Vaccine freeze-drying protective agent and freeze-drying method thereof

Abstract

The present invention relates to the field of biological products, and in particular to a vaccine freeze-drying protective agent and a freeze-drying method thereof. The present invention provides a freeze-drying protective agent for a live attenuated Japanese encephalitis vaccine, comprising: one or a combination of two or more of lactose, thiourea, sodium glutamate, L-arginine, sorbitol, glycine, human serum albumin or sodium hyaluronate. The live attenuated Japanese encephalitis vaccine prepared by the present invention has a stable appearance and good solubility, and can be stored at 37°C for 21 days and at 2-8°C for 36 months.

Description

Technical Field

[0001] The present invention relates to the field of biological products, and in particular to a vaccine freeze-drying protectant and a freeze-drying method thereof. Background Art

[0002] Epidemic Japanese encephalitis, abbreviated as JE, is a natural epidemic disease caused by the JE virus. It is transmitted by mosquitoes and is prevalent in summer and autumn. After people are bitten by infected mosquitoes, most people become latently infected and only a few develop encephalitis. The incidence rate is generally between 0.02 and 0.1%, and the mortality rate is relatively high, about 10%. This disease mainly affects children, especially school-age children. JE not only has a high mortality rate, but also serious sequelae. About 30% of patients have residual sequelae of varying degrees after the disease. Therefore, JE is an acute infectious disease that seriously threatens human health. The disease is mainly distributed in Asia. There are about 2,000 cases in my country each year, and the death toll is about 200.

[0003] Vaccination is an effective preventive measure against Japanese encephalitis. Currently, the vaccines on the market mainly include inactivated Japanese encephalitis vaccine, live attenuated Japanese encephalitis vaccine, etc. The live attenuated Japanese encephalitis vaccine has the advantages of long-lasting and strong immune effect, small local adverse reactions, wide vaccination routes and few vaccination times. Currently, the live attenuated Japanese encephalitis vaccines sold on the market are mainly freeze-dried products. Most of the publicly available freeze-dried formulas of live attenuated Japanese encephalitis vaccines contain gelatin and dextran. A large number of studies have shown that the vast majority of allergic reactions are related to gelatin and dextran. Therefore, removing gelatin and dextran from the freeze-dried formula is a development trend of vaccine formula improvement. The freeze-dried live attenuated Japanese encephalitis vaccine products currently on the market generally have low heat resistance, which makes their storage conditions harsh, their validity period is short, and their immune effect is greatly reduced.

[0004] The vacuum freeze-drying process of live attenuated vaccines also has a great impact on their shelf life. In the conventional vacuum freeze-drying process, the live attenuated vaccines need to be frozen at ultra-low temperatures, and the ice crystals formed are uneven in size, affecting the survival rate of the virus and the solubility of the freeze-dried products. Therefore, in the conventional freeze-drying process, although the formula of the lyoprotectant has been improved, the heat resistance of the product has been limited. Summary of the invention

[0005] In view of this, the present invention provides a vaccine freeze-drying protective agent and a freeze-drying method thereof. The purpose of the present invention is to provide a heat-resistant freeze-drying protective agent and a freeze-drying method for attenuated live Japanese encephalitis vaccines in view of the problems of poor heat resistance, short validity period and poor resolubility of existing attenuated live Japanese encephalitis vaccines. The prepared attenuated live Japanese encephalitis vaccines have stable appearance and good solubility, and can store the attenuated live Japanese encephalitis vaccines at 37°C for 21 days and at 2-8°C for 36 months.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a freeze-drying protective agent, which comprises one or a combination of two or more of lactose, thiourea, sodium glutamate, L-arginine, sorbitol, glycine, human serum albumin or sodium hyaluronate.

[0008] In some specific embodiments of the present invention, the above-mentioned lyoprotectant, in parts by mass, includes:

[0009]

[0010] In some specific embodiments of the present invention, the above-mentioned lyoprotectant, in terms of concentration, comprises:

[0011]

[0012] The present invention also provides a method for preparing the above-mentioned lyophilized protective agent, which comprises mixing one or a combination of two or more of lactose, thiourea, sodium glutamate, L-arginine, sorbitol, glycine, human serum albumin or sodium hyaluronate with PBS having a pH value of 7.4 to 7.6 to obtain the lyophilized protective agent.

[0013] The present invention also provides the use of the above lyophilization protectant or the lyophilization protectant prepared by the above preparation method in reducing the damage to the attenuated live vaccine during the lyophilization process.

