continuously lyse for 90 min to fully disrupt the viral envelope structure and form viral lysate containing hemagglutinin (HA) and neuraminidase (NA); S5.3 Quantification and dilution: Quantify the above viral lysate by determining the hemagglutinin content using the one-way immunodiffusion method (SRID), and dilute to a final concentration of 30 μg / mL according to the formula of 15 μg hemagglutinin per 0.5 mL dose; S5.4 Preparation of monovalent virus stock solution: 0.02 mol / L pH 7.4 phosphate buffer, 1 mg / mL gelatin, 0.5 mg / mL sodium alginate, filter at 0.2 μm under aseptic conditions, and dispense into 0.5 mL pre-filled syringes to obtain the monovalent virus stock solution.

[0038] The serum-free culture medium in steps S1.3 and S2.3 is based on FreeStyle™ F17 serum-free culture medium and also includes the following components: 200 mg / L pea protein hydrolysate, 0.3 g / L glutamine, 0.2 g / L arginine, 0.2 g / L isoleucine, 0.1 g / L valine, 0.1 g / L aspartic acid, 0.25 mg / mL nicotinamide, and 0.25 mg / mL ectoine.

[0039] The method for preparing an influenza virus vaccine using Vero cells in Comparative Example 1 is basically the same as that in Example 1, except that the serum-free culture medium in steps S1.3 and S2.3 is based on FreeStyle™ F17 serum-free culture medium and also includes the following components: 200 mg / L pea protein hydrolysate, 0.3 g / L glutamine, 0.2 g / L arginine, 0.2 g / L isoleucine, 0.1 g / L valine, 0.1 g / L aspartic acid, 0.25 mg / mL N-acetylcysteine, and 0.25 mg / mL ectoine.

[0040] Example 6 describes a method for preparing an influenza virus vaccine using Vero cells, as follows: Step S1, Cell Culture and Expansion S1.1 Cell Resuscitation and Culture: Vero cells were resuscitated, with an initial inoculation density of 2.5 × 10⁻⁶ cells. 5 Cells / mL were seeded in roller bottles containing MEM medium with 10 wt% fetal bovine serum and cultured at 37°C and 5% CO2. S1.2 Cell digestion and passage: When cells reached over 80% confluence, the culture medium was aspirated, and the cells were washed with 0.01 mol / L sterile phosphate buffer solution (pH 7.4). Cells were then passaged at 0.1 mL / cm³. 2 Add trypsin-EDTA digestion solution and digest at 37℃ until most cells become rounded and detach. Add serum-containing complete culture medium to stop digestion, disperse by pipetting to form a single-cell suspension, and passage at a ratio of 1:3-4 for a total of 3 passages to obtain sufficient cell mass for inoculation into the bioreactor; S1.3 Microcarrier culture: Seed the expanded Vero cells into a bioreactor loaded with Cytodex-1 microcarriers at a concentration of 3 g / L, and culture in serum-free medium. Set the rotation speed to 100 rpm, pH to 7.4, and dissolved oxygen >40% to allow cells to attach to the microcarriers and grow to a density of 1.5-2.0 × 10⁶ cells / year. 6 cells / mL.

[0041] Step S2, Virus Inoculation and Culture S2.1 Cell Washing: Discard the culture medium in the reactor and wash the cells three times with a preheated (37°C) pH 7.4 0.01 mol / L phosphate buffer solution to remove residual serum; S2.2 Virus Inoculation: Inoculate with pre-diluted to 10...4 TCID 50 / mL of working seed batch of influenza virus, using influenza A H3N2 strain A / New York / 32 / 2020, with a multiplicity of infection (MOI) of 0.01; S2.3 Virus maintenance culture: serum-free medium was added, and 2μg / mL of TPCK-trypsin was added to lyse viral hemagglutinin and promote viral replication. Culture was continued at 35℃; S2.4 Process monitoring and harvest: samples were taken periodically for monitoring during culture; culture was terminated after 96 hours when the HA titer reached its peak; Step S3, virus clarification and concentration: S3.1 Clarification: three-stage depth filtration was performed using 0.65μm, 0.45μm, and 0.22μm chromatography filters to remove cell debris and microcarriers, obtaining a clarified virus solution; S3.2 Concentration: concentration was performed using a tangential flow ultrafiltration system with a molecular weight cutoff of 300kDa, and the above... The clarified virus solution was concentrated to 1 / 10 of its original volume; Step S4, Virus Purification S4.1 Density Gradient Centrifugation: A 15-60 wt% continuous sucrose gradient was prepared using 0.01 mol / L phosphate buffer solution at pH 7.4. The concentrated virus solution was placed on top of the gradient and pre-centrifuged at 5000 rpm for 15 min at 4°C. The supernatant was then centrifuged at 30000 rpm for 2.5 h. Bands formed in the 20-30% sucrose range, and the bands rich in virus particles were collected; S4.2 Buffer Replacement: The collected antigen bands were replaced by ultrafiltration at 4°C using a molecular weight cutoff of 100 kDa. The buffer solution was 0.01 mol / L phosphate buffer solution at pH 7.4. S4.3 Purification: Using a molecular sieve column, the solution was equilibrated with a 0.01 mol / L phosphate buffer solution at pH 7.4 to obtain a purified virus suspension; S5. Inactivation, lysis and preparation: S5.1 Inactivation: β-propiolactone was added dropwise to the purified virus suspension at 4℃ and 200 rpm to a final concentration of 0.1 vol%, and stirring was maintained for 36 h. Then, the suspension was allowed to stand in a 37℃ water bath for 2 h to ensure that the virus completely lost its infectivity, resulting in an inactivated virus solution; S5.2 Lysis: Sodium dodecyl sarcosinate was added to the inactivated virus solution at 4℃ and 200 rpm to a final concentration of 0. 1 wt% of the virus was continuously lysed for 90 min to fully disrupt the viral envelope structure and form a viral lysate containing hemagglutinin (HA) and neuraminidase (NA); S5.3 Quantification and dilution: The viral lysate was quantified, and the hemagglutinin content was determined by one-way immunodiffusion (SRID). The lysate was diluted to a final concentration of 30 μg / mL according to the formula of 15 μg hemagglutinin per 0.5 mL dose; S5.4 Preparation of monovalent viral stock solution: 0.02 mol / L pH 7.4 phosphate buffer and 1.5 mg / mL poloxamer-PLGA blended microspheres were added. The mixture was filtered through a 0.2 μm filter under sterile conditions and dispensed into 0.5 mL pre-filled syringes to obtain the monovalent viral stock solution.

