A novel coronavirus serum-free Vero cell inactivated vaccine and preparation method thereof

Abstract

This invention discloses a serum-free Vero cell inactivated vaccine for novel coronavirus and its preparation method. The preparation method includes the following steps: cell resuscitation, cell expansion, seed batch preparation, cell culture in a bioreactor, virus culture, inactivation of virus harvest fluid, clarification, ultrafiltration concentration, enzyme digestion, molecular sieve chromatography, semi-finished product preparation, and packaging. This invention improves product quality and production efficiency by optimizing culture process parameters, using serum-free culture medium during cell culture to reduce the risk of exogenous factor contamination, and by reducing purification steps to increase antigen recovery rate. The result is a serum-free Vero cell inactivated vaccine for novel coronavirus with high antigen recovery rate and low impurity content.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine preparation, and in particular to a novel coronavirus serum-free Vero cell inactivated vaccine and a preparation method thereof. Background Art

[0002] As an effective way to target and prevent the new coronavirus, vaccines have become the focus of global drug research and development.

[0003] Inactivated vaccines are vaccines that have been prepared by physically or chemically making the virus lose its infectivity and ability to replicate, but retaining the activity of the virus in inducing an immune response in the human body. Inactivated vaccines are the most effective vaccine development approach for emerging infectious diseases. They have the advantages of mature production technology, controllable quality standards, and a wide range of protection. They can be used for large-scale vaccination, and there are internationally accepted standards to judge the safety and effectiveness of vaccines.

[0004] At present, in the production of inactivated vaccines for the new coronavirus, serum-containing culture media are usually used to culture and expand cells. Serum can provide cells with growth factors necessary for survival and proliferation, matrix components for cell adhesion and spreading, carrier proteins and substances that neutralize toxicity, and also provide a good buffer system and protease inhibitors for the culture medium. However, the composition of serum is complex and not completely clear, and there is a risk of contamination by pathogenic factors such as exogenous viruses. Large differences are likely to occur between batches, and the source of serum is easily affected by market factors. Serum-containing culture media make cell culture standardization and product purification more difficult, and the safety risk of allergic reactions caused by residual heterologous serum in vaccines always exists.

[0005] In recent years, mature serum-free culture media have been launched on the market. Serum-free culture media can avoid or improve the defects of serum-containing culture media. The ECVAM Scientific Advisory Committee (ESAC) and the European Society of Toxicology Invitro (ESTIV) and other organizations have recommended reducing the addition of animal-derived substances such as serum during in vitro cell culture. For safety and stability reasons, serum-free culture media should be used as much as possible. Serum-free cell culture technology has gradually become the mainstream and trend in the field of in vitro cell culture.

[0006] There are currently no reports on the use of serum-free Vero cells to prepare inactivated new coronavirus vaccines. Summary of the invention

[0007] The technical problem to be solved by the present invention is to overcome the technical defects of the background technology and provide a novel coronavirus serum-free Vero cell inactivated vaccine and a preparation method thereof. The present invention optimizes the culture process parameters, uses serum-free culture medium in the cell culture process, reduces the risk of contamination by exogenous factors, and reduces the purification steps to improve the antigen recovery rate, thereby improving product quality and production efficiency, and prepares a novel coronavirus serum-free Vero cell inactivated vaccine with high antigen recovery rate and low impurity content.

[0008] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0009] A method for preparing a novel coronavirus serum-free Vero cell inactivated vaccine comprises the following steps:

[0010] (1) Cell recovery: Take an appropriate amount of Vero cells from the working cell bank, thaw them in a water bath, inoculate them into a culture bottle containing cell culture medium, and culture them in a carbon dioxide incubator;

[0011] (2) Cell expansion: Cells are cultured in serum-free medium and expanded in a cell factory as required; after subculturing, the cells are placed in a constant temperature chamber and cultured into a uniform monolayer of cells;

[0012] (3) Seed batch preparation: Delta purified strains are separated as the original seed bank. The virus liquid harvested after the original seed strains are continuously propagated through Vero cells is the main seed batch virus seed. The virus liquid harvested after the main seed batch virus seed is continuously propagated through Vero cells is the working seed batch virus seed.