[0014] The present invention also provides the use of the lyophilized protective agent or the lyophilized protective agent prepared by the preparation method in the preparation of live attenuated Japanese encephalitis vaccine.

[0015] The present invention also provides a freeze-drying method for a live attenuated Japanese encephalitis vaccine, and the freeze-drying procedure includes: a pre-freezing stage with a temperature of -60 to -50°C, maintained for 2 to 6 hours; after heating to -25 to -20°C, maintained for 2 to 8 hours; after cooling again to -60 to -50°C, maintained for 2 to 6 hours; a sublimation drying stage with a temperature increase of 10 to 30 hours to -30 to -20°C, and a vacuum pressure of 0.05 to 0.2 mbar; and a desorption drying stage with a temperature increase of 6 to 12 hours to 20 to 25°C, and a vacuum pressure of 0.001 to 0.01 mbar.

[0016] The present invention also provides a method for preparing a live attenuated Japanese encephalitis vaccine, comprising mixing a stock solution of the live attenuated Japanese encephalitis vaccine with the above-mentioned freeze-drying protective agent to obtain a semi-finished product; freeze-drying the semi-finished product to obtain a live attenuated Japanese encephalitis vaccine;

[0017] The freeze-drying procedure includes: a pre-freezing stage with a temperature of -60 to -50°C, maintained for 2 to 6 hours; heating to -25 to -20°C and maintaining for 2 to 8 hours; cooling again to -60 to -50°C and maintaining for 2 to 6 hours; a sublimation drying stage with a temperature increase of 10 to 30 hours to -30 to -20°C and a vacuum pressure of 0.05 to 0.2 mbar; and a desorption drying stage with a temperature increase of 6 to 12 hours to 20 to 25°C and a vacuum pressure of 0.001 to 0.01 mbar.

[0018] In some specific embodiments of the present invention, the ratio of the Japanese encephalitis live attenuated vaccine concentrate to the lyophilization protective agent in the semi-finished product in the above preparation method is 1:2; and the pH value of the semi-finished product is 7.2-7.8.

[0019] The present invention also provides a live attenuated Japanese encephalitis vaccine, comprising the above-mentioned freeze-drying protective agent; or prepared by the above-mentioned freeze-drying method; or prepared by the above-mentioned preparation method.

[0020] In some specific embodiments of the present invention, the above-mentioned live attenuated Japanese encephalitis vaccine further includes acceptable adjuvants; the adjuvants include aluminum hydroxide, mineral oil, Tween-80, Freund's adjuvant, cytidylic acid or liposomes.

[0021] The present invention also provides the use of the lyophilized protective agent or the lyophilized protective agent prepared by the above preparation method in the preparation of attenuated varicella live vaccine, attenuated mumps live vaccine, attenuated measles live vaccine and / or attenuated rubella live vaccine.

[0022] The present invention has the following effects:

[0023] The freeze-dried protective agent provided by the present invention has a reasonable combination of ingredients and content, which can inhibit the formation of ice crystals during the freeze-drying process, thereby enhancing the heat resistance of the attenuated live vaccine freeze-dried products; the freeze-drying method provided by the present invention reduces the damage to the attenuated live vaccine during the freeze-drying process. Compared with the existing freeze-dried products of the attenuated live encephalitis Japanese encephalitis vaccine, the freeze-dried product of the attenuated live encephalitis Japanese encephalitis prepared by the present invention has the characteristics of good thermal stability and long validity period. The vaccine has a shelf life of 21 days at 37°C and a shelf life of 36 months at 2-8°C. Specifically:

[0024] The results of the long-term storage experiment and the stability results at 37°C show that the virus titer of the freeze-dried vaccine of the present invention is greater than 5.7lg PFU / mL after 21 days of storage at 37°C; after 36 months of storage in the long-term storage experiment at 2-8°C, the virus titer result is greater than 5.7lg PFU / mL, both of which meet the standards of the 2020 edition of the "Chinese Pharmacopoeia"; while the current market's live attenuated Japanese encephalitis vaccine products have a virus titer result of less than 5.7lg PFU / mL after 21 days of storage at 37°C; after 36 months of storage in the long-term storage experiment at 2-8°C, the virus titer result is less than 5.7lg PFU / mL, which does not meet the standards of the 2020 edition of the "Chinese Pharmacopoeia". DETAILED DESCRIPTION

[0025] The present invention discloses a vaccine freeze-drying protective agent and a freeze-drying method thereof. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the same. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0026] The present invention aims to solve the problems of heat intolerance, short validity period and poor thermal stability of the live attenuated Japanese encephalitis vaccine currently on the market during transportation, and invents this formula and a corresponding freeze-drying process to improve the heat resistance of the live attenuated Japanese encephalitis vaccine and extend its validity period.

