A hepatitis A inactivated vaccine and preparation method thereof

Abstract

The present invention relates to the technical field of biomedicine, and specifically to an inactivated hepatitis A vaccine and a preparation method thereof. The steps include: cell amplification, virus culture, virus harvesting, cell disruption, ultrafiltration concentration, inactivation, purification, and finished product; the virus culture step is: isolating from the feces of hepatitis A patients and obtaining virus strains through adaptive passage; adding a carrier to the bioreactor for sterilization and standby use, inoculating the virus strain into the collected cell suspension and transferring it to the bioreactor for culture; the cell disruption step is: ultrasonically vibrating the collected virus harvest liquid to obtain a cell disruption liquid; adding chloroform for extraction, centrifuging, and collecting the supernatant virus liquid. The present invention provides a preparation method for an inactivated hepatitis A vaccine, which prepares an efficient, safe, and stable inactivated hepatitis A vaccine by optimizing the virus culture and inactivation process. The method has the advantages of high virus yield and low production cost, and is suitable for large-scale production and application.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an inactivated hepatitis A vaccine and a preparation method thereof. Background Art

[0002] Hepatitis A is an acute infectious disease caused by the hepatitis A virus (HAV), primarily transmitted through the fecal-oral route. The virus is widely distributed worldwide and is particularly common in areas with poor sanitation. Symptoms of hepatitis A infection include fever, fatigue, loss of appetite, nausea, vomiting, abdominal pain, and jaundice. While most patients recover fully, in rare cases, hepatitis A can lead to acute liver failure and even death. Therefore, preventing the spread of hepatitis A is crucial, and vaccination is one of the most effective preventive measures.

[0003] Currently, hepatitis A vaccines primarily include inactivated and live attenuated vaccines. Inactivated vaccines inactivate the virus through chemical or physical methods, rendering it infective but retaining immunogenicity. Inactivated vaccines are highly safe and suitable for widespread use, but they typically require multiple doses to maintain long-term immunity. Live attenuated vaccines weaken the virus through genetic engineering or continuous culture, while still stimulating an immune response. Live attenuated vaccines typically require only a single dose, but their safety profile is relatively low and they may cause adverse reactions in some populations. Inactivated vaccines, on the other hand, are widely used due to their safety and stability.

[0004] Although existing hepatitis A vaccines have achieved remarkable results in preventing the disease, their preparation methods still have some limitations: (1) Low virus culture efficiency: The replication efficiency of hepatitis A virus in cell culture is low, resulting in insufficient vaccine production and difficulty in meeting the needs of large-scale vaccination; (2) Challenges in the inactivation process: The inactivation process may destroy the immunogenicity of the virus, resulting in a decrease in viral immunogenicity and affecting the effectiveness of the vaccine. In addition, incomplete inactivation may lead to vaccine safety issues; (3) Complex purification steps: The existing purification process usually includes multiple steps of centrifugation, ultrafiltration and chromatography, which are cumbersome and costly; (4) Insufficient vaccine stability: The vaccine may be inactivated during storage and transportation due to factors such as temperature changes or light, affecting its effectiveness.

[0005] Therefore, it is of great significance to develop a method for preparing an inactivated hepatitis A vaccine that is efficient, safe, highly immunoprotective and economical. Summary of the Invention

[0006] In response to the above-mentioned deficiencies in the prior art, the present application provides an inactivated hepatitis A vaccine and a preparation method thereof.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] A method for preparing an inactivated hepatitis A vaccine comprises the following steps: cell amplification, virus culture, virus harvesting, cell disruption, ultrafiltration concentration, inactivation, purification, and finished product;

[0009] The virus culture steps include: isolating and adaptively passaged from feces of hepatitis A patients to obtain virus strains, establishing original seed batches, master seed batches, and working seed batches of the virus strains; adding a carrier into a bioreactor for sterilization and standby use; inoculating the virus strain into the collected cell suspension at an MOI of 0.2-1 and transferring the collected cell suspension to the bioreactor; controlling the bioreactor to continue culturing at 32-37° C., allowing the cultured cells to gradually adhere to the bioreactor carrier and continue to amplify, and allowing the virus to replicate within the cells; supplementing the culture medium during the culture period according to the monitored sugar content and pH value of the culture medium; and culturing at 32-37° C. for 22-28 days.

