A virus-like particle vaccine lyoprotectant and method of making same

Abstract

The application discloses a virus-like particle vaccine freeze-drying protective agent and a preparation method thereof. The freeze-drying protective agent comprises A liquid and B liquid mixed in a volume ratio of 1:1 to 3:1; the A liquid comprises the following components with terminal concentrations: 0.01-0.5 mol / L base liquid I, 0.5-2 mmol / L base liquid II, 0.01-0.1% polysorbate 80, 5-20% sucrose and 1-5% trypsin; and the B liquid comprises the following components with terminal concentrations: 0.1-5% trehalose, 0.1-5% oligogalactose and 0.1-2% PEG6000. Through the compounding of the A liquid and the B liquid, the application can effectively enhance the immunization effect of the vaccine, prolong the storage time of the vaccine and keep the virus-like particle form intact.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological products, and particularly relates to a virus-like particle vaccine freeze-drying protective agent, a preparation method and application. BACKGROUND

[0002] Traditional vaccines are mainly attenuated vaccines and inactivated vaccines, which can well stimulate the immune response of the body and resist the spread of diseases, but these traditional vaccines also have some obvious defects, such as the safety and the difficulty in preparation, which restrict the use of vaccines in some diseases. Virus-like particles (VLPs) are hollow particles self-assembled by one or more structural proteins of viruses in vivo or in vitro, and cannot replicate and amplify because they do not contain the genetic material of viruses and do not have the ability to infect and cause diseases. VLPs are a kind of multi-protein molecules, most of which are composed of several identical proteins to form a icosahedron or helical structure, which is similar to the structure of live viruses while lacking the genome of viruses, so it is very safe. At the same time, VLPs are similar in morphological structure to natural virus particles, well simulating the antigen epitopes of natural viruses, and the size of dozens of nanometers and the highly ordered epitope structure are also conducive to the phagocytosis and antigen processing of dendritic cells, thereby effectively inducing the collective immune protective response to viruses. In addition, in the production of VLPs vaccines, prokaryotic expression systems such as Escherichia coli or other eukaryotic expression systems can be used for vaccine production, and live viruses can be avoided in the production process, which also reduces the biological safety risk involved in the production process of vaccines. Compared with traditional vaccines, VLPs have better safety, and also have the immunogenicity of traditional vaccines, so more and more pathogenic vaccines are prepared by using VLPs technology.

[0003] The production and preparation process of VLPs vaccines includes four main steps, namely lysis, crude extraction, purification and assembly, so that the VLPs are kept in a solution state during the preparation process. However, when VLPs are stored in liquid form, even if low-temperature conditions are used for storage, aggregation, denaturation and precipitation of virus-like particles are still likely to occur, which will greatly reduce the immune effect of VLPs vaccines. Therefore, in the preparation of VLPs vaccines, the VLPs sample after purification and assembly needs to be prepared into a liquid-state vaccine in a very short time, which limits the batch production efficiency of the vaccine, increases the quality instability factor of the vaccine product, and also increases the cost of the vaccine. At the same time, the liquid-state vaccine has more stringent transportation conditions and requirements.

[0004] Freeze-drying technology is the most commonly used technology in the production of biological products. It can not only maintain the biological characteristics of the product, but also prolong the effective period of the biological product. Freeze-drying is to freeze the solution or suspension into solid state, and then sublimate the ice under low temperature and high vacuum degree, leaving dry matter. Because freeze-drying is carried out under low temperature and high vacuum degree, the sample does not bubble, does not boil, the dry matter does not stick to the wall, is easy to take out, and becomes loose powder, which is easy to dissolve in water. Due to the above advantages of freeze-drying, it is suitable for products that are sensitive to heat, easy to absorb moisture, easy to oxidize and easy to produce bubbles during solvent evaporation, such as proteins, enzymes, nucleic acids, antibiotics, hormones, etc. Freeze-drying can dehydrate proteins, making it difficult for chemical reactions that destroy proteins to occur, and greatly extending their effective period. However, the freeze-drying process can cause damage to the biological activity, resulting in decreased solubility and potency. Since the safety and immunological efficacy of VLPs vaccine depend on various factors such as the configuration, morphology, proportion of VLPs in it, therefore, in the freeze-drying process of virus-like particles, it is crucial to develop a suitable freeze-drying protective agent in the freeze-drying process of biological products. Only a suitable freeze-drying protective agent can ensure that the configuration, morphology, proportion and other characteristics of VLPs are basically the same as before freeze-drying, and do not affect the safety and immunological efficacy of the final vaccine product. SUMMARY