[0027] The present invention aims at the shortcomings of the conventional freeze-drying process, introduces annealing technology, increases the glass transition temperature of the product, increases the sublimation temperature, reduces the influence of the vacuum freeze-drying process on the activity of the vaccine, and improves the heat resistance of the product.

[0028] The heat-resistant Japanese encephalitis attenuated live vaccine freeze-dried protective agent of the present invention has the following contents of various components in the semi-finished product:

[0029]

[0030]

[0031] The solvent is PBS with a pH of 7.4 to 7.6, and the semi-finished vaccine product refers to a liquid vaccine before freeze-drying.

[0032] Preparation of lyophilized protective agent: Dissolve lactose, thiourea, sodium glutamate, L-arginine, sorbitol, glycine, human serum albumin and sodium hyaluronate in PBS solution, and filter and sterilize with a 0.22 μm microporous filter membrane.

[0033] Preparation of semi-finished products: Take the original liquid of attenuated live encephalitis vaccine and mix it with the freeze-dried protective agent in a ratio of 1:2, adjust the pH to 7.2-7.8, and determine the virus titer according to the method of the 2020 edition of the "Chinese Pharmacopoeia". The virus titer of the vaccine semi-finished product is higher than 7.2lg PFU / mL.

[0034] In the above preparation method, the semi-finished vaccine product is packaged at 0.5 mL / vial.

[0035] Freeze-drying method: The lowest temperature in the pre-freezing stage is -60 to -50°C, and the lowest temperature is maintained for 2 to 6 hours. The temperature is then raised to -25 to -20°C, maintained for 2 to 8 hours, and then lowered to -60 to -50°C, and the lowest temperature is maintained for 2 to 6 hours. The final temperature of sublimation drying is -30 to -20°C, and the time to reach the final temperature is 10 to 30 hours. The vacuum pressure is controlled at 0.05 to 0.2 mbar. The final temperature of analytical drying is 20 to 25°C, the vacuum pressure is controlled at 0.001 to 0.01 mbar, and the operation is 6 to 12 hours.

[0036] The freeze-drying protective agent and freeze-drying method of the present invention are aimed at the live attenuated Japanese encephalitis vaccine. By providing a heat-resistant freeze-drying protective agent and a freeze-drying method thereof, the freeze-dried product of the live attenuated Japanese encephalitis vaccine has a stable appearance, good resolubility and good thermal stability. The live attenuated Japanese encephalitis vaccine can be stored at 37°C for 21 days and at 2-8°C for 36 months, thereby extending the shelf life of the vaccine.

[0037] The raw materials and reagents of the present invention can be purchased from the market.

[0038] The present invention will be further described below in conjunction with embodiments:

[0039] Example 1: Preparation method of the stock solution of Japanese encephalitis live attenuated vaccine

[0040] 1) The production cells of the live attenuated Japanese encephalitis vaccine are hamster kidney cells, and the production strain used is the attenuated strain of Japanese encephalitis virus SA14-14-2;

[0041] 2) Cell preparation: Inoculate microcarriers at 20 g / L in a 30L bioreactor, take 500 mL of mixed cell solution from the upper tank, adjust the parameters, set the pH to 7.4, the temperature to 37°C, and perfuse culture for 5 to 7 days;

[0042] 3) Virus inoculation: When the cell density in the reaction tank reaches 1.0×10 7 / mL, lower the temperature of the reaction tank to 33°C and stop perfusion. Take the working seed and inoculate the virus at 0.002 MOI, and perfuse after adsorption for 4 to 5 hours;

[0043] 4) Virus harvesting: Virus fluid was harvested 72 hours after virus inoculation and continued for 13 days;

[0044] 5) Clarify with a 0.65 μm filter membrane and concentrate 30 to 50 times with a 300 kD cut-off membrane ultrafiltration. The protein content of the concentrate should be 15 to 40 mg / mL;

[0045] 6) Purification: The virus solution was purified by column chromatography. The chromatographic medium was Sepharose 4FF, the elution equilibrium solution was PBS at pH 7.6, the detection UV wavelength for collecting the purified solution was 280 nm, and the collection range started from 20 mV and ended when the absorbance value dropped back to 50 mV. This was the original solution of the Japanese encephalitis attenuated live vaccine. The virus titer of the original solution was determined according to the method of the 2020 edition of the "Chinese Pharmacopoeia".