[0010] The cell disruption step comprises: ultrasonically vibrating the collected virus harvest solution to obtain a cell disruption solution; adding chloroform for extraction, centrifuging at 3000-5000 rpm for 15-30 minutes, and collecting the supernatant virus solution.

[0011] Preferably, the cultured cells are human embryonic lung diploid cells KMB 17 ; The virus strain is hepatitis A virus strain 8.

[0012] To further illustrate the technical solution of the present invention, the S1 cell expansion steps used in the embodiment are as follows: human embryonic lung diploid cells KMB 17 The cell culture medium is a mixture containing 5-10% (V / V) newborn calf serum, MEM medium and 1-3% (V / V) glutamine. The cells are revived and passaged to a cell factory for large-scale expansion culture. After the above cells have expanded to a certain number and grown densely, the cells are digested and collected to obtain a cell suspension.

[0013] Preferably, the virus harvesting step comprises: discarding the culture medium in the virus culture step, adding 0.3-1% (V / V) trypsin solution according to the amount of the vector to digest the cells, and then eluting and collecting the cells attached to the vector with PBS buffer having a concentration of 5-15 mmol / L and a pH value of 6.8-7.2 to obtain a virus harvest solution;

[0014] Preferably, the ultrafiltration concentration step comprises: concentrating the supernatant virus solution 40-60 times using a 300KD pore size membrane to form a virus concentrate;

[0015] Preferably, the purification step comprises: passing the inactivated solution through a 4FF molecular sieve packing to purify and remove impurities of different particle sizes, collecting effective antigens to form a purified solution, adding 1-5 mg / ml of glycine protective agent to the purified solution to maintain antigen stability, and forming a stock solution, that is, obtaining a hepatitis A inactivated vaccine stock solution;

[0016] The present invention aims to effectively shorten the time and workload associated with cell passage, while simultaneously increasing viral yield and improving production efficiency. This invention utilizes cell passage in cell flasks and cell factories, followed by cell amplification using vectors. This not only reduces the number of cell passages and workload, but also produces a high-yield, high-quality hepatitis A virus harvest.

[0017] Fish glue is a complex natural collagen whose molecular structure contains a large number of hydrophilic and hydrophobic functional groups such as amino, hydroxyl, carboxyl, and thiol groups. Therefore, fish glue has strong amphiphilicity and good biocompatibility, which is suitable for the attachment and growth of cells and viruses. In addition, fish glue can be degraded by enzymes secreted by cells. The degradation products are non-toxic and can be used by cells, making them suitable for long-term culture, low cost, and suitable for large-scale production.

[0018] Chitosan is a natural high-molecular polysaccharide with excellent biocompatibility and biodegradability. It has a positive charge on its surface and can interact with the negative charge on the cell membrane surface, providing a good growth environment for cells and promoting cell attachment and proliferation. It has natural antibacterial properties, which can inhibit the growth of bacteria and fungi, reduce the risk of contamination during cell culture, and ensure the safety of vaccines. When mixed with fish glue, it can form a more uniform porous structure, increase the specific surface area of the carrier, provide more attachment sites for cells, and promote cell growth and viral replication.

[0019] Genipin is a cross-linking agent. It is the product of Gardenia jasminoides hydrolysis by β-glucosidase. It is an excellent natural biological cross-linking agent that can be cross-linked with proteins, collagen, gelatin and chitosan to produce biomaterials. Its toxicity is much lower than glutaraldehyde and other commonly used chemical cross-linking agents.

[0020] The present invention uses fish glue solution to modify the surface of the chitosan carrier, thereby increasing the protein content on the carrier surface and improving the adhesion of cells. Fish glue, as a polypeptide mixture, contains multiple bioactive groups and can promote the interaction between cells and the chitosan carrier. The use of a cross-linking agent further strengthens the binding force between fish glue and the chitosan carrier, improves the mechanical strength of the carrier, makes it more stable during subsequent cell culture and operation, and reduces cell loss.

[0021] The carrier preparation method is as follows: fish glue, chitosan and solvent are stirred by a biological cross-linking agent, and freeze-dried to obtain the carrier.