[0005] In view of the above deficiencies in the prior art, the present application provides a virus-like particle vaccine freeze-drying protective agent and a preparation method thereof, to reduce the special requirements for the environment during the storage of VLPs, improve the production efficiency, and reduce the production cost and transportation cost.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application to solve its technical problems is:

[0007] A virus-like particle vaccine freeze-drying protective agent, comprising A liquid and B liquid mixed in a volume ratio of 1:1 to 3:1;

[0008] In the A liquid, the components include the following final concentrations:

[0009] 0.01-0.5mol / L of base solution I, 0.5-2mmol / L of base solution II, 0.01-0.1% of polysorbate 80, 5-20% of sucrose, and 1-5% of tryptone;

[0010] In the B liquid, the components include the following final concentrations:

[0011] 0.1-5% of trehalose, 0.1-5% of galactose oligosaccharide, and 0.1-2% of PEG6000.

[0012] Further, the volume ratio of the A liquid and the B liquid is 2:1.

[0013] Further, the A liquid comprises components with the following final concentrations:

[0014] 0.01 mol / L base liquid I, 1 mmol / L base liquid II, 0.05% polysorbate 80, 10% sucrose, and 2.5% tryptone.

[0015] Further, the base liquid I is a Tris solution with a pH value of 8.0; and the base liquid II is an EDTA solution with a pH value of 8.0.

[0016] Further, the B liquid comprises components with the following final concentrations:

[0017] 2% trehalose, 2% oligogalactose, and 1% PEG6000.

[0018] The preparation method of the virus-like particle vaccine freeze-drying protective agent comprises the following steps:

[0019] (1) preparing the base liquid I and the base liquid II;

[0020] (2) dissolving and mixing the components of the A liquid according to the formula, and sterilizing at 100-150 DEG C and 80-100 KPa;

[0021] (3) dissolving and mixing the components of the B liquid according to the formula, and filtering sterilizing;

[0022] (4) after the A liquid is cooled to room temperature, the A liquid and the B liquid are mixed uniformly at a volume ratio of 1:1-3:1.

[0023] Further, the sterilization temperature of the A liquid is 116 DEG C, and the pressure is 100 KPa.

[0024] Further, the freeze-drying protective agent is mixed with the virus-like particle solution at a ratio of 1:1-1:2, and then freeze-drying is performed.

[0025] The beneficial effects of the present application are:

[0026] 1. The freeze-drying protective agent in the present application is divided into the A liquid and the B liquid, the A liquid is mainly buffer solution, excipient, protein and other substances, which can keep the virus-like particles at a fixed pH value and always maintain their inherent morphology during freeze-drying. The B liquid contains trehalose, oligogalactose and PEG6000.

[0027] Wherein, trehalose and sucrose are both reducing disaccharides, and can complement each other, and the two can be used together as a cryoprotectant, and can play a role of a dehydration protective agent in the process of drying and dehydration; in addition to the protective effect, galacto-oligosaccharide also has the effect of an immune enhancer, and can improve the immune effect of the VLPs vaccine in the later stage. In addition, the trehalose and other substances in the B liquid can further improve the shape maintaining effect of the sucrose and other substances in the A liquid on the virus-like particles, and the sucrose and other substances in the A liquid can protect the immunogenicity of the virus-like particles, thereby helping the immune enhancing effect of the galacto-oligosaccharide in the B liquid.

[0028] 2. When the virus-like particle semi-finished product and finished product are stored in a liquid state, there are disadvantages of large space occupation and quality reduction with storage time, and after the virus-like particles are freeze-dried by using the freeze-drying protective agent, the storage time of the virus-like particles can be greatly prolonged under the premise of maintaining the characteristics and immunogenicity of the virus-like particles, thereby reducing the production of finished products and improving the quality and stability of the products.

[0029] 3. When the virus-like particle semi-finished product and finished product are in a liquid state, in order to prepare a multi-link multi-valent virus-like particle vaccine, the virus-like particle content and other factors will be affected, the production technology and production quality of the semi-finished product are required to be higher, and the production cost of the vaccine is also greatly increased. By using the freeze-drying protective agent, the preparation of the multi-link multi-valent virus-like particle vaccine can be more convenient and faster under the existing production technology and conditions.

