Preparation method of freeze-dried varicella attenuated live vaccine and freeze-dried varicella attenuated live vaccine

Abstract

The invention relates to the technical field of biological agents, and particularly discloses a preparation method of a freeze-dried varicella attenuated live vaccine and the freeze-dried varicella attenuated live vaccine. The preparation method of the freeze-dried varicella attenuated live vaccine provided by the invention comprises the following steps: culturing cells, infecting the cells with viruses, and harvesting the cells. Wherein the specific steps of harvesting the cells are as follows: adding a stabilizer into the washed cells, then harvesting a cell culture by using an ultrasonic cell harvesting device, and filtering the cell culture to obtain a stock solution; the process parameters in the ultrasonic cell harvesting device are as follows: the ultrasonic power is 60-90%, the ultrasonic time is 1.0-2.5 s, and the ultrasonic frequency is 1-2; the invention also provides the freeze-dried varicella attenuated live vaccine obtained by the preparation method. The preparation method of the freeze-dried varicella attenuated live vaccine provided by the invention has the advantages of simple process steps, short preparation period, high process stability and the like, and the obtained freeze-dried varicella attenuated live vaccine is good in quality consistency and good in clinical medication safety.

Description

Technical Field

[0001] The present application relates to the technical field of biological preparations, and in particular to a method for preparing a freeze-dried live attenuated varicella vaccine and a freeze-dried live attenuated varicella vaccine. Background Art

[0002] As an important biological product for preventing varicella, freeze-dried live attenuated varicella vaccine plays an irreplaceable role in the global public health system. Varicella is a highly contagious disease caused by varicella-zoster virus (VZV). VZV has only one serotype and humans are the only natural host of the virus. Therefore, early prevention of VZV is extremely important. With the advancement of vaccine technology and the rapid development of bioengineering technology, especially the introduction of new cell lines and bioreactors, the production efficiency and protective efficacy of freeze-dried live attenuated varicella vaccine have been improved.

[0003] At present, the method for preparing freeze-dried varicella live attenuated vaccine in the industry mainly includes the following steps: cell culture, virus infection, cell harvesting and stock solution filtration. Among them, the cell harvesting step requires the use of effective means to release and recover virus particles from host cells. The current traditional methods of cell harvesting mainly include ultrasonic disruption, chemical reagents or enzyme treatment. Among them, methods such as chemical reagents or enzyme treatment have problems such as cumbersome and complicated processes, long preparation cycles, and low virus yields, and cannot be widely promoted and used; ultrasonic disruption of cell harvesting is the most widely used due to its advantages such as simple processes and short preparation cycles. However, the existing ultrasonic equipment may have improper power settings or insufficient action time, resulting in incomplete destruction of the cell structure, which in turn reduces the efficiency and quality of subsequent virus harvesting, making it difficult to achieve efficient cell disruption and virus release.

[0004] Therefore, how to simplify the production process of freeze-dried varicella live attenuated vaccine, shorten the production cycle, and increase the virus harvest rate are still key technical problems that need to be urgently solved in the industry at this stage. Summary of the invention

[0005] In order to simplify the production process of freeze-dried varicella live attenuated vaccine, shorten the production cycle, and improve the virus harvest rate, the present application provides a preparation method of freeze-dried varicella live attenuated vaccine and a freeze-dried varicella live attenuated vaccine.

[0006] In the first aspect, the present application provides a method for preparing a freeze-dried live attenuated varicella vaccine, using the following technical scheme: A method for preparing a freeze-dried live attenuated varicella vaccine, comprising the following steps: (1) Cell culture: MRC-5 human diploid cells were expanded and subcultured within 35 generations; (2) Virus infection of cells: After the MRC-5 human diploid cells grow into a uniform and dense monolayer of cells, add the Oka strain virus seed to infect the cells, and then infect, culture, and wash; (3) harvesting cells: adding a stabilizer to the washed cells, then using an ultrasonic cell harvester to harvest the cell culture, and filtering the cell culture to obtain a stock solution; The process parameters of the ultrasonic cell harvesting device are as follows: ultrasonic power 60-90%, ultrasonic times 1-2 times, and ultrasonic time of each time 1.0-2.5 seconds.