[0022] Preferably, the carrier preparation method is as follows: mixing fish glue and water to prepare a 15-30wt% fish glue aqueous solution; mixing 8-12 parts by weight of a 10-20wt% fish glue aqueous solution with 30-70 parts by weight of cyclohexane, stirring at 25-35°C and 400-800rpm for 30-60min to obtain fish glue solution; adding 50-80 parts by weight of chitosan solution, continuing to stir for 3-10min to obtain a milky white solution; adding 3-8 parts by weight of a 5-10wt% biocrosslinking agent ethanol solution, continuing to stir for 1-4h, adding to n-hexane for separation, filtering under reduced pressure, washing, and freeze-drying to obtain a carrier.

[0023] Furthermore, the chitosan solution is prepared as follows: 2-5 parts by weight of chitosan is mixed with 40-80 parts by weight of a 1-3 wt% acetic acid aqueous solution, and heated to dissolve at 40-50° C. and 400-800 rpm to obtain a chitosan solution.

[0024] Furthermore, the bio-crosslinking agent is at least one of genipin and tannic acid; furthermore, the bio-crosslinking agent is a mixture of genipin and tannic acid in a mass ratio of 1:(1-2).

[0025] Genipin significantly improves the carrier strength and cell adhesion by cross-linking with amino groups in fish glue and chitosan; tannic acid cross-links with amino and hydroxyl groups in the carrier through phenolic hydroxyl groups, enhancing the carrier structure, antioxidant and antibacterial properties; the two mechanisms of action are complementary, synergistically improving the virus culture microenvironment and significantly increasing the viral antigen content and vaccine potency.

[0026] The vector prepared by the present invention, when used in the viral culture process of an inactivated hepatitis A vaccine, can effectively promote cell growth and viral proliferation, thereby improving vaccine production efficiency. Compared to existing hepatitis A vaccine preparations, the vector prepared by the present invention has the following advantages: it can provide a three-dimensional porous structure for viral culture, increasing the cell attachment area and providing space for cell adhesion, proliferation, and differentiation. This reduces the risk of fungal and bacterial infection, reduces impurities, optimizes the cell growth environment, reduces environmental damage to the virus, and enhances viral replication efficiency, thereby increasing virus yield, improving vaccine stability, purity, and shelf life, and enhancing safety and effectiveness.

[0027] Preferably, the culture medium is a mixture containing 1-10% (V / V) inactivated newborn calf serum and MEM culture medium.

[0028] Furthermore, the components of the MEM are 0.03-0.1wt% Glu and 0.05-0.3wt% NaHCO3.

[0029] Preferably, the inactivator is β-propiolactone, and the inactivation conditions are an inactivation temperature of 2-8° C. and an inactivation time of 18-36 hours.

[0030] Preferably, the ultrasonic power is 1000-2000 W, the ultrasonic frequency is 15-40 kHz, and the ultrasonic oscillation time is 20-50 min; and the extraction times are 3-5 times.

[0031] Preferably, a protective agent glycine is added during the purification.

[0032] Glycine is a small molecule amino acid with good stability and buffering capacity. When used during the purification and storage process, it can interact with the antigen surface through its zwitterionic properties (both hydrophilic and hydrophobic groups), reduce the aggregation between antigen molecules, and reduce the damage to the antigen structure caused by ice crystals formed during the freezing process. It can work synergistically with aluminum hydroxide adjuvant to prevent antigen degradation or inactivation during storage, improve the stability of the antigen, maintain the dispersion state of the antigen, and improve the uniformity and effectiveness of the vaccine.

[0033] Preferably, the finished product is added with an adjuvant aluminum hydroxide and a stabilizer glycine.

[0034] An inactivated hepatitis A vaccine is obtained by the above-mentioned preparation method.

[0035] The beneficial effects of the present invention are as follows: 1. The preparation method of the present invention adopts a carrier for culture, which not only improves the cell adhesion of the carrier, but also enhances its stability during the cell culture process, realizes the controllable separation between cells and the carrier, reduces the loss during the cell harvesting process, improves the overall efficiency and quality of cell culture, reduces the workload of liquid replacement and harvesting, and at the same time obtains a hepatitis A virus harvest liquid with high yield and good quality, improves production efficiency, is suitable for large-scale production, and reduces the cost of vaccine production.