[0042] The serum-free culture medium in steps S1.3 and S2.3 is based on FreeStyle™ F17 serum-free culture medium and also includes the following components: 200 mg / L pea protein hydrolysate, 0.3 g / L glutamine, 0.2 g / L arginine, 0.2 g / L isoleucine, 0.1 g / L valine, 0.1 g / L aspartic acid, 0.25 mg / mL nicotinamide, and 0.25 mg / mL ectoine.

[0043] The preparation method of the poloxamer-PLGA blend microspheres is as follows: 1g of polyvinyl alcohol is added to 99mL of water and stirred at 200rpm for 5min in an 80℃ water bath. The mixture is then cooled to 25℃ to obtain a polyvinyl alcohol aqueous solution. The solution is refrigerated at 4℃ overnight to ensure full hydration before use. 0.45g of polylactic acid-glycolic acid copolymer and 0.05g of poloxamer are added to 10mL of dichloromethane to a final concentration of 5wt%. The mixture is stirred at 200rpm for 5min at room temperature to obtain the oil phase. 1mL of the prepared oil phase and 10mL of the prepared polyvinyl alcohol aqueous solution are mixed. The mixture was sheared at 10,000 rpm for 5 min, and then 50 mL of prepared polyvinyl alcohol aqueous solution was added. The mixture was stirred at 500 rpm for 6 h at room temperature to allow the dichloromethane to fully evaporate and the polymer microspheres to solidify. Subsequently, the mixture was centrifuged at 8,000 rpm for 15 min at 4 °C, and the microsphere precipitate was collected. The precipitate was washed 5 times with pre-cooled water at 4 °C to remove residual polyvinyl alcohol, solvent, and unencapsulated free polymer. The washed microsphere precipitate was redispersed in 2 mL of 2 wt% mannitol aqueous solution. The mixture was then dried at -60 °C and 100 Pa for 8 h to obtain the final product.

[0044] Example 7 describes a method for preparing an influenza virus vaccine using Vero cells, as follows: Step S1, Cell Culture and Expansion S1.1 Cell Resuscitation and Culture: Vero cells were resuscitated, with an initial inoculation density of 2.5 × 10⁻⁶ cells. 5 Cells / mL were seeded in roller bottles containing MEM medium with 10 wt% fetal bovine serum and cultured at 37°C and 5% CO2. S1.2 Cell digestion and passage: When cells reached over 80% confluence, the culture medium was aspirated, and the cells were washed with 0.01 mol / L sterile phosphate buffer solution (pH 7.4). Cells were then passaged at 0.1 mL / cm³. 2Add trypsin-EDTA digestion solution and digest at 37°C until most cells become rounded and detach. Add serum-containing complete culture medium to stop digestion, disperse by pipetting to form a single-cell suspension, and passage at a ratio of 1:3-4 for a total of 3 passages to obtain sufficient cell mass for inoculation into the bioreactor. S1.3 Microcarrier Culture: Inoculate the expanded Vero cells into a bioreactor loaded with Cytodex-1 microcarriers at a concentration of 3 g / L. Culture in serum-free medium at a rotation speed of 100 rpm, pH 7.4, and dissolved oxygen >40%, allowing cells to adhere to the microcarriers and grow to a density of 1.5-2.0 × 10⁶ cells / year. 6 cells / mL.