[0007] The present application provides a method for preparing a freeze-dried live attenuated varicella vaccine, which has the advantages of simple process steps, short preparation cycle, high virus harvest rate, stable process, etc. The obtained freeze-dried live attenuated varicella vaccine has good quality consistency and good clinical drug safety. The above-mentioned preparation method first subcultures the cells to within 35 generations, ensures the activity and consistency of MRC-5 human diploid cells, reduces the possibility of genetic variation, and improves the safety and stability of the vaccine; secondly, the Oka strain virus is used to infect the cells, and the virus is added at the appropriate time point to ensure the effective infection of the virus and improve the titer of the virus. Finally, a new type of ultrasonic cell harvesting device is used for cell harvesting. By controlling the ultrasonic power, ultrasonic time and ultrasonic number, the active components of the virus can be retained to the maximum extent while effectively destroying the cell structure, reducing the degree of damage to the virus particles, and obtaining highly active varicella virus. In summary, the preparation method of the freeze-dried live attenuated varicella vaccine provided by the present application improves the virus harvesting efficiency while simplifying the preparation process, and provides effective technical support for the large-scale production of freeze-dried live attenuated varicella vaccine.

[0008] Optionally, the ultrasonic power is 70-85%.

[0009] In some embodiments, the ultrasound power may be 60-70%, 60-80%, 60-85%, 60-90%, 70-80%, 70-85%, 70-90%, 80-85%, 80-90%, or 85-90%.

[0010] In a specific embodiment, the ultrasound power may also be 60%, 70%, 80%, 85% or 90%.

[0011] Optionally, the number of ultrasounds is 2 times.

[0012] Optionally, the pore size of the filter used for the filtration is 40 to 105 μm.

[0013] Optionally, the stabilizer comprises the following components: sucrose 60-75 g / L, trehalose 20-25 g / L, sodium glutamate 10-12 g / L, mannitol 15-20 g / L and small molecule peptide 10-50 mL / L, and the balance is water; The small molecule peptide is prepared from glutamic acid and lysine in a molar ratio of 1:(0.27-0.43).

[0014] In this application, sucrose, trehalose, sodium glutamate, mannitol and small molecule peptides are used as stabilizers, which are added to cells before cell harvesting, thereby playing a key protective role in cell harvesting and subsequent processing, significantly improving the stability of intracellular virus particles, and ensuring the virus titer in the freeze-dried varicella live attenuated vaccine. Among them, the use of small molecule peptides can effectively avoid the polymerization of varicella virus caused by ultrasonic vibration, high temperature and high concentration, greatly improving the effectiveness of varicella virus antigens. In addition, the stabilizer provided in this application also helps to reduce the loss of active ingredients of the vaccine during subsequent freeze-drying and re-dissolution, effectively ensuring the biological activity of the final vaccine product and its long-term storage stability, and further improving the effectiveness and safety of the vaccine.

[0015] Optionally, the preparation method of the small molecule peptide is: dissolving glutamic acid in water and heating to 100±5°C; then adding lysine and adjusting the pH to 6.5-7.0; finally adding water to dilute to a final concentration of glutamic acid of 0.4-0.6M to obtain the small molecule peptide.

[0016] Optionally, the addition amount of the small molecule peptide is 10-35 mL / L.

[0017] In some embodiments, the addition amount of the small molecule peptide can be 10 mL / L, 20 mL / L, 35 mL / L or 50 mL / L.

[0018] In a specific embodiment, the addition amount of the small molecule peptide can also be 10-20 mL / L, 10-35 mL / L, 10-50 mL / L, 20-35 mL / L, 20-50 mL / L or 35-50 mL / L.

[0019] In some embodiments, the molar ratio of glutamic acid to lysine can be 1:(0.27-0.33), 1:(0.27-0.38), 1:(0.33-0.38), 1:(0.33-0.43) or 1:(0.38-0.43).

[0020] In a specific embodiment, the molar ratio of glutamic acid to lysine can also be 1:0.27, 1:0.33, 1:0.38 or 1:0.43.