[0013] (4) Bioreactor cell culture: Serum-free cell growth medium is added to the bioreactor and Vero cells are inoculated for culture;

[0014] (5) Virus culture: After the cells consume glucose and reach a stable period, replace the maintenance medium; inoculate the working seed batch virus into the production cells at an MOI of 0.005 to 0.01, and the same working seed batch virus is inoculated at the same MOI;

[0015] (6) Inactivation of virus harvest fluid: pre-cool the virus harvest fluid, add β-propiolactone and mix well to inactivate; after the inactivation is completed, hydrolyze;

[0016] (7) Clarification: After the inactivation verification inspection is passed, use a filter membrane for clarification filtration;

[0017] (8) Ultrafiltration and concentration: The clarified liquid is ultrafiltered and concentrated using an ultrafiltration membrane with a molecular weight cutoff of 100 to 300 kD, and the ultrafiltration concentrate is transferred to a cold storage for storage;

[0018] (9) Enzyme digestion: add nuclease, shake well, and digest by enzyme;

[0019] (10) Molecular sieve chromatography: the enzyme digestion solution is purified, the molecular sieve chromatography medium is Sepharose 4FF, and the first peak is collected as the stock solution; the stock solution is sampled and tested as required;

[0020] (11) Preparation of semi-finished products: Add the stock solution to the preparation container, then add aluminum hydroxide adjuvant, add buffer, make up to the volume of the semi-finished product preparation liquid, adjust the pH value, and stir; take samples and test the semi-finished products as required;

[0021] (12) Packaging: Packaging the semi-finished product into pre-filled bottles;

[0022] (13) Through-the-lens inspection: Conduct through-the-lens inspection and sampling inspection as required.

[0023] Preferably, in step (1), the Vero cells are P136 generation CCL-81 Vero cells.

[0024] Preferably, in step (1), the temperature of the water bath is 40±1°C.

[0025] Preferably, in step (1), the volume of the cell culture medium is 30-50 ml.

[0026] Preferably, in step (1), the temperature of the carbon dioxide incubator is 37±1°C.

[0027] Preferably, in the step (1), during culturing in the carbon dioxide incubator, the liquid is replaced every 6 to 8 hours, and the culturing is continued in the carbon dioxide incubator.

[0028] Preferably, in step (2), the serum-free culture medium is VP SFM.

[0029] Preferably, in step (2), the cells are expanded to 144 generations.

[0030] Preferably, in step (2), the temperature of the temperature chamber is 37±2°C.

[0031] Preferably, in step (3), the virus liquid harvested after the original seed strain is propagated for two generations in CCL-81 Vero cells is used as the main seed batch virus seed.

[0032] Preferably, in step (3), the virus liquid harvested after the main seed batch of virus seeds is propagated through CCL-81 Vero cells for three generations is the working seed batch of virus seeds.

[0033] Preferably, in step (3), the seed batch is stored below -60°C.

[0034] Preferably, in step (4), the bioreactor is a basket-type bioreactor filled with sheet-like carriers.

[0035] Preferably, in step (4), the Vero cells are CCL-81 Vero cells.

[0036] Preferably, in the step (4), the specific parameters for culturing the inoculated Vero cells are: sheet carrier: 20-40 g / L, culture medium: VP SFM AGT, culture time: 12-14 days, stirring: 80-100 rpm, culture temperature: 37°C, culture pH: 7.0-7.2, DO: 30%-80%.

[0037] Preferably, in step (5), the cells are cultured for 12 to 14 days until the cells reach a stable glucose consumption phase.

[0038] Preferably, in step (5), the maintenance solution is DMEM solution.

[0039] Preferably, in step (5), the parameters of the virus culture are: stirring: 80-100 rpm, temperature: 34±1° C., pH: 7.4-7.7, DO: 30%-80%.

[0040] Preferably, in step (5), the virus harvest fluid is harvested 2 to 3 days after the virus culture.