[0045] Step S2, Virus Inoculation and Culture S2.1 Cell Washing: Discard the culture medium in the reactor and wash the cells three times with a preheated (37°C) pH 7.4 0.01 mol / L phosphate buffer solution to remove residual serum; S2.2 Virus Inoculation: Inoculate with pre-diluted to 10... 4 TCID 50 / mL of working seed batch of influenza virus, using influenza A H3N2 strain A / New York / 32 / 2020, with a multiplicity of infection (MOI) of 0.01; S2.3 Virus maintenance culture: serum-free medium was added, and 2μg / mL of TPCK-trypsin was added to lyse viral hemagglutinin and promote viral replication. Culture was continued at 35℃; S2.4 Process monitoring and harvest: samples were taken periodically for monitoring during culture; culture was terminated after 96 hours when the HA titer reached its peak; Step S3, virus clarification and concentration: S3.1 Clarification: three-stage depth filtration was performed using 0.65μm, 0.45μm, and 0.22μm chromatography filters to remove cell debris and microcarriers, obtaining a clarified virus solution; S3.2 Concentration: concentration was performed using a tangential flow ultrafiltration system with a molecular weight cutoff of 300kDa, and the above... The clarified virus solution was concentrated to 1 / 10 of its original volume; Step S4, Virus Purification S4.1 Density Gradient Centrifugation: A 15-60 wt% continuous sucrose gradient was prepared using 0.01 mol / L phosphate buffer solution at pH 7.4. The concentrated virus solution was placed on top of the gradient and pre-centrifuged at 5000 rpm for 15 min at 4°C. The supernatant was then centrifuged at 30000 rpm for 2.5 h. Bands formed in the 20-30% sucrose range, and the bands rich in virus particles were collected; S4.2 Buffer Replacement: The collected antigen bands were replaced by ultrafiltration at 4°C using a molecular weight cutoff of 100 kDa. The buffer solution was 0.01 mol / L phosphate buffer solution at pH 7.4. S4.3 Purification: Using a molecular sieve column, the solution was equilibrated with a 0.01 mol / L phosphate buffer solution at pH 7.4 to obtain a purified virus suspension; S5. Inactivation, lysis and preparation: S5.1 Inactivation: β-propiolactone was added dropwise to the purified virus suspension at 4℃ and 200 rpm to a final concentration of 0.1 vol%, and stirring was maintained for 36 h. Then, the suspension was allowed to stand in a 37℃ water bath for 2 h to ensure that the virus completely lost its infectivity, resulting in an inactivated virus solution; S5.2 Lysis: Sodium dodecyl sarcosinate was added to the inactivated virus solution at 4℃ and 200 rpm to a final concentration of 0. 1 wt% of the virus was continuously lysed for 90 min to fully disrupt the viral envelope structure and form a viral lysate containing hemagglutinin (HA) and neuraminidase (NA); S5.3 Quantification and dilution: The viral lysate was quantified, and the hemagglutinin content was determined by one-way immunodiffusion (SRID). The lysate was diluted to a final concentration of 30 μg / mL according to the formula of 15 μg hemagglutinin per 0.5 mL dose; S5.4 Preparation of monovalent viral stock solution: 0.02 mol / L pH 7.4 phosphate buffer and 1.5 mg / mL poloxamer-PLGA blended microspheres were added. The mixture was filtered through a 0.2 μm filter under sterile conditions and dispensed into 0.5 mL pre-filled syringes to obtain the monovalent viral stock solution.

[0046] The serum-free culture medium in steps S1.3 and S2.3 is based on FreeStyle™ F17 serum-free culture medium and also includes the following components: 200 mg / L pea protein hydrolysate, 0.3 g / L glutamine, 0.2 g / L arginine, 0.2 g / L isoleucine, 0.1 g / L valine, 0.1 g / L aspartic acid, 0.25 mg / mL nicotinamide, and 0.25 mg / mL ectoine.

[0047] The preparation method of the poloxamer-PLGA blend microspheres is as follows: Dissolve 2g of polyvinyl alcohol in 200mL of water, stir at 200rpm for 5min in an 80℃ water bath, and cool to 25℃ to obtain the polyvinyl alcohol aqueous phase; refrigerate at 4℃ overnight to allow for full hydration before use; add 10mg of chitosan to 5mL of 1vol% acetic acid aqueous solution, stir at 200rpm for 5min, filter through 0.22μm to remove bacteria to obtain chitosan acetic acid solution, and use at 4℃; add 0.45g of polylactic acid-glycolic acid copolymer and 0.05g of poloxamer to 10mL of dichloromethane. The mixture was stirred at 200 rpm for 5 min at room temperature to obtain the oil phase. 1 mL of chitosan acetate solution was added to the oil phase and homogenized at 6000 rpm for 2 min at 0 °C to form a water / oil type primary emulsion. The emulsion was then transferred to 200 mL of a spare polyvinyl alcohol aqueous phase and stirred at 600 rpm for 8 h at room temperature to allow the dichloromethane to fully evaporate and the microspheres to solidify. The precipitate was then collected by centrifugation at 8000 rpm for 15 min at 4 °C. The precipitate was washed 5 times with pre-cooled water at 4 °C and then resuspended in 5 mL of 5 wt% trehalose aqueous solution and dried at -60 °C and 100 Pa for 8 h to obtain the final product.