[0030] Figure description

[0031] Figure 1 It is an electron micrograph of the porcine foot-and-mouth disease O type virus-like particles before freeze-drying;

[0032] Figure 2 It is an electron micrograph of the porcine foot-and-mouth disease O type virus-like particles after freeze-drying;

[0033] Figure 3 It is an electron micrograph of the porcine foot-and-mouth disease O type virus-like particles stored at-15℃ for 24 months;

[0034] Figure 4 It is the particle size detection results of the porcine foot-and-mouth disease O type virus-like particles before and after freeze-drying and after being stored for 24 months;

[0035] Figure 5 It is the assembly rate detection results of the porcine foot-and-mouth disease O type virus-like particles before and after freeze-drying and after being stored for 24 months. DETAILED DESCRIPTION

[0036] The specific embodiments of the present application are described below to facilitate the understanding of the present application for those skilled in the art, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are within the scope of protection.

[0037] Example 1

[0038] A freeze-drying protective agent for a virus-like particle vaccine is prepared as follows:

[0039] 1. Preparation of a base solution

[0040] (1) Preparation of 0.1 mol / L Tris solution (pH 8.0): Dissolve 12.1 g of Tris base in 800 mL of water, adjust the pH of the solution to 8.0 with hydrochloric acid, and add water to make up to 1000 mL.

[0041] (2) Preparation of 50 mmol / L EDTA solution (pH 8.0): Dissolve 18.6 g of EDTA disodium salt in 800 mL of water, adjust the pH to 8.0 with sodium hydroxide solution, and add water to make up to 1000 mL.

[0042] 2. Preparation of A solution

[0043] Each liter of A solution contains 100 mL of 0.1 mol / L Tris solution (pH 8.0), 20 mL of 50 mmol / L EDTA solution (pH 8.0), 0.05% w / v of polysorbate 80, 10% w / v of sucrose, and 2.5% w / v of tryptone, all dissolved in an appropriate amount of water, and made up to 1000 mL. Sterilize at a temperature of 116°C and a pressure of 100 kilopascals for standby use.

[0044] 3. Preparation of B solution

[0045] Each liter of B solution contains 2% w / v of trehalose, 2% w / v of galactose oligosaccharide, and 1% w / v of PEG6000, all mixed and dissolved in an appropriate amount of water, made up to 1000 mL, and sterilized by filtration for standby use.

[0046] 4. Preparation of a freeze-drying protective agent

[0047] After the A solution is cooled to room temperature, the A solution and the B solution are mixed in a ratio of 1:1 to 3:1 to obtain a freeze-drying protective agent for a virus-like particle.

[0048] Example 2

[0049] A freeze-drying protective agent for a virus-like particle vaccine is prepared as follows:

[0050] 1. Preparation of base solution

[0051] (1) Preparation of 0.1 mol / L Tris solution (pH 8.0): dissolve 12.1 g of Tris base in 800 mL of water, adjust the pH of the solution to 8.0 with hydrochloric acid, and dilute to 1000 mL with water.

[0052] (2) Preparation of 50 mmol / L EDTA solution (pH 8.0): dissolve 18.6 g of disodium EDTA in 800 mL of water, adjust the pH to 8.0 with sodium hydroxide solution, and dilute to 1000 mL with water.

[0053] 2. Preparation of solution A

[0054] Each liter of solution A contains 100 mL of 0.1 mol / L Tris solution (pH 8.0), 20 mL of 50 mmol / L EDTA solution (pH 8.0), 0.01% w / v polysorbate 80, 5% w / v sucrose, and 1% w / v trypsin, all dissolved in an appropriate amount of water, and diluted to 1000 mL. Sterilize at 110°C and 100 kPa pressure for standby use.

[0055] 3. Preparation of solution B

[0056] Each liter of solution B contains 0.1% w / v trehalose, 0.1% w / v galactose oligosaccharide, and 0.1% w / v PEG6000, all mixed and dissolved in an appropriate amount of water, and diluted to 1000 mL. Filter sterilize for standby use.

[0057] 4. Preparation of freeze-drying protectant

[0058] After the solution A is cooled to room temperature, mix the solution A and solution B in a ratio of 3:1 to obtain a freeze-drying protectant for virus-like particle vaccine.