[0041] Preferably, in step (6), the precooling temperature is 2-8°C.

[0042] Preferably, in step (6), the volume ratio of β-propiolactone to virus harvest solution is 1:4000.

[0043] Preferably, in step (6), the inactivation temperature is 2-8°C and the inactivation time is 24h.

[0044] Preferably, in step (6), the hydrolysis temperature is 37° C. and the hydrolysis time is 2 h.

[0045] Preferably, in step (7), the pore size of the filter membrane is 0.65 μm.

[0046] Preferably, in step (8), the concentrated solution is concentrated to 20 to 30 times during ultrafiltration.

[0047] Preferably, in step (8), the temperature of the cold storage is 2-8°C.

[0048] Preferably, in step (9), the nuclease is a non-restriction endonuclease.

[0049] More preferably, the concentration of the non-restrictive endonuclease is 20-80 U / ml.

[0050] Preferably, in step (9), the enzymatic hydrolysis temperature is 37±1° C., and the enzymatic hydrolysis time is 6 to 10 hours.

[0051] Preferably, in step (10), the eluent for the molecular sieve chromatography is a phosphate buffer solution with a pH of 7.4 to 7.6, the linear flow rate is 0.1 to 0.5 cm / min, the sample loading amount is 2% to 6% of the column volume, and the detection wavelength is 280 nm.

[0052] Preferably, in step (11), the stock solution is added to the preparation container at a final antigen concentration of 1600 RU / ml.

[0053] Preferably, in step (11), the final concentration of the aluminum hydroxide adjuvant is 1 mg / ml (aluminum content).

[0054] Preferably, in step (11), the buffer is a phosphate buffer with a pH of 7.4 to 7.6.

[0055] Preferably, in the step (11), the pH value is 7.0 to 8.0.

[0056] Preferably, in the step (11), the stirring is carried out on a magnetic stirrer.

[0057] Preferably, in the step (11), the stirring temperature is room temperature.

[0058] Preferably, in step (11), the stirring speed is 400-500 rpm, and the stirring time is 2 hours.

[0059] Preferably, in step (12), the semi-finished product is packaged into prefilled bottles at a rate of 0.5 ml / dose.

[0060] Preferably, in step (12), each dose of antigen contains 800 RU.

[0061] A novel coronavirus serum-free Vero cell inactivated vaccine is prepared using the method for preparing a novel coronavirus serum-free Vero cell inactivated vaccine as described above.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] The present invention optimizes the culture process parameters and uses serum-free culture medium in the cell culture process to reduce the risk of contamination by exogenous factors. At the same time, it reduces the purification steps and increases the antigen recovery rate, thereby improving product quality and production efficiency, and preparing a new coronavirus serum-free Vero cell inactivated vaccine with high antigen recovery rate and low impurity content. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 This is a spectrum of the novel coronavirus purified solution after one-step purification using Sepharose 4FF in Example 1 of the present invention, identified by polyacrylamide gel electrophoresis (SDS-PAGE) method;

[0065] Figure 2 This is a graph of the purified solution of the new coronavirus after one-step purification using Sepharose 4FF in Example 1 of the present invention, which was identified by the Western Blot method (from left to right: the results of immunoblotting of S protein of three batches of stock solutions, and the results of immunoblotting of N protein of three batches of stock solutions);

[0066] Figure 3 This is an electron microscopic image of the purified solution of the new coronavirus after one-step purification using Sepharose 4FF in Example 1 of the present invention. DETAILED DESCRIPTION

[0067] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments and accompanying drawings. It should be understood that these embodiments are only used to further illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art may make some non-essential changes or adjustments to the present invention, which still belong to the protection scope of the present invention.