[0048] Comparative Example 2 describes a method for preparing an influenza virus vaccine using Vero cells, as follows: Step S1, Cell Culture and Expansion S1.1 Cell Resuscitation and Culture: Vero cells were resuscitated, with an initial inoculation density of 2.5 × 10⁻⁶ cells. 5 Cells / mL were seeded in roller bottles containing MEM medium with 10 wt% fetal bovine serum and cultured at 37°C and 5% CO2. S1.2 Cell digestion and passage: When cells reached over 80% confluence, the culture medium was aspirated, and the cells were washed with 0.01 mol / L sterile phosphate buffer solution (pH 7.4). Cells were then passaged at 0.1 mL / cm³. 2Add trypsin-EDTA digestion solution and digest at 37°C until most cells become rounded and detach. Add serum-containing complete culture medium to stop digestion, disperse by pipetting to form a single-cell suspension, and passage at a ratio of 1:3-4 for a total of 3 passages to obtain sufficient cell mass for inoculation into the bioreactor. S1.3 Microcarrier Culture: Inoculate the expanded Vero cells into a bioreactor loaded with Cytodex-1 microcarriers at a concentration of 3 g / L. Culture in serum-free medium at a rotation speed of 100 rpm, pH 7.4, and dissolved oxygen >40%, allowing cells to adhere to the microcarriers and grow to a density of 1.5-2.0 × 10⁶ cells / year. 6 cells / mL.

[0049] Step S2, Virus Inoculation and Culture S2.1 Cell Washing: Discard the culture medium in the reactor and wash the cells three times with a preheated (37°C) pH 7.4 0.01 mol / L phosphate buffer solution to remove residual serum; S2.2 Virus Inoculation: Inoculate with pre-diluted to 10... 4 TCID 50 / mL of working seed batch of influenza virus, using influenza A H3N2 strain A / New York / 32 / 2020, with a multiplicity of infection (MOI) of 0.01; S2.3 Virus maintenance culture: serum-free medium was added, and 2μg / mL of TPCK-trypsin was added to lyse viral hemagglutinin and promote viral replication. Culture was continued at 35℃; S2.4 Process monitoring and harvest: samples were taken regularly during culture for monitoring; culture was terminated after 96 hours when the HA titer reached its peak; Step S3, virus clarification and concentration S3.1 Clarification: three-stage depth filtration was performed using 0.65μm, 0.45μm, and 0.22μm chromatography filters to remove cell debris and microcarriers, obtaining a clarified virus solution; S3.2 Concentration: concentration was performed using a tangential flow ultrafiltration system with a molecular weight cutoff of 300kDa. The clarified virus solution was concentrated to 1 / 10 of its original volume; Step S4, Virus Purification S4.1 Density Gradient Centrifugation: A 15-60 wt% continuous sucrose gradient was prepared using 0.01 mol / L phosphate buffer solution at pH 7.4. The concentrated virus solution was placed on top of the gradient and pre-centrifuged at 5000 rpm for 15 min at 4°C. The supernatant was then centrifuged at 30000 rpm for 2.5 h. Bands formed in the 20-30% sucrose range, and the bands rich in virus particles were collected; S4.2 Buffer Replacement: The collected antigen bands were replaced using ultrafiltration with a molecular weight cutoff of 100 kDa at 4°C. The buffer solution was 0.01 mol / L pH 7.4. Phosphate buffer solution was used to concentrate the solution to a target protein concentration of 1 mg / mL; S4.3 Purification: A molecular sieve column was used, equilibrated with 0.01 mol / L phosphate buffer solution at pH 7.4, and the sample was loaded to obtain a purified virus suspension; Step S5, Inactivation, Lysis and Preparation S5.1 Inactivation: β-propiolactone was added dropwise to the purified virus suspension at 4℃ and 200 rpm to a final concentration of 0.1 vol%, and stirring was maintained for 36 h. Then, the suspension was allowed to stand in a 37℃ water bath for 2 h to ensure that the virus completely lost its infectivity, thus obtaining an inactivated virus solution; S5.2 Lysis: Sodium dodecyl sarcosinate was added to the inactivated virus solution at 4℃ and 200 rpm to a final concentration of 0.1 vol%. The concentration was 0.1 wt%, and the virus was continuously lysed for 90 min to fully destroy the viral envelope structure and form a viral lysate containing hemagglutinin (HA) and neuraminidase (NA); S5.3 Quantification and dilution: The viral lysate was quantified, and the hemagglutinin content was determined by one-way immunodiffusion (SRID). The solution was diluted to a final concentration of 30 μg / mL according to the formula of 15 μg hemagglutinin per 0.5 mL dose; S5.4 Preparation of monovalent virus stock solution: 0.02 mol / L pH 7.4 phosphate buffer and 1.5 mg / mL PLGA microspheres were added. The solution was filtered through a 0.2 μm filter under sterile conditions and dispensed into 0.5 mL pre-filled syringes to obtain the monovalent virus stock solution.