[0046] Example 2

[0047] The components and contents of the freeze-dried protective agent in this example (final concentration in the semi-finished vaccine product) are as follows:

[0048]

[0049] The above-mentioned protective agent solvent is PBS with a pH of 7.2, which is filtered and sterilized with a 0.22μm microporous filter membrane. The Japanese encephalitis attenuated live vaccine stock solution is mixed with the freeze-dried protective agent in a ratio of 1:2. The stock solution used is the same as that in Example 1. The pH is adjusted to 7.2. The virus titer is determined according to the method of the 2020 edition of the "Chinese Pharmacopoeia". The virus titer of the vaccine semi-finished product is 7.3lg PFU / mL.

[0050] The semi-finished vaccine product is packaged into vials, 0.5 mL / vial, and freeze-dried to obtain the finished product of the Japanese encephalitis live attenuated vaccine.

[0051] The freeze-drying method is as follows: the lowest temperature in the pre-freezing stage is -50°C, the lowest temperature is maintained for 6 hours, the temperature is raised to -25°C, maintained for 2 hours, and the temperature is lowered to -50°C again, the lowest temperature is maintained for 6 hours; the final temperature of sublimation drying is -30°C, the time to reach the final temperature is 30 hours, and the vacuum pressure is controlled at 0.05mbar; the final temperature of analytical drying is 20°C, the vacuum pressure is controlled at 0.001mbar, and it runs for 12 hours.

[0052] Example 3

[0053] The components and contents of the freeze-dried protective agent in this example (final concentration in the semi-finished vaccine product) are as follows:

[0054]

[0055] The above-mentioned protective agent solvent is PBS with a pH of 7.3, which is filtered and sterilized with a 0.22μm microporous filter membrane. The Japanese encephalitis attenuated live vaccine stock solution is mixed with the freeze-dried protective agent in a ratio of 1:2. The stock solution used is the same as that in Example 1. The pH is adjusted to 7.5. The virus titer is determined according to the method of the 2020 edition of the "Chinese Pharmacopoeia". The virus titer of the vaccine semi-finished product is 7.3lg PFU / mL.

[0056] The semi-finished vaccine product is packaged into vials, 0.5 mL / vial, and freeze-dried to obtain the finished product of the Japanese encephalitis live attenuated vaccine.

[0057] The freeze-drying method is as follows: the lowest temperature in the pre-freezing stage is -55°C, the lowest temperature is maintained for 4 hours, the temperature is raised to -22°C, maintained for 8 hours, and the temperature is lowered to -55°C again, the lowest temperature is maintained for 4 hours; the final temperature of sublimation drying is -25°C, the time to reach the final temperature is 20 hours, and the vacuum pressure is controlled at 0.1mbar; the final temperature of analytical drying is 25°C, the vacuum pressure is controlled at 0.005mbar, and it runs for 10 hours.

[0058] Example 4

[0059] The components and contents of the freeze-dried protective agent in this example (final concentration in the semi-finished vaccine product) are as follows:

[0060]

[0061]

[0062] The above-mentioned protective agent solvent is PBS with a pH of 7.4, which is filtered and sterilized with a 0.22μm microporous filter membrane. The Japanese encephalitis attenuated live vaccine stock solution is mixed with the freeze-dried protective agent in a ratio of 1:2. The stock solution used is the same as that in Example 1. The pH is adjusted to 7.8. The virus titer is determined according to the method of the 2020 edition of the "Chinese Pharmacopoeia". The virus titer of the vaccine semi-finished product is 7.3lg PFU / mL.

[0063] The semi-finished vaccine product is packaged into vials, 0.5 mL / vial, and freeze-dried to obtain the finished product of the Japanese encephalitis live attenuated vaccine.

[0064] The freeze-drying method is as follows: the lowest temperature in the pre-freezing stage is -60°C, the lowest temperature is maintained for 2 hours, the temperature is raised to -20°C, maintained for 4 hours, and the temperature is lowered to -60°C again, the lowest temperature is maintained for 2 hours; the final temperature of sublimation drying is -20°C, the time to reach the final temperature is 10 hours, and the vacuum pressure is controlled at 0.2mbar; the final temperature of analytical drying is 22°C, the vacuum pressure is controlled at 0.01mbar, and it runs for 6 hours.