[0036] 2. The present invention significantly improves virus yield, simplifies the process, shortens the production cycle, stabilizes the vaccine formula, and prolongs the shelf life by optimizing virus culture conditions, thereby realizing large-scale production and storage of inactivated hepatitis A vaccines.

[0037] 3. The inactivated hepatitis A vaccine prepared by the present invention has high immunogenicity, is safe and reliable, more efficient and has long-lasting immunity, and has wide clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0039] Figure 1 This is a picture of cell morphology when the virus was inoculated in Example 2. The picture is a 10×10 photo.

[0040] Figure 2 The hepatitis A virus reproduction activity curves of Example 2-11 after different culture days.

[0041] Figure 3 This is a picture of cell morphology when the virus was harvested in Example 2. The picture is a 10×10 photo.

[0042] Figure 4 This is the virus inactivation titer kinetic curve of the hepatitis A virus strain Lv 8 in Example 2 after passage in KMB17 cells.

[0043] Figure 5 This is a trend diagram showing the effect of adding different glycine concentrations on antigen stability in Examples 12-14. DETAILED DESCRIPTION

[0044] The above content of the present invention will be further described in detail below in conjunction with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments.

[0045] The following are the introductions of some raw materials in this application. Other raw materials not mentioned are commercially available:

[0046] Fish maw was purchased from Jiangsu Caiwei Biotechnology Co., Ltd.

[0047] Gelatin was purchased from Guangdong Mingcheng Biotechnology Co., Ltd., mesh size: 120 mesh.

[0048] Chitosan was purchased from Qingdao Haihui Biological Co., Ltd. with a molecular weight of 550,000 g / mol, a degree of deacetylation of 92.3%, and was food grade.

[0049] The commercially available vector was purchased from Bailinke Pharmaceutical Technology (Shanghai) Co., Ltd., model number Puredex® Cyto-1.

[0050] Example 1

[0051] A method for preparing an inactivated hepatitis A vaccine comprises the following steps:

[0052] S1 cell expansion: human embryonic lung diploid cells KMB 17 The cell culture medium is a mixture containing 8% (v / v) newborn calf serum, MEM medium, and 2% (v / v) glutamine. The cells are revived and passaged to a cell factory for large-scale expansion culture. The composition of the MEM is 0.05wt% Glu and 0.1wt% NaHCO3.

[0053] S2 virus culture: Hepatitis A virus strain 8 was isolated from the feces of hepatitis A patients and obtained through adaptive passage, and the original seed batch, master seed batch and working seed batch of hepatitis A virus were established; after the above cells grew densely in the cell factory, the hepatitis A virus strain 8 was inoculated at an MOI of 0.5 and continued to be cultured at 35℃. 17 The cells were infected and virus replication occurred; during the culture period, culture medium was added according to the monitored sugar content and pH value of the culture medium; the culture medium was a mixture of 2% (v / v) inactivated newborn calf serum and MEM medium; the cells were cultured at 35°C for 22 days; the composition of the MEM medium was the same as that in S1;

[0054] S3 Virus Harvest: After discarding the culture medium, add 0.5% (v / v) trypsin solution to each layer of the cell factory to digest the cells. Then, elute and collect the cells attached to the cell factory with 10 mmol / L PBS buffer, pH 7.0, to obtain the virus harvest solution;

[0055] S4 cell disruption: The collected virus harvest liquid was placed under ultrasonic vibration at a power of 1500W and an ultrasonic frequency of 20kHz for 30 minutes to obtain a cell disruption liquid; chloroform was added for extraction 4 times, and the supernatant virus liquid was collected by centrifugation at 4000 rpm for 20 minutes;

[0056] S5 ultrafiltration concentration: The supernatant virus solution is concentrated 50 times using a 300KD pore size membrane to form a virus concentrate;

[0057] S6 inactivation: Add an inactivator to the concentrate at a ratio of 1:4000 (V / V) and inactivate the virus at 4°C for 24 hours. After inactivation, place the concentrate at 37°C for 3 hours to form a virus inactivation solution; the inactivator is β-propiolactone;

[0058] S7 purification: The inactivated solution is purified by passing it through a 4FF molecular sieve packing to remove impurities of different particle sizes, and the effective antigen is collected to form a purified solution. 3 mg / ml of glycine protective agent is added to the purified solution to maintain antigen stability to form a stock solution, i.e., the hepatitis A inactivated vaccine stock solution;

[0059] S8 finished product: Add the adjuvant aluminum hydroxide and the stabilizer glycine at 1 mg / ml to the above-mentioned inactivated hepatitis A vaccine stock solution, mix thoroughly, and then divide and package to obtain the inactivated hepatitis A vaccine.