[0050] The serum-free culture medium in steps S1.3 and S2.3 is based on FreeStyle™ F17 serum-free culture medium and also includes the following components: 200 mg / L pea protein hydrolysate, 0.3 g / L glutamine, 0.2 g / L arginine, 0.2 g / L isoleucine, 0.1 g / L valine, 0.1 g / L aspartic acid, 0.25 mg / mL nicotinamide, and 0.25 mg / mL ectoine.

[0051] The preparation method of the PLGA microspheres is as follows: 1g of polyvinyl alcohol is added to 99mL of water and stirred at 200rpm for 5min in an 80℃ water bath. The mixture is then cooled to 25℃ to obtain a polyvinyl alcohol aqueous solution. The solution is then refrigerated at 4℃ overnight to ensure full hydration before use. 0.45g of polylactic acid-glycolic acid copolymer is added to 10mL of dichloromethane to a final concentration of 5wt%. The mixture is stirred at 200rpm for 5min at room temperature to obtain the oil phase. 1mL of the prepared oil phase and 10mL of the prepared polyvinyl alcohol aqueous solution are mixed and stirred at 10000rpm. The mixture was subjected to high-speed shearing at PM for 5 min, followed by the addition of 50 mL of prepared polyvinyl alcohol aqueous solution. The mixture was stirred at 500 rpm for 6 h at room temperature to allow the dichloromethane to fully evaporate and the polymer microspheres to solidify. Subsequently, the microspheres were centrifuged at 8000 rpm for 15 min at 4 °C and the precipitate was collected. The precipitate was washed 5 times with pre-cooled water at 4 °C to remove residual polyvinyl alcohol, solvent, and unencapsulated free polymer. The washed microsphere precipitate was redispersed in 2 mL of 2 wt% mannitol aqueous solution and dried at -60 °C and 100 Pa for 8 h to obtain the final product.

[0052] Comparative Example 3 describes a method for preparing an influenza virus vaccine using Vero cells, as follows: Step S1, Cell Culture and Expansion S1.1 Cell Resuscitation and Culture: Vero cells were resuscitated, with an initial inoculation density of 2.5 × 10⁻⁶ cells. 5 Cells / mL were seeded in roller bottles containing MEM medium with 10 wt% fetal bovine serum and cultured at 37°C and 5% CO2. S1.2 Cell digestion and passage: When cells reached over 80% confluence, the culture medium was aspirated, and the cells were washed with 0.01 mol / L sterile phosphate buffer solution (pH 7.4). Cells were then passaged at 0.1 mL / cm³. 2Add trypsin-EDTA digestion solution and digest at 37°C until most cells become rounded and detach. Add serum-containing complete culture medium to stop digestion, disperse by pipetting to form a single-cell suspension, and passage at a ratio of 1:3-4 for a total of 3 passages to obtain sufficient cell mass for inoculation into the bioreactor. S1.3 Microcarrier Culture: Inoculate the expanded Vero cells into a bioreactor loaded with Cytodex-1 microcarriers at a concentration of 3 g / L. Culture in serum-free medium at a rotation speed of 100 rpm, pH 7.4, and dissolved oxygen >40%, allowing cells to adhere to the microcarriers and grow to a density of 1.5-2.0 × 10⁶ cells / year. 6 cells / mL; Step S2, Virus Inoculation and Culture S2.1 Cell Washing: Discard the culture medium in the reactor, and wash the cells three times with a 0.01 mol / L phosphate buffer solution at pH 7.4 (preheated to 37°C) to remove residual serum; S2.2 Virus Inoculation: Inoculate with pre-diluted to 10 4 TCID 50The working seed batch of influenza virus was prepared in mL, using strain A H3N2 (A / New York / 32 / 2020) with a multiplicity of infection (MOI) of 0.01. S2.3 Virus maintenance culture: Serum-free medium was added, and 2 μg / mL of TPCK-trypsin was supplemented to lyse viral hemagglutinin and promote viral replication. Culture was continued at 35°C. S2.4 Process monitoring and harvesting: Samples were taken periodically during culture for monitoring. Culture was terminated after 96 hours when the HA titer reached its peak. Step S3: Virus clarification and concentration. S3.1 Clarification: Three-stage depth filtration was performed using 0.65 μm, 0.45 μm, and 0.22 μm chromatography filters to remove cell debris and microcarriers, obtaining a clear virus solution. S3.2 Concentration: Concentration was performed using a tangential flow ultrafiltration system with a molecular weight cutoff of 300 kDa. Concentrate the clarified virus solution to 1 / 10 of its original volume; Step S4, Virus Purification S4.1 Density Gradient Centrifugation: Prepare a 15-60 wt% continuous sucrose gradient using 0.01 mol / L phosphate buffer solution at pH 7.4. Spread the concentrated virus solution on top of the gradient and pre-centrifuge at 5000 rpm for 15 min at 4°C. Centrifuge the supernatant at 30000 rpm for 2.5 h. Bands will form in the 20-30% sucrose range; collect the bands rich in virus particles; S4.2 Buffer Replacement: Replace the collected antigen bands using ultrafiltration with a molecular weight cutoff of 100 kDa at 4°C. The buffer solution is 0.01 mol / L phosphate buffer solution at pH 7.4. S4.3 Purification: Using a molecular sieve column, the solution was equilibrated with 0.01 mol / L phosphate buffer solution at pH 7.4 to obtain a purified virus suspension; S5. Inactivation, lysis and preparation: S5.1 Inactivation: β-propiolactone was added dropwise to the purified virus suspension at 4℃ and 200 rpm to a final concentration of 0.1 vol%, and stirring was maintained for 36 h. Then, the solution was allowed to stand in a 37℃ water bath for 2 h to ensure complete loss of infectivity, resulting in an inactivated virus solution; S5.2 Lysis: Sodium dodecyl sarcosinate was added to the inactivated virus solution at 4℃ and 200 rpm until... The final concentration was 0.1 wt%. Lysis was continued for 90 minutes to fully disrupt the viral envelope structure, forming a viral lysate containing hemagglutinin (HA) and neuraminidase (NA). S5.3 Quantification and Dilution: The viral lysate was quantified using the single-path immunodiffusion (SRID) method to determine the hemagglutinin content. It was diluted to a final concentration of 30 μg / mL according to a formulation of 15 μg hemagglutinin per 0.5 mL dose. S5.4 Preparation of Monovalent Virus Stock Solution: The hemagglutinin was diluted to 30 μg / mL using only 0.02 mol / L pH 7.4 phosphate buffer. The solution was filtered through a 0.2 μm filter under aseptic conditions and dispensed into 0.5 mL pre-filled syringes to obtain the monovalent virus stock solution.