[0021] Optionally, the preparation method also includes freeze-drying, and the specific steps are: pre-cooling the freeze-dried protective agent at 2-8°C, and then adding it to the original solution for freeze-drying to obtain a freeze-dried dosage form of varicella live attenuated vaccine; the freeze-dried protective agent includes the following components: sucrose 40-60g / L, trehalose 15-20g / L, sodium glutamate 5-10g / L, mannitol 10-15g / L, human serum albumin 10-30g / L, and the balance is water.

[0022] In the present application, the pre-cooled lyoprotectant is added to the stock solution for lyophilization, which can significantly improve the stability and biological activity of the lyophilized live attenuated varicella vaccine. Specifically, by selecting the above-mentioned components and their contents of lyoprotectants, the active ingredients of the vaccine can be effectively protected during the lyophilization process, and the activity loss caused by freeze drying can be avoided, thereby ensuring the stability of the lyophilized vaccine during storage and transportation. At the same time, the lyoprotectant formula can quickly form a uniform and stable solution during reconstitution, which is beneficial to improving the safety and immune effect of the vaccine.

[0023] Optionally, step (2) is specifically as follows: after the MRC-5 human diploid cells grow into a uniform and dense monolayer of cells, the medium is replaced with a MEM medium having a pH of 7.3 to 7.5 and containing 1 to 3% newborn calf serum, and the Oka strain virus is added to infect the cells, with an infection multiplicity MOI of 0.01 to 0.05. After 48 to 60 hours of infection, the culture medium is discarded, the cells are washed with PBS buffer, and then replaced with a MEM medium having a pH of 7.3 to 7.5 and without newborn calf serum, and the cells are cultured at 33 to 35°C. When more than 75% of the MRC-5 human diploid cells show typical lesions, the original maintenance medium is discarded, and the cell surface is washed with a PBS buffer precooled at 2 to 8°C.

[0024] Optionally, the culture medium used for the amplification subculture is: L-15 culture medium with a pH of 7.3 to 7.5 and containing 6 to 10% newborn calf serum.

[0025] In a second aspect, the present application provides a freeze-dried live attenuated varicella vaccine, which is prepared using the above-mentioned method for preparing the freeze-dried live attenuated varicella vaccine.

[0026] In summary, this application has the following beneficial effects: 1. The present application provides a method for preparing a freeze-dried live attenuated varicella vaccine, which uses a specific ultrasonic cell harvesting device to harvest cells, and by controlling the ultrasonic power to 60-90%, the ultrasonic time to 1.0-2.5s, and the number of ultrasonic times to 1-2 times, it can effectively destroy the cell structure while retaining the active components of the virus to the maximum extent, reduce the degree of damage to the virus particles, and greatly improve the virus harvesting efficiency.

[0027] 2. The preparation method of the freeze-dried varicella live attenuated vaccine provided in this application has the advantages of simple process steps, short preparation cycle, and high process stability. The obtained freeze-dried varicella live attenuated vaccine has good quality consistency and good clinical drug safety, which provides effective technical support for the large-scale production of freeze-dried varicella live attenuated vaccine.

[0028] 3. This application uses sucrose, trehalose, sodium glutamate, mannitol and small molecule peptides as stabilizers, and further controls the content of small molecule peptides within the range of 10-35mL / L, so that the obtained freeze-dried varicella live attenuated vaccine has better thermal stability and storage stability. DETAILED DESCRIPTION

[0029] The present application provides a method for preparing a freeze-dried live attenuated varicella vaccine, comprising the following steps: (1) Cell culture: MRC-5 human diploid cells were cultured at 36-38°C for 4-5 days in L-15 medium with a pH of 7.3-7.5 and 6-10% newborn calf serum. The subculture ratio was 1:(2-4) and the number of subcultures was within 35.

[0030] (2) Virus infection of cells: After the MRC-5 human diploid cells grow into a uniform and dense monolayer of cells, the medium is replaced with MEM medium at pH 7.3-7.5 and containing 1-3% newborn calf serum, and the Oka strain virus is added to infect the cells at an infection multiplicity of infection (MOI) of 0.01-0.05. After 48-60 hours of infection, the culture medium is discarded and the cells are washed with PBS buffer. The medium is then replaced with MEM medium at pH 7.3-7.5 and without newborn calf serum and cultured at 33-35°C. When more than 75% of the MRC-5 human diploid cells show typical lesions, the original maintenance medium is discarded and the cell surface is washed with PBS buffer precooled at 2-8°C.