[0060] Example 2

[0061] A method for preparing an inactivated hepatitis A vaccine comprises the following steps:

[0062] S1 cell expansion: human embryonic lung diploid cells KMB 17The cell culture medium is a mixture containing 8% (v / v) newborn calf serum, MEM medium, and 2% (v / v) glutamine. The cells are revived and passaged to a cell factory for large-scale expansion culture. After the cells have expanded to a certain number and grown densely, the cells are digested and collected to obtain a cell suspension. The components of the MEM are 0.05wt% Glu and 0.1wt% NaHCO3.

[0063] S2 virus culture: Hepatitis A virus strain 8 was isolated from the feces of hepatitis A patients and obtained through adaptive passage, and the original seed batch, main seed batch and working seed batch of hepatitis A virus were established; the carrier was added to the bioreactor for sterilization and standby use, and the hepatitis A virus strain 8 was inoculated into the collected cell suspension at an MOI of 0.5 and transferred to the bioreactor, and the bioreactor was controlled to continue culturing at 35℃. KMB 17 The cells gradually adhered to the bioreactor carrier and continued to proliferate, and the virus replicated within the cells. During the culture period, culture medium was added according to the monitored sugar content and pH value of the culture medium. The culture medium was a mixture of 2% (v / v) inactivated newborn calf serum and MEM medium. The cells were cultured at 35°C for 22 days. The composition of the MEM medium was the same as that in S1.

[0064] S3 Virus Harvest: After discarding the culture medium, add 0.5% (v / v) trypsin solution to each layer of the cell factory to digest the cells. Then, elute and collect the cells attached to the cell factory with 10 mmol / L PBS buffer, pH 7.0, to obtain the virus harvest solution;

[0065] S4 cell disruption: The collected virus harvest liquid was placed under ultrasonic vibration at a power of 1500W and an ultrasonic frequency of 20kHz for 30 minutes to obtain a cell disruption liquid; chloroform was added for extraction 4 times, and the supernatant virus liquid was collected by centrifugation at 4000 rpm for 20 minutes;

[0066] S5 ultrafiltration concentration: The supernatant virus solution is concentrated 50 times using a 300KD pore size membrane to form a virus concentrate;

[0067] S6 inactivation: Add an inactivator to the concentrate at a ratio of 1:4000 (V / V) and inactivate the virus at 4°C for 24 hours. After inactivation, place the concentrate at 37°C for 3 hours to form a virus inactivation solution; the inactivator is β-propiolactone;

[0068] S7 purification: The inactivated solution is purified by passing it through a 4FF molecular sieve packing to remove impurities of different particle sizes, and the effective antigen is collected to form a purified solution. 3 mg / ml of glycine protective agent is added to the purified solution to maintain antigen stability to form a stock solution, i.e., the hepatitis A inactivated vaccine stock solution;

[0069] S8 finished product: Add the adjuvant aluminum hydroxide and the stabilizer glycine at 1 mg / ml to the above-mentioned inactivated hepatitis A vaccine stock solution, mix thoroughly, and then divide and package to obtain the inactivated hepatitis A vaccine.

[0070] The carrier preparation method in S2 is as follows: fish glue and water are mixed to form a 20wt% fish glue aqueous solution; 10 parts by weight of a 15wt% fish glue aqueous solution are mixed with 40 parts by weight of cyclohexane, and stirred at 30°C and 600rpm for 40min to obtain fish glue solution; 60 parts by weight of chitosan solution are added, and stirring is continued for 5min to obtain a milky white solution; 5 parts by weight of an 8wt% genipin ethanol solution are added, and stirring is continued for 2h, and the solution is added into n-hexane for separation, filtered under reduced pressure, washed, and freeze-dried to obtain a carrier; the chitosan solution is prepared as follows: 3 parts by weight of chitosan are mixed with 60 parts by weight of a 1.5wt% acetic acid aqueous solution, and heated to dissolve at 45°C and 600rpm to obtain a chitosan solution.