[0053] The serum-free culture medium in steps S1.3 and S2.3 is FreeStyle™ F17 serum-free culture medium.

[0054] Test Example 1: Monitoring of culture medium samples during step S2 of Examples 1-5 and Comparative Examples 1 and 3 during the culture period; monitoring cytopathic effect (CPE), hemagglutination titer (HA), and viral titer (TCID). 50 Following viral infection, specifically at 24h, 48h, 72h, and 96h after viral inoculation in step S2.2, aseptic samples were taken from the bioreactors or culture systems of each group. Cytopathic Effect (CPE) Observation: A small amount of sample was taken, and the morphological changes of Vero cells were observed under an inverted optical microscope. The proportion of cells exhibiting typical lesions such as rounding and detachment was assessed and recorded, categorized into five levels: 0% (no lesions), 25% (mild lesions), 50% (moderate lesions), 75% (significant lesions), and 100% (complete lesions, cell layer disintegration). Hemagglutination titer (HA) determination: A microhemagglutination assay was used. Using 96-well V-plates, samples were serially diluted with physiological saline at ratios of 1:2, 1:4, and so on up to 1:2048. 25 μL of diluted sample was added to each well, followed by an equal volume of 0.5 vol% chicken erythrocyte suspension. Agglutination was observed after standing at room temperature for 30 min. The HA titer is the reciprocal of the highest dilution that causes complete agglutination, and the result is expressed as Log2(HA Titer).

[0055] Viral titer TCID 50 Assay: The endpoint dilution method was used, and the samples were serially diluted 10-fold with cell maintenance medium. -1 Up to 10 -8 Each dilution was seeded into 96-well plates, with 100 μL of well-grown Vero cells in 8 replicate wells. The plates were incubated at 35°C in a 5% CO2 incubator for 7 days, and cell proliferation efficacy (CPE) was observed daily. The half-maximal infectious dose (TCID) was calculated using the Reed-Muench method. 50 ( / mL), results are expressed in Log 10 TCID 50 / mL is used to express the results. The results are shown in Tables 1 and 2.

[0056] Table 1. Observation results of cytopathic effect (CPE) during culture.

[0057] Table 2 Hemagglutination titer (HA) and viral titer (TCID) at harvest 50

[0058] Compared with Comparative Example 1, the viral replication efficiency gradients of Examples 1-5 were all improved, and the viral titer TCID was reduced.50 The overall upward trend in hemagglutination titer (HA) confirms that the optimization of the serum-free culture medium components improves the culture environment of Vero cells, thereby increasing the production efficiency of influenza virus. Compared to Comparative Example 3, which used only basic serum-free culture medium, Example 1 added pea protein hydrolysate and a specific amino acid mixture, resulting in improvements in both virus titer and hemagglutination titer. The addition of this specific component is not simply a nutritional supplement; rather, the pea protein hydrolysate provides small molecule peptides and amino acids, and the amino acid mixture further supplements cell metabolism, helping Vero cells maintain higher viability and functional integrity under serum-free conditions and throughout the viral infection cycle, thus supporting more efficient and sustained virus production.