[0065] Comparative Example 1: Freeze-drying process comparison

[0066] The components and contents of the freeze-dried protective agent in this comparative example (final concentration in the semi-finished vaccine product):

[0067]

[0068] The above-mentioned protective agent solvent is PBS with a pH of 7.2, which is filtered and sterilized with a 0.22μm microporous filter membrane. The Japanese encephalitis attenuated live vaccine stock solution is mixed with the freeze-dried protective agent in a ratio of 1:2. The stock solution used is the same as that in Example 1. The pH is adjusted to 7.2. The virus titer is determined according to the method of the 2020 edition of the "Chinese Pharmacopoeia". The virus titer of the vaccine semi-finished product is 7.3lg PFU / mL.

[0069] The semi-finished vaccine product is packaged into vials, 0.5 mL / vial, and freeze-dried to obtain the finished product of the Japanese encephalitis live attenuated vaccine.

[0070] The freeze-drying method is as follows: the lowest temperature in the pre-freezing stage is -60°C, the lowest temperature is maintained for 7 hours, the final temperature of sublimation drying is -40°C, the time to reach the final temperature is 40 hours, and the vacuum pressure is controlled at 0.05mbar; the final temperature of analytical drying is 20°C, the vacuum pressure is controlled at 0.001mbar, and it runs for 15 hours.

[0071] Comparative Example 2: Formula Comparison

[0072] The components and contents of the freeze-dried protective agent in this comparative example (final concentration in the semi-finished vaccine product):

[0073]

[0074] The above-mentioned protective agent solvent is PBS with a pH of 7.4, which is filtered and sterilized with a 0.22μm microporous filter membrane. The Japanese encephalitis attenuated live vaccine stock solution is mixed with the freeze-dried protective agent in a ratio of 1:2. The stock solution used is the same as that in Example 1. The pH is adjusted to 7.8. The virus titer is determined according to the method of the 2020 edition of the "Chinese Pharmacopoeia". The virus titer of the vaccine semi-finished product is 7.3lg PFU / mL.

[0075] The semi-finished vaccine product is packaged into vials, 0.5 mL / vial, and freeze-dried to obtain the finished product of the Japanese encephalitis live attenuated vaccine.

[0076] The freeze-drying method is as follows: the lowest temperature in the pre-freezing stage is -60°C, the lowest temperature is maintained for 2 hours, the temperature is raised to -20°C, maintained for 4 hours, and the temperature is lowered to -60°C again, the lowest temperature is maintained for 2 hours; the final temperature of sublimation drying is -20°C, the time to reach the final temperature is 10 hours, and the vacuum pressure is controlled at 0.2mbar; the final temperature of analytical drying is 22°C, the vacuum pressure is controlled at 0.01mbar, and it runs for 6 hours.

[0077] Comparative Example 3: Formula Comparison

[0078] Components and contents of the freeze-dried protective agent in this comparative example (final concentration in the semi-finished vaccine product)

[0079]

[0080] The above-mentioned protective agent solvent is PBS with a pH of 7.4, which is filtered and sterilized with a 0.22μm microporous filter membrane. The Japanese encephalitis attenuated live vaccine stock solution is mixed with the freeze-dried protective agent in a ratio of 1:2. The stock solution used is the same as that in Example 1. The pH is adjusted to 7.7. The virus titer of the stock solution is determined according to the method of the 2020 edition of the "Chinese Pharmacopoeia". The virus titer of the vaccine semi-finished product is 7.3lg PFU / mL.

[0081] The freeze-drying method is as follows: the lowest temperature in the pre-freezing stage is -60°C, the lowest temperature is maintained for 7 hours, the final temperature of sublimation drying is -40°C, the time to reach the final temperature is 40 hours, and the vacuum pressure is controlled at 0.05mbar; the final temperature of analytical drying is 20°C, the vacuum pressure is controlled at 0.001mbar, and it runs for 15 hours.

[0082] Example 5

[0083] The finished products of the Japanese encephalitis live attenuated vaccine of the above Examples 2, 3, 4 and Comparative Examples 1, 2, 3 were placed at 37° C. and 2-8° C. for different time periods, and samples were taken for appearance, moisture and virus titer determination. The quality standards were in accordance with the 2020 edition of the Chinese Pharmacopoeia.