[0031] (3) Harvesting cells: adding a stabilizer to the cells obtained in step (2) in an amount of 1-2 L / cell factory, then harvesting the cell culture using an ultrasonic cell harvesting device, and filtering the cell culture to obtain a stock solution; The stabilizer comprises the following components: 60-75 g / L sucrose, 20-25 g / L trehalose, 10-12 g / L sodium glutamate, 15-20 g / L mannitol and 10-50 mL / L small molecule peptide, and the balance is water; the small molecule peptide is prepared from glutamic acid and lysine in a molar ratio of 1:(0.27-0.43); further, the preparation method of the small molecule peptide is as follows: dissolving glutamic acid in water and heating to 100±5°C; then adding lysine and adjusting the pH to 6.5-7.0; finally adding water to dilute to a final concentration of glutamic acid of 0.4-0.6 M to obtain the small molecule peptide.

[0032] The process parameters of the ultrasonic cell harvesting device are as follows: ultrasonic power 60-90%, ultrasonic times 1-2 times, and ultrasonic time of each time 1.0-2.5s; the pore size of the filter used in the filtration is 40-105 μm.

[0033] (4) Freeze-drying: pre-cool the freeze-drying protective agent at 2-8°C, then add it to the stock solution for freeze-drying to obtain a freeze-dried dosage form of the live attenuated varicella vaccine; The freeze-drying protective agent comprises the following components: 40-60 g / L sucrose, 15-20 g / L trehalose, 5-10 g / L sodium glutamate, 10-15 g / L mannitol, 10-30 g / L human albumin, and the balance is water.

[0034] In the present application, MRC-5 diploid cells were derived from ATCC; Oka strain virus was derived from ATCC; ultrasonic cell harvesting device was purchased from Wuhan Minzhao Biotechnology Co., Ltd.; the remaining raw materials, reagents, solvents, etc. used in the present application can be obtained commercially.

[0035] The present application is further described in detail below in combination with preparation examples, embodiments, and performance testing experiments.

[0036] Preparation Example 1-5 Preparation Examples 1-5 each provide a stabilizer.

[0037] The difference between the above preparation examples is that the amount of small molecule peptide added in the stabilizer is shown in Table 1 below.

[0038] The preparation method of the stabilizer provided in Preparation Example 1-5 comprises the following steps: (1) Preparation of small molecule peptide: 7.36 g of glutamic acid was dissolved in 50 mL of water and heated to 100°C; then 5.56 g of lysine was added and the pH was adjusted to 6.7; finally, water was added to dilute the mixture to a final concentration of glutamic acid of 0.5 M to obtain a small molecule peptide. In the above small molecule peptide, the molar ratio of glutamic acid to lysine was 1:(0.38).

[0039] (2) Dissolve 70 g of sucrose, 20 g of trehalose, 12 g of sodium glutamate, 18 g of mannitol and a small molecule peptide in 1 L of water and stir well to obtain a stabilizer.

[0040] Table 1 Addition amount of small molecule peptide in the stabilizer provided in Preparation Examples 1-5 Preparation Example Addition amount of small molecule peptide (mL) 1 0 2 10 3 20 4 35 5 50 Preparation Example 6-8 Preparation Examples 6-8 each provide a stabilizer.

[0041] The difference of the above-mentioned Preparation Example 4 is that the molar ratio of glutamic acid to lysine in the small molecule peptide is specifically shown in Table 2 below.

[0042] Table 2 Molar ratio of glutamic acid to lysine in small molecule peptides of Preparation Example 4 and Preparation Examples 6-8 Preparation Example 9 Preparation Example 9 provides a stabilizer.

[0043] The difference of the above Preparation Example 4 is that the small molecule peptide is replaced by 2.57 g of glutamic acid.

[0044] Preparation Example 10 Preparation Example 10 provides a stabilizer.

[0045] The difference of the above Preparation Example 4 is that the small molecule peptide is replaced by 0.9 g of lysine.

[0046] Examples 1-10 Examples 1-10 respectively provide a method for preparing a freeze-dried live attenuated varicella vaccine.