[0071] Example 3

[0072] The same as Example 1, except that the S2 virus culture step is as follows: the hepatitis A virus strain 8 is isolated from the feces of hepatitis A patients and adaptively passaged to obtain the hepatitis A virus strain 8, establish the hepatitis A virus strain original seed batch, master seed batch and working seed batch; add the carrier into the bioreactor for sterilization, inoculate the hepatitis A virus strain 8 into the collected cell suspension at an MOI of 0.5 and transfer it to the bioreactor, control the bioreactor to continue culturing at 35°C, KMB 17 The cells gradually adhered to the bioreactor carrier and continued to proliferate, and the virus replicated within the cells. During the culture period, culture medium was added according to the monitored sugar content and pH value of the culture medium. The culture medium was a mixture of 2% (v / v) inactivated newborn calf serum and MEM culture medium. The cells were cultured at 35°C for 20 days. The components of the MEM were the same as those in S1.

[0073] Example 4

[0074] The same as Example 1, except that the S2 virus culture step is as follows: the hepatitis A virus strain 8 is isolated from the feces of hepatitis A patients and adaptively passaged to obtain the hepatitis A virus strain 8, establish the hepatitis A virus strain original seed batch, master seed batch and working seed batch; add the carrier into the bioreactor for sterilization, inoculate the hepatitis A virus strain 8 into the collected cell suspension at an MOI of 0.5 and transfer it to the bioreactor, control the bioreactor to continue culturing at 35°C, KMB 17The cells gradually adhered to the bioreactor carrier and continued to proliferate, and the virus replicated within the cells. During the culture period, culture medium was added according to the monitored sugar content and pH value of the culture medium. The culture medium was a mixture of 2% (v / v) inactivated newborn calf serum and MEM culture medium. The cells were cultured at 35°C for 24 days. The components of the MEM were the same as those in S1.

[0075] Example 5

[0076] The method is basically the same as Example 2, except that the culture days are 14 days.

[0077] Example 6

[0078] The method is basically the same as Example 2, except that the culture days are 16 days.

[0079] Example 7

[0080] The method is basically the same as Example 2, except that the culture days are 18 days.

[0081] Example 8

[0082] The method is basically the same as Example 2, except that the culture days are 26 days.

[0083] Example 9

[0084] The method is basically the same as Example 2, except that the culture days are 28 days.

[0085] Example 10

[0086] The method is basically the same as Example 2, except that the culture days are 30 days.

[0087] Example 11

[0088] The method is basically the same as Example 2, except that the culture days are 32 days.

[0089] Example 12

[0090] The process is basically the same as Example 2, except that in S7 purification: the inactivated liquid is passed through a 4FF molecular sieve packing to purify and remove impurities of different particle sizes, and the effective antigens are collected to form a purified liquid, thereby obtaining the hepatitis A inactivated vaccine stock solution.

[0091] Example 13

[0092] The method is basically the same as Example 2, except that S7 purification is performed: the inactivated liquid is passed through a 4FF molecular sieve filler to purify impurities of different particle sizes, and the effective antigen is collected to form a purified liquid. 1 mg / ml glycine protective agent is added to the purified liquid to maintain the stability of the antigen to form a stock solution, that is, the hepatitis A inactivated vaccine stock solution is obtained.

[0093] Example 14

[0094] The method is basically the same as Example 2, except that S7 purification is performed: the inactivated liquid is passed through a 4FF molecular sieve filler to purify impurities of different particle sizes, the effective antigen is collected to form a purified liquid, and 5 mg / ml glycine protective agent is added to the purified liquid to maintain the stability of the antigen to form a stock solution, that is, the hepatitis A inactivated vaccine stock solution is obtained.

[0095] Example 15

[0096] Basically the same as Example 2, the fish glue in the carrier preparation method in S2 is replaced by gelatin.