[0059] Example 3: The use of N-acetylcysteine ​​alone resulted in a faster progression of the cytopathic effect, suggesting that it may have promoted the early advancement of the viral replication cycle through an antioxidant mechanism. Example 4: The use of ectoine alone, as a highly effective stress protectant, protected Vero cells from osmotic pressure and heat stress caused by large-scale viral replication and the accumulation of metabolic byproducts, reduced non-specific cell loss, and enabled more cells to continuously support viral replication.

[0060] Furthermore, in Example 5, the combination of nicotinamide and ectoine effectively increased viral yield. This is likely because nicotinamide acts as an NAD precursor and metabolic regulator, while ectoine acts as a stress protectant; their synergistic effect promotes high viral production and maintains healthy cell culture conditions. The lower performance of Comparative Example 1 compared to Examples 2-5, and even the lower performance compared to the baseline Example 1, suggests that the combination of N-acetylcysteine ​​and ectoine may stem from unexpected, mutually canceling effects on the intracellular target sites, metabolic pathways, or redox states of these two additives. Example 5, in a serum-free culture system, achieved an optimal balance between influenza virus proliferation efficiency, antigen yield, and culture stability in Vero cells. Its synergistic and complementary mechanisms created an ideal cellular microenvironment for efficient and stable virus production.

[0061] Test Example 2: Neuraminidase Activity Assay. A neuraminidase activity assay kit (Sigma-Aldrich, catalog number MAK121) was used. 20 µL of 10 mmol / L standard was added to 480 µL of water to prepare a 400 µmol / L standard working solution. 0, 15, 30, and 50 µL of the standard working solution were added to 480 µL of water to prepare a series of standard solutions with concentrations of 0, 120, 240, and 400 µmol / L. The monovalent viral stock solution prepared in Examples 1-7 and Comparative Examples 1-3 (i.e., lysed virus containing HA and NA) was diluted to a uniform protein concentration of 30 µg with 0.1 mol / L phosphate buffer solution at pH 7.4. HA / mL; 20µL of each sample was added to a 96-well plate, with duplicate wells, sample activity wells, and sample blank wells for each sample; the culture dish was kept away from light during incubation; the absorbance was measured at 570nm at 20min, and M20min was determined colorimetrically; 80µL of reaction solution containing substrate and buffer was added to each well; 20µL of standard solution of corresponding concentration was added to the standard wells; under light-protected conditions, the plate was incubated at 37℃ for 20min, and the absorbance was immediately measured at 570nm, recorded as M20min; incubation was continued for 50min, for a total of 50min, and the absorbance was measured again, recorded as M50min; according to the kit instructions, the neuraminidase activity U / L of each sample was calculated based on the standard curve. The results are shown in Table 3.

[0062] Table 3 Results of neuraminidase activity assay

[0063] The vaccine stock solutions prepared in Examples 1-7 all exhibited high neuraminidase activity, indicating that the lysis process effectively preserved the neuraminidase structure on the surface of the virus, possessing good enzyme activity, which is beneficial for inducing the body to produce an immune response against neuraminidase NA.

[0064] Compared to the combination of N-acetylcysteine ​​and ectoine in Comparative Example 1, the corresponding neuraminidase N activity was significantly lower than in other Examples 1-7, further indicating that this combination of cell homeostasis regulators has an antagonistic effect, which is detrimental to the integrity of viral structural proteins or the preservation of enzyme activity. In Comparative Example 2, the use of traditional PLGA microspheres showed no significant advantage compared to Examples 1-6, indicating that the PLGA carrier alone has no significant promoting effect on maintaining neuraminidase activity.

[0065] Furthermore, Example 7 used PLGA / chitosan blended microspheres, which showed good activity corresponding to neuraminidase (NA). Chitosan, as a positively charged polysaccharide, can bind to negatively charged mucosal surfaces or antigen-presenting cell membranes through electrostatic interactions, thereby enhancing antigen adhesion and endocytosis and improving antigen presentation efficiency. In addition, the microspheres prepared by the double emulsion method have a denser and more stable internal structure and encapsulation efficiency, which is beneficial for protecting the neuraminidase (NA) protein and maintaining its native spatial conformation and enzyme activity during storage and delivery. This invention, by optimizing the serum-free culture medium formulation and introducing a composite microsphere adjuvant system, not only improved the viral proliferation titer in Vero cells but also effectively maintained the biological activity of neuraminidase, providing an important technological basis for developing influenza vaccines with more comprehensive immune protection.