[0047] The difference between the above embodiments is that in step (3) of the method for preparing freeze-dried live attenuated varicella vaccine, the stabilizer is derived from Preparation Examples 1-10 respectively.

[0048] The preparation method of the freeze-dried live attenuated varicella vaccine provided in Examples 1-10 comprises the following steps: (1) Cell culture: Human diploid MRC-5 cells were cultured at a subculture ratio of 1:4 in L-15 medium (pH 7.5) containing 6% newborn calf serum at 36°C for 5 days for amplification and subculture within 35 generations.

[0049] (2) Virus infection of cells: After the MRC-5 human diploid cells grow into a uniform and dense monolayer of cells, replace the MEM culture medium with a pH of 7.5 and containing 3% newborn calf serum, and add the varicella virus Oka strain to infect the cells at an infection multiplicity of infection (MOI) of 0.03 in the cell factory; after the MRC-5 human diploid cells are infected with the virus for 60 hours, discard the culture medium, wash the cell surface with a PBS solution equivalent to 2 times the volume of the culture medium, wash away the calf serum, replace the maintenance medium with a MEM culture medium with a pH of 7.5 and without newborn calf serum, and continue to culture at 33°C; when the MRC-5 human diploid cells show more than 75% typical lesions, pour out the original maintenance medium, wash the cell surface with a PBS buffer solution precooled at 2-8°C equivalent to 1 times the volume of the maintenance medium, and wash away the calf serum again.

[0050] (3) Harvesting cells: Add a stabilizer (Preparation Example 1-10) pre-cooled at 5°C to the cells obtained in step (2) in an amount of 1.5 L / cell factory, and then use an ultrasonic cell harvesting device to harvest the cell culture. The ultrasonic power is 80%, the ultrasonic frequency is 2 times, and the ultrasonic time is 1 s each time. The cell culture is filtered and clarified using a disposable filter with a pore size of 70 μm to obtain a stock solution.

[0051] (4) Freeze-drying: precooling the freeze-drying protective agent at 5° C., and then adding it to the stock solution obtained in step (3) for freeze-drying to obtain a freeze-dried dosage form of the live attenuated varicella vaccine; The freeze-drying protective agent includes the following components: 50 g / L sucrose, 15 g / L trehalose, g / L sodium glutamate, 10 g / L mannitol, 20 g / L human albumin, and the balance is water.

[0052] Examples 11-14 Examples 11-14 respectively provide a method for preparing a freeze-dried live attenuated varicella vaccine.

[0053] The difference between the above embodiment and embodiment 4 is that the ultrasonic power in step (3) of the preparation method of freeze-dried live attenuated varicella vaccine is specifically shown in Table 3 below.

[0054] Table 3 Ultrasonic power in step (3) of the preparation method of Example 4 and Examples 11-14 Example Ultrasonic power (%) 4 80 11 60 12 70 13 85 14 90 Embodiment 15 Example 15 provides a method for preparing a freeze-dried live attenuated varicella vaccine.

[0055] The difference between the above embodiment and embodiment 4 is that the number of ultrasonic times in step (3) of the preparation method of freeze-dried live attenuated varicella vaccine is 1 time and the ultrasonic time is 2 seconds.

[0056] Comparative Example 1 Comparative Example 1 provides a method for preparing a freeze-dried live attenuated varicella vaccine.

[0057] The difference between the comparative example and Example 4 is that the ultrasonic power in step (3) of the preparation method of freeze-dried live attenuated varicella vaccine is 50%.

[0058] Comparative Example 2 Comparative Example 2 provides a method for preparing a freeze-dried live attenuated varicella vaccine.

[0059] The difference between the comparative example and Example 4 is that the ultrasonic power in step (3) of the preparation method of freeze-dried live attenuated varicella vaccine is 100%.

[0060] Comparative Example 3 Comparative Example 3 provides a method for preparing a freeze-dried live attenuated varicella vaccine.

[0061] The difference between the comparative example and Example 4 is that in step (3) of the preparation method of freeze-dried live attenuated varicella vaccine, the number of ultrasonications is 1 and the ultrasonication time is 4 s.