[0068] Example 1

[0069] A method for preparing a novel coronavirus serum-free Vero cell inactivated vaccine comprises the following steps:

[0070] (1) Cell recovery: Take one tube of P136 generation CCL-81 Vero cells from the working cell bank according to production requirements, thaw quickly in a 40±1°C water bath, inoculate into a T75 bottle containing 30-50 ml of cell culture medium after thawing, and culture in a carbon dioxide incubator (37±1°C). After 6-8 hours, change the medium and continue culturing in a carbon dioxide incubator (37±1°C);

[0071] (2) Cell expansion: Cells were cultured in VP SFM serum-free medium and expanded in a cell factory as required, with cells expanded to 144 generations; after subculturing, cells were placed in a constant temperature room (37 ± 2°C) and cultured into a uniform monolayer of cells;

[0072] (3) Seed batch preparation: Delta purified strains were separated as the original seed bank. The virus liquid harvested after the original seed strains were continuously propagated for 2 generations through CCL-81 Vero cells was used as the main seed batch virus stock. The virus liquid harvested after the main seed batch virus stock was continuously propagated for 3 generations through CCL-81 Vero cells was used as the working seed batch virus stock. The seed batch was stored below -60°C.

[0073] (4) Bioreactor cell culture: Serum-free cell growth medium was added to a BioCore QC Pro260L basket bioreactor filled with sheet carriers, and CCL-81 Vero cells were inoculated for culture; the specific parameters were as follows: sheet carrier: 20-40 g / L, culture medium: VP SFM AGT, culture time: 12-14 days, stirring: 80-100 rpm, culture temperature: 37°C, culture pH: 7.0-7.2, DO: 30%-80%;

[0074] (5) Virus culture: After the cells have been cultured for 12 to 14 days and the cells have consumed glucose to reach a stable period, the maintenance medium is replaced with DMEM solution; the working seed batch virus strain is inoculated into the production cells at an MOI of 0.005 to 0.01, and the same working seed batch virus strain should be inoculated at the same MOI; culture parameters: stirring: 80 to 100 rpm, temperature: 34 ± 1 ° C, pH: 7.4 to 7.7, DO: 30% to 80%; the virus harvest fluid is harvested 2 to 3 days after virus culture;

[0075] (6) Inactivation of virus harvest fluid: precool the virus harvest fluid to 2-8°C, add β-propiolactone at a ratio of 1:4000, mix well, and inactivate at 2-8°C for 24 h. After the inactivation is complete, hydrolyze at 37°C for 2 h.

[0076] (7) Clarification: After the inactivation verification inspection is passed, clarify and filter using a 0.65 μm filter membrane;

[0077] (8) Ultrafiltration concentration: The clarified liquid is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 100 to 300 kD and concentrated to 20 to 30 times, and the ultrafiltration concentrate is transferred to a cold storage at 2 to 8°C for storage;

[0078] (9) Enzyme digestion: Add non-restriction endonuclease at a concentration of 20-80 U / ml as required, shake well, and digest at 37±1°C for 6-10 hours;

[0079] (10) Molecular sieve chromatography: the enzyme digestion solution is used for purification, the molecular sieve chromatography medium is Sepharose 4FF, the eluent is phosphate buffered saline (PBS) at pH 7.4-7.6, the linear flow rate is 0.1-0.5 cm / min, the sample load is 2%-6% of the column volume, the detection wavelength is 280 nm, and the first peak is collected as the stock solution; the stock solution is sampled and tested as required;

[0080] (11) Preparation of semi-finished product: Add the stock solution to the preparation container at a final antigen concentration of 1600 RU / ml, then add aluminum hydroxide adjuvant at a final concentration of 1 mg / ml (aluminum content), add phosphate buffer (PBS) at pH 7.4-7.6, make up to the volume of the semi-finished product preparation, adjust the pH value to 7.0-8.0, place on a magnetic stirrer, stir at a speed of 400-500 rpm, and stir at room temperature for 2 hours; take samples of the semi-finished product for testing as required;

[0081] (12) Packaging: The semi-finished product is packaged into prefilled bottles at a rate of 0.5 ml / dose, i.e., each dose contains 800 RU of antigen;

[0082] (13) Through-the-lens inspection: Conduct through-the-lens inspection and sampling inspection as required.