[0097] Example 16

[0098] Basically the same as Example 2, the biocrosslinking agent in the carrier preparation method in S2 is replaced by tannic acid.

[0099] Example 17

[0100] Basically the same as Example 2, the biocrosslinking agent in the carrier preparation method in S2 is a mixture of genipin and tannic acid in a mass ratio of 2:3.

[0101] Comparative Example 1

[0102] Basically the same as Example 2, the carrier is commercially available.

[0103] Performance testing

[0104] (1) Comparison of Vaccine Potency The inactivated hepatitis A vaccines prepared in Examples 1-2 and 12-14 were tested for potency. The potency was tested according to the 2020 edition of the Pharmacopoeia. The specific test results are shown in Table 1.

[0105] Table 1 Test results of the potency of inactivated hepatitis A vaccine

[0106]

[0107] From the above embodiments and combined Figure 5 It can be seen that the inactivated hepatitis A vaccine prepared in Example 2 has a high titer, with the titer being above 1.7. In particular, after the addition of the carrier in Example 2, the titer was significantly improved, reaching 2.068, demonstrating that the addition of the carrier can significantly increase the vaccine titer, and the vaccine titer is stably maintained at a high level.

[0108] (2) Viral antigen content analysis and detection method: According to the enzyme-linked immunosorbent assay (ELISA), the hepatitis A virus harvest sample to be tested in the example was serially diluted 2-fold with PBS buffer. The dilutions of appropriate dilutions were used for ELISA detection, and replicate wells were set for each dilution. Hepatitis A antigen determination wells, positive and negative control wells, and blank control wells were set. The ELISA test was performed according to the kit instructions to detect the antigen content in the hepatitis A virus harvest. The results are shown in Table 2.

[0109] Table 2 Detection results of inactivated hepatitis A virus antigen content

[0110]

[0111] From the above results, it can be seen that the virus culture can be obtained by using the carrier used in the present invention.

[0112] A higher antigen content indicates that the prepared virus harvest liquid has a higher efficacy. As can be seen from Example 2 and Example 15, the antigen content is higher. The possible reason is that fish glue has higher biocompatibility and cell adhesion properties than gelatin, so a higher antigen content can be obtained during the virus culture process. Further comparison of Example 2 and Examples 16-17 shows that the use of biocrosslinkers such as genipin and tannic acid has better antigen content and higher titer. The possible reason is that genipin significantly improves the carrier strength and cell adhesion by crosslinking with amino groups in fish glue and chitosan; tannic acid crosslinks with amino groups and hydroxyl groups in the carrier through phenolic hydroxyl groups to enhance the carrier structure, antioxidant and antibacterial properties; the two mechanisms of action complement each other, synergistically improve the virus culture microenvironment, and significantly improve the viral antigen content and vaccine titer.

[0113] (3) Testing the prepared cells and viruses

[0114] Figure 1 This is a cell morphology diagram obtained after the cell expansion culture in Example 2. It can be seen from the figure that the cell morphology is good when inoculated with the virus, the density is moderate, the cells are evenly attached to the wall, and there are no obvious plaques or detachment, indicating that the KMB17 cells have been fully expanded before inoculation with the virus and have reached a growth state suitable for viral infection.

[0115] Figure 2 In Example 2, the virus reproduction activity was tested for different days in the virus culture step. As can be seen from the figure, the reproduction activity showed a trend of gradual increase over time during the culture process from 0 to 22 days, indicating that the virus replicated stably in the cells without obvious replication inhibition or cytotoxicity. The reproduction activity showed a downward trend from 22 days to 32 days, with a peak of reproduction occurring on the 22nd day of culture. The virus reproduction activity reached a peak on the 20th to 24th day of culture, indicating that the virus was stable in KMB. 17 The cells replicate more efficiently.

[0116] Figure 3 The cell morphology diagram of the virus harvest in Example 2 shows that the cell morphology has changed significantly, and some cells have shrunk, fallen off or lysed, indicating that the virus replication has caused cell pathological changes. The changes in cell morphology are consistent with the virus reproduction activity curve ( Figure 2 ) peak time is consistent, further verifying that the virus successfully replicated in the cells and reached a peak, indicating that the virus is sensitive to KMB 17 The cells have a high infection efficiency and are suitable for virus harvesting.