Claims

1. A method for preparing an influenza virus vaccine using Vero cells, characterized in that, Includes the following steps: Step S1: After resuscitation, Vero cells are seeded into a bioreactor containing microcarriers and passaged, and high-density amplification is performed using serum-free medium. Step S2: Inoculated with a working seed batch of influenza virus, the virus is maintained in serum-free medium supplemented with TPCK-trypsin until the virus fluid is harvested. Step S3: The virus fluid is clarified and concentrated. Step S4: The concentrated virus fluid is purified to obtain a purified virus suspension. Step S5: The purified virus suspension is sequentially inactivated and lysed, and polymer microspheres and buffer are added. After sterile filtration, a monovalent vaccine stock solution is prepared. The serum-free medium contains pea protein hydrolysate, an amino acid mixture, and at least one cell homeostasis regulator. The polymer microspheres are either poloxamer-PLGA blend microspheres or PLGA / chitosan composite microspheres.

2. The method for preparing an influenza virus vaccine using Vero cells according to claim 1, characterized in that, In step S1, the process of reviving and passaged Vero cells is as follows: after reviving Vero cells, they are cultured in a medium containing fetal bovine serum at 35-37°C and 5% CO2. When the cells grow to 80-90% confluence, they are digested and passaged using trypsin-EDTA solution.

3. The method for preparing an influenza virus vaccine using Vero cells according to claim 1, characterized in that, In step S2, before virus inoculation, the cells are washed 2-3 times with a 0.01 mol / L pH 7.4 phosphate buffer solution preheated to 37°C to remove residual serum.

4. The method for preparing an influenza virus vaccine using Vero cells according to claim 1, characterized in that, The cell homeostasis regulator is at least one of nicotinamide, N-acetylcysteine, and ectoine.

5. The method for preparing an influenza virus vaccine using Vero cells according to claim 1, characterized in that, The preparation method of poloxamer-PLGA blended microspheres in step S5 is as follows: Add 0.5-1g of polyvinyl alcohol to 50-100mL of water, stir at 100-300rpm for 3-5min in a water bath at 70-80℃, and cool to room temperature to obtain the aqueous phase; refrigerate overnight for later use; add 0.1-1g of polylactic acid-glycolic acid copolymer and 0.01-0.1g of poloxamer to 5-10mL of dichloromethane, and stir at 100-300rpm at room temperature. Stir for 3-5 minutes to obtain the oil phase; mix the prepared oil phase and the aqueous phase at 8000-10000 rpm for 3-5 minutes, then add 20-50 mL of polyvinyl alcohol aqueous solution, stir at 300-500 rpm for 6 hours at room temperature to allow the dichloromethane to fully evaporate and the polymer microspheres to solidify; then centrifuge at 4℃ and 5000-8000 rpm for 10-20 minutes to collect the microsphere precipitate; wash and disperse with water; and freeze-dry under vacuum to obtain the final product.

6. The method for preparing an influenza virus vaccine using Vero cells according to claim 1, characterized in that, The preparation method of the poloxamer-PLGA blend microspheres in step S5 is as follows: 1-10 mg of chitosan is added to 5-10 mL of acetic acid aqueous solution, stirred at 100-300 rpm for 3-5 min, filtered for sterilization, and stored at 0-4℃; 0.1-1 g of polylactic acid-glycolic acid copolymer and 0.01-0.1 g of poloxamer are dissolved in 5-10 mL of dichloromethane, stirred at 100-300 rpm for 3-5 min at room temperature to obtain the oil phase; the reserved chitosan acetic acid solution is added to the reserved oil phase, homogenized at 5000-6000 rpm for 1-3 min at 0-4℃ to form a water / oil type primary emulsion; then transferred to 100-300 mL of polyvinyl alcohol aqueous solution, stirred at 400-800 rpm for 6-8 h at room temperature to allow the dichloromethane to fully evaporate and the microspheres to solidify; then centrifuged to precipitate; washed with water and resuspended, then vacuum freeze-dried to obtain the final product.

7. The method for preparing an influenza virus vaccine using Vero cells according to claim 5, characterized in that, The water washing and dispersion treatment involves washing the precipitate 3-5 times with pre-cooled water at 0-4℃ to remove residual polyvinyl alcohol, solvent, and unencapsulated free polymer; and redispersing the washed microsphere precipitate in 1-5 mL of 2wt% mannitol aqueous solution.

8. The method for preparing an influenza virus vaccine using Vero cells according to claim 6, characterized in that, The centrifugation precipitation, water washing and resuspending treatment is as follows: centrifuge at 0-4℃ and 5000-8000rpm for 10-20min, collect the precipitate; wash with pre-cooled 0-4℃ water 3-5 times; and then resuspend in 3-5mL of 5wt% trehalose aqueous solution.

9. The method for preparing an influenza virus vaccine using Vero cells according to claim 6, characterized in that, The vacuum freeze-drying process involves drying at a temperature of -60 to -40°C and a vacuum of 80-100 Pa for 6-8 hours.

10. A method for preparing an influenza virus vaccine using Vero cells, characterized in that, It is prepared by the method described in any one of claims 1-9.

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