[0062] Comparative Example 4 Comparative Example 4 provides a method for preparing a freeze-dried live attenuated varicella vaccine.

[0063] The difference between the comparative example and Example 4 is that the number of ultrasonications in step (3) of the preparation method of freeze-dried live attenuated varicella vaccine is 3 times, and the duration of each ultrasonication is 1 s.

[0064] Performance testing The freeze-dried live attenuated varicella vaccines obtained in Examples 1-15 and Comparative Examples 1-4 were subjected to various tests, as follows: (1) Identification test: The freeze-dried live attenuated varicella vaccine was subjected to an identification test. The identification method was as follows: the virus solution diluted to 500-1000 PFU / ml was mixed with an appropriately diluted varicella virus-specific immune serum in equal amounts, then placed in a 37°C water bath for neutralization for 60 minutes, inoculated with human diploid MRC-5 cells, and cultured at 37°C±1°C and 5% CO2 for 7-10 days. The varicella virus should be completely neutralized. Serum and cell controls were set up at the same time, both of which should be negative, and the virus titer of the virus control should not be less than 500 PFU / ml.

[0065] The results show that the freeze-dried live attenuated varicella vaccines obtained in Examples 1-15 and Comparative Examples 1-4 all comply with the provisions of the 2020 edition of the "Chinese Pharmacopoeia".

[0066] (2) Appearance: Observe the appearance of the freeze-dried varicella live attenuated vaccine. It should be milky white or white loose body. After reconstitution, it will be a clear liquid with a slight opalescent luster and no foreign matter.

[0067] The results show that the freeze-dried live attenuated varicella vaccines obtained in Examples 1-15 and Comparative Examples 1-4 all comply with the provisions of the 2020 edition of the "Chinese Pharmacopoeia".

[0068] (3) pH: The pH of the freeze-dried live attenuated varicella vaccine shall be tested and inspected in accordance with the law (General Rule 0631).

[0069] The results show that the pH of the freeze-dried live attenuated varicella vaccine obtained in Examples 1-15 and Comparative Examples 1-4 is within the approved range and complies with the provisions of the 2020 edition of the "Chinese Pharmacopoeia".

[0070] (4) Abnormal toxicity test: The freeze-dried live attenuated varicella vaccine shall be subjected to abnormal toxicity test and inspected in accordance with the law (General Rule 1141).

[0071] The results show that the freeze-dried live attenuated varicella vaccines obtained in Examples 1-15 and Comparative Examples 1-4 all comply with the provisions of the 2020 edition of the "Chinese Pharmacopoeia".

[0072] (5) Bacterial endotoxin content detection: The bacterial endotoxin content of freeze-dried varicella live attenuated vaccine shall be tested in accordance with the law (General Rule 1143).

[0073] The results show that the bacterial endotoxin content of the freeze-dried varicella live attenuated vaccine obtained in Examples 1-15 and Comparative Examples 1-4 complies with the requirements of the 2020 edition of the "Chinese Pharmacopoeia".

[0074] (6) Virus titration: The virus titration of the freeze-dried live attenuated varicella vaccine obtained in Examples 1-15 and Comparative Examples 1-4 was tested. The results are shown in Table 4 below.

[0075] Titer detection method: Use the plaque method to titrate the virus. Take the test sample and make a suitable multiple serial dilution. Inoculate each dilution into the human diploid cell MRC-5 strain, and culture at 37℃±1℃, 5% CO2 for 7-10 days to determine the result.

[0076] (7) Accelerated stability test: The freeze-dried live attenuated varicella vaccines obtained in Examples 1-15 and Comparative Examples 1-4 were placed at 37° C. for 28 days. The titers of the live vaccines were tested on the 7th, 14th, 21st and 28th days, respectively. The results are shown in Table 4 below.

[0077] (8) Storage stability test: The freeze-dried live attenuated varicella vaccines obtained in Examples 1-15 and Comparative Examples 1-4 were placed at 5°C for 24 months. The titers of the live vaccines were tested at the 3rd, 6th, 9th, 12th, 18th and 24th months, respectively. The results are shown in Table 4 below.