[0083] Effect Example

[0084] In Example 1 of the present invention, in the preparation method of the novel coronavirus serum-free Vero cell inactivated vaccine, the vaccine research process is controlled by the following detection methods to ensure batch stability, product safety and effectiveness. The detection methods are summarized as follows:

[0085] Protein content: refer to (General Rules 0731 Folin phenol method 1 of the current edition of the Pharmacopoeia of the People's Republic of China).

[0086] Antigen content: detected by enzyme-linked immunosorbent assay.

[0087] The reference product is a qualified finished product with qualified safety evaluation and an antigen content of 1600RU / ml. After the reference product is reconstituted, it is diluted with the sample diluent with the test product, and 100μl is added to each well of the ELISA plate, and 2 wells are made in parallel for each dilution. The sample diluent is a negative control. The ELISA plate is coated, incubated at room temperature for 60min, the liquid in the plate is discarded, the washing solution is added, and the plate is washed 4 times, 50μl of ELISA working solution is added, the ELISA plate is coated, incubated at room temperature for 60min, the liquid in the plate is discarded, the washing solution is added, and the plate is washed 4 times, 50μl of substrate solution A and substrate solution B are added to each well successively, the ELISA plate is coated, incubated at room temperature in the dark for 15min, 50μl of stop solution is added to each well, and the ELISA reader reads the OD value of each well at a wavelength of 450nm. Accurately enter the concentration values ​​of the reference product and its corresponding OD value into the Excel worksheet, the concentration values ​​of the reference product are the horizontal axis, and the corresponding OD value is the vertical axis to establish a standard equation, enter the logarithm of the OD value of the test product into the standard equation, and calculate the concentration of the test product.

[0088] Nuclease content: Detected by enzyme-linked immunosorbent assay; add reference and test samples to the ELISA plate, 50 μl / well, and make two parallel wells. Add 100 μl / well of enzyme-labeled antibody solution, cover the ELISA plate, and incubate at 24±4℃ with 200-600rpm / min for 60 minutes. Discard the liquid in the plate, add washing solution, and repeat the plate washing 4 times. Add 100 μl / well of TMB Substrate colorimetric solution. Put the ELISA plate in a sealed bag and let it stand at 24±4℃ in the dark for 30 minutes. Add 100 μl of stop solution and read the OD value of each well at a wavelength of 450nm using an ELISA reader. Accurately input the concentration values ​​of the reference substance and its corresponding OD value into the Excel worksheet, calculate the average OD value of each concentration, take the concentration values ​​of the reference substance as the horizontal axis, and the corresponding OD value of each concentration as the vertical axis, use ELISACalc software to establish a standard curve according to four-parameter fitting, calculate the standard equation, and substitute the logarithmic value of the test substance OD value into the standard equation for calculation; the test results show that the nuclease residues in each batch of samples after one-step purification in Example 1 are all below the detection limit, which meets the national limit standard.

[0089] Residual bovine serum albumin: detected by enzyme-linked immunosorbent assay (General Rule 3411 of Part III of the current edition of the Pharmacopoeia of the People's Republic of China); the test results show that the residual bovine serum albumin in each batch of samples after one-step purification in Example 1 is less than 10 ng / dose, which meets the national limit standard.

[0090] Residual amount of Vero cell DNA: refer to the quantitative PCR method (General Rules 3407 Method 3 of the current edition of the Pharmacopoeia of the People's Republic of China); the test results show that the residual amount of Vero cell DNA in each batch of samples after one-step purification in Example 1 is less than 100pg / dose, which meets the national limit standard.

[0091] Residual Vero cell protein: Detection was performed in accordance with (General Rules 3429 of Part III of the current edition of the Pharmacopoeia of the People's Republic of China); the test results showed that the residual Vero cell protein in each batch of samples after one-step purification in Example 1 was less than 100 ng / dose, which met the national limit standard.

[0092] Example 1 The test data of the virus harvest solution and stock solution of each batch of novel coronavirus serum-free Vero cell inactivated vaccine are shown in Table 1.