[0117] Figure 4 In Example 2, when the inactivator β-propiolactone was used for inactivation, the inactivation curve showed that the virus titer dropped rapidly in the early stage of inactivation and then stabilized, indicating that β-propiolactone can effectively inactivate the virus at 2-8°C. It completely lost its activity 14 hours after inactivation and significantly reduced the virus titer, indicating that its inactivation effect is significant, verifying the reliability of the inactivation process and ensuring the safety of the vaccine.

[0118] Figure 5 This is a trend graph of antigen stability when different glycines are used as protective agents in Example 2. It can be seen from the graph that glycine concentration exceeding 3 mg / ml may have limited effect on improving stability, and may even have a negative impact. When the glycine concentration is 3 mg / ml, the antigen stability is significantly improved, indicating that glycine can effectively protect the antigen from degradation or denaturation, and can maintain the stability of the antigen during purification and storage, thereby ensuring the effectiveness of the vaccine.

[0119] The above embodiments are merely examples for clarity of explanation and are not intended to limit the embodiments. Those skilled in the art will appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all embodiments here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for preparing an inactivated hepatitis A vaccine, characterized in that: The following steps are involved: Cell expansion, virus culture, virus harvesting, cell disruption, ultrafiltration concentration, inactivation, purification, finished product; The virus culture steps include: isolating and adaptively passaged from feces of hepatitis A patients to obtain virus strains, establishing original seed batches, master seed batches, and working seed batches of the virus strains; adding a carrier into a bioreactor for sterilization and standby use; inoculating the virus strain into the collected cell suspension at an MOI of 0.2-1 and transferring the collected cell suspension to the bioreactor; controlling the bioreactor to continue culturing at 32-37° C., allowing the cultured cells to gradually adhere to the bioreactor carrier and continue to amplify, and allowing the virus to replicate within the cells; supplementing the culture medium during the culture period according to the monitored sugar content and pH value of the culture medium; and culturing at 32-37° C. for 22-28 days. The cell disruption step comprises: ultrasonically vibrating the collected virus harvest solution to obtain a cell disruption solution; adding chloroform for extraction, centrifuging at 3000-5000 rpm for 15-30 minutes, and collecting the supernatant virus solution; The carrier preparation method is as follows: mixing fish glue and water to prepare a 15-30wt% fish glue aqueous solution; mixing 8-12 parts by weight of a 10-20wt% fish glue aqueous solution with 30-70 parts by weight of cyclohexane, stirring at 25-35°C and 400-800 rpm for 30-60 minutes to obtain fish glue solution; adding 50-80 parts by weight of chitosan solution, continuing to stir for 3-10 minutes to obtain a milky white solution; adding 3-8 parts by weight of a 5-10wt% biocrosslinking agent ethanol solution, continuing to stir for 1-4 hours, adding n-hexane for separation, filtering under reduced pressure, washing, and freeze-drying to obtain the carrier; The biological cross-linking agent is a mixture of genipin and tannic acid in a mass ratio of 1:(1-2).

2. The method for preparing the inactivated hepatitis A vaccine according to claim 1, wherein The cultured cells are human embryonic lung diploid cells KMB17; the virus strain is hepatitis A virus strain Lv 8.

3. The method for preparing the inactivated hepatitis A vaccine according to claim 1, wherein The culture medium is a mixture containing 1-10% (V / V) inactivated newborn calf serum and MEM culture medium.

4. The method for preparing the inactivated hepatitis A vaccine according to claim 1, wherein The inactivation agent is β-propiolactone, and the inactivation conditions are an inactivation temperature of 2-8°C and an inactivation time of 18-36h.

5. The method for preparing the inactivated hepatitis A vaccine according to claim 1, wherein The ultrasonic power is 1000-2000W, the ultrasonic frequency is 15-40kHz, and the ultrasonic oscillation time is 20-50min; and the extraction times are 3-5 times.

6. The method for preparing the inactivated hepatitis A vaccine according to claim 1, wherein Glycine was added during the purification.

7. The method for preparing the inactivated hepatitis A vaccine according to claim 1, wherein Aluminum hydroxide and glycine are added to the finished product.

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