[0084] The experimental results are shown in Table 1 and Table 2:

[0085] Table 1 37℃ thermal stability test results of freeze-dried products (lg PFU / mL)

[0086]

[0087] Table 2 Virus titer results of lyophilized products at 2-8°C long-term storage experiment (lg PFU / mL)

[0088]

[0089]

[0090] Based on the above results:

[0091] (1) The long-term storage experiment results and 37°C stability results of Examples 2, 3, and 4 showed that after 21 days of storage at 37°C, the virus titer result was greater than 5.7 lg PFU / mL; after 36 months of storage in the 2-8°C long-term storage experiment, the virus titer result was greater than 5.7 lg PFU / mL, both of which met the standards of the 2020 edition of the Chinese Pharmacopoeia.

[0092] (2) For the comparative example and the live attenuated Japanese encephalitis vaccine products currently on the market, the virus titer results were greater than 5.7lg PFU / mL after being stored at 37°C for 7 days; and the virus titer results were greater than 5.7lg PFU / mL after being stored in a long-term storage experiment at 2-8°C for 18 months, both of which met the standards of the 2020 edition of the Chinese Pharmacopoeia. However, after being stored at 37°C for 21 days, the virus titer results were less than 5.7lg PFU / mL; after being stored in a long-term storage experiment at 2-8°C for 36 months, the virus titer results were less than 5.7lg PFU / mL, which did not meet the standards of the 2020 edition of the Chinese Pharmacopoeia.

[0093] The above results confirm that the freeze-dried product of the present invention has the advantages of good heat stability, storage resistance and long shelf life compared with other freeze-dried product formulations and freeze-drying processes currently on the market.

[0094] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A freeze-dried protective agent for live attenuated Japanese encephalitis vaccine, characterized in that: In parts by mass, the composition is: The preparation method of the freeze-dried protective agent is as follows: lactose, thiourea, sodium glutamate, L-arginine, sorbitol, glycine, human serum albumin and sodium hyaluronate are mixed with PBS having a pH value of 7.4 to 7.6 to obtain the freeze-dried protective agent; The freeze-drying procedure of the freeze-drying protective agent includes: the temperature of the pre-freezing stage is -60 to -50°C, maintained for 2 to 6 hours; after heating to -25 to -20°C, maintained for 2 to 8 hours; after cooling to -60 to -50°C again, maintained for 2 to 6 hours; the temperature is increased to -30 to -20°C for 10 to 30 hours in the sublimation drying stage, and the vacuum pressure is 0.05 to 0.2 mbar; the temperature is increased to 20 to 25°C for 6 to 12 hours in the analytical drying stage, and the vacuum pressure is 0.001 to 0.01 mbar.

2. The lyoprotectant according to claim 1, characterized in that In terms of concentration, the composition is:

3. The method for preparing the lyoprotectant according to claim 1 or 2, characterized in that: Lactose, thiourea, sodium glutamate, L-arginine, sorbitol, glycine, human serum albumin and sodium hyaluronate are mixed with PBS having a pH value of 7.4 to 7.6 to obtain a lyophilization protective agent.

4. Use of the lyoprotectant according to claim 1 or 2 or the lyoprotectant prepared by the preparation method according to claim 3 in reducing damage to live attenuated Japanese encephalitis vaccine during freeze-drying.

5. Use of the lyoprotectant according to claim 1 or 2 or the lyoprotectant prepared by the preparation method according to claim 3 in the preparation of a live attenuated Japanese encephalitis vaccine.

6. A method for preparing a live attenuated Japanese encephalitis vaccine, characterized in that: Mixing the Japanese encephalitis attenuated live vaccine stock solution with the lyophilization protective agent as claimed in claim 1 or 2 to obtain a semi-finished product; Freeze-drying the semi-finished product to obtain a live attenuated Japanese encephalitis vaccine; The freeze-drying procedure includes: a pre-freezing stage at a temperature of -60 to -50°C for 2 to 6 hours; After heating to -25~-20℃, maintain for 2~8 hours; cool down to -60~-50℃ again, maintain for 2~6 hours; during the sublimation drying stage, heat up to -30~-20℃ for 10~30 hours, and the vacuum pressure is 0.05~0.2mbar; during the analytical drying stage, heat up to 20~25℃ for 6~12 hours, and the vacuum pressure is 0.001~0.01mbar; The ratio of the Japanese encephalitis attenuated live vaccine concentrate to the lyophilization protective agent in the semi-finished product is 1:2; and the pH value of the semi-finished product is 7.2-7.

8.

7. A live attenuated Japanese encephalitis vaccine, characterized in that: Prepared by the preparation method as claimed in claim 6.

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