[0093] Table 1 Test data of virus harvested liquid and stock solution of each batch of novel coronavirus serum-free Vero cell inactivated vaccine in Example 1

[0094]

[0095]

[0096] The data of antigen recovery rate and impurity removal rate of each batch of novel coronavirus serum-free Vero cell inactivated vaccine in Example 1 are shown in Table 2.

[0097] Table 2 Antigen recovery rate and impurity removal rate of each batch of novel coronavirus serum-free Vero cell inactivated vaccine in Example 1

[0098]

[0099] As can be seen from Table 2, the antigen recovery rate of each batch of stock solution in Example 1 is 75% to 78%, the Vero cell protein (HCP) removal rate is more than 99.80%, the Vero cell DNA removal rate is 99.99% and above, and the cumulative nuclease removal rate is more than 99.90%. The antigen content and impurity content of each batch of stock solution meet the quality standards and have good batch consistency.

[0100] It can be seen from the detection indicators of each batch of samples after purification in Table 2 that the purification method of the present invention has excellent purification effect on the new coronavirus, and the purification method is stable and repeatable; the present invention uses the medium Sepharose 4FF to purify the new coronavirus, which not only retains the effective antigen, but also effectively removes impurities such as nucleases, HCP and DNA, and is suitable for the rapid development and production of serum-free Vero cell inactivated vaccines for the new coronavirus.

[0101] Further, by Figure 1 , Figure 2 It can be seen that the purified solution of the new coronavirus in Example 1 of the present invention (Batch 1) was subjected to SDS-PAGE electrophoresis, and corresponding bands were detected near 50KD, near 180KD and greater than 250KD. By immunoblotting, they were combined with corresponding antibodies and exposed for color development, confirming that they were N protein, S protein monomer and S protein polymer, respectively.

[0102] Depend on Figure 3 It can be seen that: electron microscopic observation of the purified novel coronavirus stock solution (batch 1) in Example 1 of the present invention found that the novel coronavirus virus particles are mostly spherical or elliptical, polymorphic, and have a diameter of about 60 to 150 nm; there are relatively typical spine wrappings on the viral envelope, and the spines are different, and their size and shape are consistent with the typical characteristics of the novel coronavirus.

[0103] In addition, most of the inactivated new coronavirus vaccines currently sold on the market use a two-step purification process, but the more purification steps there are, the lower the antigen recovery rate, and the greater the shearing of the viral protein, which may affect the integrity of the virus.

[0104] The one-step purified novel coronavirus solution prepared in Example 1 was compared with the two-step purified novel coronavirus solution (Batch 4 and Batch 5 first used the molecular sieve chromatography of Sepharose 4FF described in Example 1 and then used the ion exchange chromatography of Capto Q). The final purified solutions of batches 4 and 5 collected were prepared as stock solutions, and the antigen cumulative recovery rate, HCP cumulative removal rate, and DNA cumulative removal rate were calculated. The calculated results were compared and analyzed with the one-step purified novel coronavirus solution prepared in Example 1, as shown in Table 3.

[0105] Table 3 Comparison of experimental results of one-time purification and two-time purification process of novel coronavirus liquid

[0106]

[0107] It can be seen from the experimental results in Table 3 that the cumulative recovery rate of antigens in the two-step purification process is 51% to 59%, while the cumulative recovery rate of antigens in the one-step purification process of the present invention is 75% to 78%, and the cumulative recovery rate of antigens in the two-step purification process is lower than the cumulative recovery rate of antigens in the one-time purification process of the present invention; the cumulative removal rates of HCP and DNA in the two-step purification process are not less than 99.96% and 99.99%, respectively, while the cumulative removal rates of HCP and DNA in the one-step purification process of the present invention are not less than 99.81% and 99.99%, respectively, and the HCP removal rate of the two-step purification process is slightly higher than that of the one-step purification of the present invention, but the residual amounts of HCP and DNA in the purified samples after the one-step purification of the present invention meet the national limit standards.