[0078] Table 4 Titer test results of freeze-dried varicella live attenuated vaccine of Examples 1-15 and Comparative Examples 1-4 (lg PFU / ml) According to the test results in Table 4, the initial titer of the freeze-dried live attenuated varicella vaccine obtained in Examples 1-15 of the present application is 4.4-5.0 lg PFU / mL, and the titer decreases by 0.3-0.6 lg PFU / mL after being stored at 37°C for 28 days, and decreases by 0.1-0.4 lg PFU / mL after being stored at 5°C for 24 months. Therefore, it is shown that the freeze-dried live attenuated varicella vaccine obtained by the preparation method provided in the present application has good activity, thermal stability and long-term storage stability.

[0079] From the test results of Examples 1-5, it can be seen that the initial titer of the freeze-dried varicella live attenuated vaccine obtained in Example 1 is only 4.4 lg PFU / mL; while the initial titer of the freeze-dried varicella live attenuated vaccine obtained in Examples 2-5 is 4.8-5.0 lg PFU / mL, and with the increase in the amount of small molecule peptide added in the stabilizer, the initial titer of the freeze-dried varicella live attenuated vaccine shows a trend of gradually increasing first and then remaining basically unchanged. Therefore, it is shown that the present application uses small molecule peptides to prepare stabilizers, and the addition amount of small molecule peptides is controlled within the range of 10-50mL / L, and the obtained freeze-dried varicella live attenuated vaccine has high biological activity; further comparison shows that the freeze-dried varicella live attenuated vaccine obtained in Examples 2-4 has a titer decrease of only 0.3lg PFU / mL after being stored at 37°C for 28 days, and a titer decrease of 0.1-0.2lg PFU / m after being stored at 5°C for 24 months, while the freeze-dried varicella live attenuated vaccine obtained in Example 5 has a titer decrease of only 0.6lg PFU / mL after being stored at 37°C for 28 days, and a titer decrease of 0.4lg PFU / mL after being stored at 5°C for 24 months, indicating that the present application further controls the addition amount of small molecule peptides in the stabilizer within the range of 10-35mL / L, and the obtained freeze-dried varicella live attenuated vaccine has better thermal stability and storage stability.

[0080] From the test results of Example 4 and Examples 6-10, it can be seen that the initial titer of the freeze-dried varicella live attenuated vaccine obtained in Examples 4 and Examples 6-8 is 4.8-5.0lg PFU / mL, while the initial titer of the freeze-dried varicella live attenuated vaccine obtained in Examples 9-10 is only 4.5lg PFU / mL; this indicates that the stabilizer of the small molecule peptide containing glutamic acid and lysine is used in the cell harvesting procedure, which can achieve efficient protection of varicella virus antigens, avoid aggregation, and ensure the activity of the antigen.

[0081] From the test results of Example 4, Examples 11-14, and Comparative Examples 1-2, it can be seen that with the increase of the ultrasonic power, the initial titer of the obtained freeze-dried varicella live attenuated vaccine shows a trend of first increasing and then decreasing; further comparison shows that the initial titer of the freeze-dried varicella live attenuated vaccine obtained in Example 4 and Examples 12-13 is 5.0 lg PFU / mL; while the initial titer of the freeze-dried varicella live attenuated vaccine obtained in Example 11 and Example 14 is 4.7-4.8 lg PFU / mL, indicating that the present application further controls the ultrasonic power in step (3) within the range of 70-85%, and the activity of the obtained freeze-dried varicella live attenuated vaccine is higher.

[0082] From the test results of Example 4, Example 15, and Comparative Examples 3-4, it can be seen that Example 4 uses 2 ultrasounds, and when the ultrasound time is 1s each time, the initial titer of the freeze-dried varicella attenuated live vaccine obtained is 5.0lg PFU / mL; while Example 15 only uses 1 ultrasound, and when the ultrasound time is 2s, the initial titer of the freeze-dried varicella attenuated live vaccine obtained is 4.6lg PFU / mL; Comparative Example 3 uses 1 ultrasound, and the ultrasound time is 4s, and the initial titer of the freeze-dried varicella attenuated live vaccine obtained is 3.9lg PFU / mL; Comparative Example 4 uses 3 ultrasounds, and the ultrasound time is 1s, and the initial titer of the freeze-dried varicella attenuated live vaccine obtained is 3.5lg PFU / mL. Therefore, it is explained that in the preparation method of the freeze-dried varicella attenuated live vaccine provided by the present application, the number of ultrasounds is further controlled to 2 times, and the ultrasound time is controlled to 1s each time, and the activity of the freeze-dried varicella attenuated live vaccine obtained is higher.