[0108] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A method for preparing a serum-free Vero cell inactivated vaccine for novel coronavirus, characterized in that: The steps include: (1) Cell recovery: Take an appropriate amount of Vero cells from the working cell bank, thaw them in a water bath, inoculate them into a culture bottle containing cell culture medium, and culture them in a carbon dioxide incubator; (2) Cell expansion: Use serum-free medium to culture cells and expand them in a cell factory as required; after subculturing, place the cells in a constant temperature chamber and culture them into a uniform monolayer of cells; (3) Seed batch preparation: Delta purified strains are separated as the original seed bank. The virus liquid harvested from the original seed strain after continuous passage through Vero cells is the main seed batch virus stock. The virus liquid harvested from the main seed batch virus stock after continuous passage through Vero cells is the working seed batch virus stock. (4) Bioreactor cell culture: Serum-free cell growth medium is added to the bioreactor and Vero cells are inoculated for culture; (5) Virus culture: After the cells consume glucose and reach a stable period, replace the maintenance medium; inoculate the working seed batch virus into the production cells at an MOI of 0.005-0.

01. The same working seed batch virus is inoculated at the same MOI; (6) Inactivation of virus harvest fluid: pre-cool the virus harvest fluid, add β-propiolactone and mix well to inactivate; after inactivation, hydrolyze; (7) Clarification: After the inactivation verification inspection is passed, use a filter membrane for clarification and filtration; (8) Ultrafiltration and concentration: The clarified liquid is ultrafiltered and concentrated using an ultrafiltration membrane with a molecular weight cutoff of 100-300 KD, and the ultrafiltration concentrate is transferred to a cold storage for storage; (9) Enzyme digestion: add nuclease, shake well, and digest by enzyme; (10) Molecular sieve chromatography: The enzyme digestion solution is purified, the molecular sieve chromatography medium is Sepharose 4FF, and the first peak is collected as the stock solution; the stock solution is sampled and tested as required; (11) Preparation of semi-finished products: Add the stock solution to the preparation container, then add aluminum hydroxide adjuvant, add buffer, make up to the volume of the semi-finished product preparation solution, adjust the pH value, and stir; take samples and test the semi-finished products as required; (12) Packaging: Packaging the semi-finished product into pre-filled bottles; In step (1), the Vero cells are P136 generation CCL-81 Vero cells; In step (2), the serum-free medium is VP SFM; In step (2), the cells are expanded to passage 144; In step (3), the virus liquid obtained after the original seed strain is continuously propagated through CCL-81 Vero cells for 2 generations is the main seed batch virus stock, and the virus liquid obtained after the main seed batch virus stock is continuously propagated through CCL-81 Vero cells for 3 generations is the working seed batch virus stock; In step (4), the bioreactor is a basket-type bioreactor filled with sheet-like carriers; In step (4), the Vero cells are CCL-81 Vero cells; the specific parameters for culturing the inoculated Vero cells are: sheet carrier: 20-40 g / L, culture medium: VP SFM AGT, culture time: 12-14 days, stirring: 80-100 rpm, culture temperature: 37°C, culture pH: 7.0-7.2, DO: 30%-80%; In step (5), the virus culture parameters are: stirring: 80-100 rpm, temperature: 34±1°C, pH: 7.4-7.7, DO: 30%-80%; virus harvest liquid is harvested 2-3 days after virus culture; In step (9), the enzymatic hydrolysis temperature is 37±1°C and the enzymatic hydrolysis time is 6 to 10 hours; In step (10), the eluent for the molecular sieve chromatography is a phosphate buffer solution with a pH of 7.4 to 7.6, the linear flow rate is 0.1 to 0.5 cm / min, the sample loading amount is 2% to 6% of the column volume, and the detection wavelength is 280 nm; In step (11), the final concentration of the aluminum hydroxide adjuvant is 1 mg / ml; In step (11), the pH value is 7.0-8.

0.

2. A serum-free Vero cell inactivated vaccine of novel coronavirus, characterized in that: The vaccine is prepared by the method for preparing a serum-free Vero cell inactivated vaccine of the new coronavirus as described in claim 1.

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