[0083] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a freeze-dried live attenuated varicella vaccine, characterized in that: The following steps are involved: (1) Cell culture: MRC-5 human diploid cells were expanded and subcultured within 35 generations; (2) Virus infection of cells: After the MRC-5 human diploid cells grow into a uniform and dense monolayer of cells, add the Oka strain virus seed to infect the cells, and then infect, culture, and wash; (3) Harvesting cells: adding a stabilizer to the washed cells, then using an ultrasonic cell harvester to harvest the cell culture, and filtering the cell culture to obtain a stock solution; The process parameters of the ultrasonic cell harvesting device are as follows: ultrasonic power 60-90%, ultrasonic times 1-2 times, and ultrasonic time of each time 1.0-2.5 s.

2. The method for preparing the freeze-dried live attenuated varicella vaccine according to claim 1, characterized in that: The ultrasonic power is 70-85%.

3. The method for preparing the freeze-dried live attenuated varicella vaccine according to claim 1, characterized in that: The number of ultrasonications is 2 times.

4. The method for preparing the freeze-dried live attenuated varicella vaccine according to claim 1, characterized in that: The pore size of the filter used in the filtration is 40-105 μm.

5. The method for preparing the freeze-dried live attenuated varicella vaccine according to claim 1, characterized in that: The stabilizer comprises the following components: sucrose 60-75 g / L, trehalose 20-25 g / L, sodium glutamate 10-12 g / L, mannitol 15-20 g / L and small molecule peptide 10-50 mL / L, and the balance is water; The small molecule peptide is prepared from glutamic acid and lysine in a molar ratio of 1:(0.27-0.43).

6. The method for preparing the freeze-dried live attenuated varicella vaccine according to claim 5, characterized in that: The preparation method of the small molecule peptide is as follows: dissolving glutamic acid in water and heating to 100±5°C; then adding lysine and adjusting the pH to 6.5-7.0; finally adding water to dilute the solution to a final concentration of glutamic acid of 0.4-0.6M to obtain the small molecule peptide.

7. The method for preparing the freeze-dried live attenuated varicella vaccine according to claim 1, characterized in that: The preparation method also includes freeze-drying, and the specific steps are: pre-cooling a freeze-dried protective agent at 2-8°C, and then adding it to the stock solution for freeze-drying to obtain a freeze-dried dosage form of a live attenuated varicella vaccine; the freeze-dried protective agent includes the following components: 40-60 g / L sucrose, 15-20 g / L trehalose, 5-10 g / L sodium glutamate, 10-15 g / L mannitol, 10-30 g / L human serum albumin, and the balance is water.

8. The method for preparing the freeze-dried live attenuated varicella vaccine according to claim 1, characterized in that: The step (2) specifically comprises: after the MRC-5 human diploid cells grow into a uniform and dense monolayer of cells, the medium is replaced with a MEM medium having a pH of 7.3-7.5 and containing 1-3% newborn calf serum, and the Oka strain virus is added to infect the cells, with an infection multiplicity MOI of 0.01-0.

05. After 48-60 hours of infection, the culture medium is discarded, the cells are washed with a PBS buffer, and then the medium is replaced with a MEM medium having a pH of 7.3-7.5 and without newborn calf serum, and the cells are cultured at 33-35°C. When more than 75% of the MRC-5 human diploid cells show typical lesions, the original maintenance medium is discarded, and the cell surface is washed with a PBS buffer precooled at 2-8°C.

9. The method for preparing the freeze-dried live attenuated varicella vaccine according to claim 1, characterized in that: The culture medium used for the amplification and subculture is: L-15 culture medium with a pH of 7.3-7.5 and containing 6-10% newborn calf serum.

10. A freeze-dried attenuated live varicella vaccine, characterized in that: The vaccine is prepared by the method for preparing the freeze-dried live attenuated varicella vaccine according to any one of claims 1 to 9.

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