[0059] Example 3

[0060] A freeze-drying protectant for virus-like particle vaccine is prepared as follows:

[0061] 1. Preparation of base solution

[0062] (1) Preparation of 0.1 mol / L Tris solution (pH 8.0): dissolve 12.1 g of Tris base in 800 mL of water, adjust the pH of the solution to 8.0 with hydrochloric acid, and dilute to 1000 mL with water.

[0063] (2) Preparation of 50 mmol / L EDTA solution (pH 8.0): 18.6 g of EDTA disodium salt was dissolved in 800 mL of water, and the pH value was adjusted to 8.0 with sodium hydroxide solution, and then water was added to 1000 mL.

[0064] 2. Preparation of A solution

[0065] Each liter of A solution contains 100 mL of 0.1 mol / L Tris solution (pH 8.0), 20 mL of 50 mmol / L EDTA solution (pH 8.0), 0.1% w / v of polysorbate 80, 20% w / v of sucrose, 5% w / v of tryptone, all of which are dissolved in an appropriate amount of water, and then diluted to 1000 mL. Sterilization is carried out at a temperature of 116°C and a pressure of 100 kilopascals for standby use.

[0066] 3. Preparation of B solution

[0067] Each liter of B solution contains 5% w / v of trehalose, 5% w / v of galactose oligosaccharide, and 2% w / v of PEG6000. All of these are mixed and dissolved in an appropriate amount of water, and then diluted to 1000 mL. Filtration is carried out for sterilization standby use.

[0068] 4. Preparation of freeze-drying protectant

[0069] After the A solution is cooled to room temperature, the A solution and the B solution are mixed uniformly at a ratio of 1:1 to 3:1, which can be used as a freeze-drying protectant for virus-like particles.

[0070] Example 4 (control group)

[0071] The preparation of the general vaccine freeze-drying protectant is as follows:

[0072] The general vaccine freeze-drying protectant is prepared according to the invention patent: A heat-resistant protectant for vaccine, a vaccine, and a preparation method of the vaccine (patent application number: 2016111534800).

[0073] Experimental example

[0074] 1. Freeze-drying

[0075] (1) Freeze-drying of virus-like particle freeze-drying protectant: The freeze-drying protectant prepared in Example 1 of the present application is mixed with the foot-and-mouth disease type O virus-like particle solution at a ratio of 1:1, and then uniformly mixed. The mixture is then divided into vials at a dose of 2 mL / vial, and the freeze-drying of the virus-like particles is completed through the steps of pre-freezing, sublimation, and drying. The foot-and-mouth disease type O virus-like particles after freeze-drying with this freeze-drying protectant are white sponge-like loose clumps, which are easy to separate from the bottle wall and quickly dissolved after adding diluent.

[0076] (2) Ordinary vaccine freeze-drying protectant freeze-drying: referring to the invention patent 2016111534800, the ordinary vaccine freeze-drying protectant is mixed with the foot-and-mouth disease O virus-like particle solution at a ratio of 1:1, and then evenly mixed. After that, it is divided into 2 mL / bottle in a Schlenk bottle through pre-freezing, sublimation and drying steps to complete the freeze-drying of the virus-like particles. The foot-and-mouth disease O virus-like particles freeze-dried by using this freeze-drying protectant are yellow, atrophic and attached to the bottom of the bottle. After adding the diluent, some of them are not dissolved.

[0077] The foot-and-mouth disease O virus-like particles freeze-dried by using the virus-like particle freeze-drying protectant meet the relevant requirements, while the ordinary vaccine freeze-drying protectant (control group) freeze-dried does not meet the basic requirements of freeze-dried products.

[0078] 2. Evaluation of freeze-drying effect of virus-like particle freeze-drying protectant

[0079] (1) Water content determination

[0080] Four bottles of freeze-dried foot-and-mouth disease O virus-like particles are taken out for water content determination according to the current Chinese Veterinary Pharmacopoeia Appendix. The results of the water content of the four bottles are 2.4%, 2.3%, 2.5% and 2.3%, respectively.

[0081] (2) Sterility test

[0082] Five bottles of freeze-dried foot-and-mouth disease O virus-like particles are taken out for sterility test according to the current Chinese Veterinary Pharmacopoeia Appendix. The results are 5 / 5 sterile growth.

[0083] (3) Electron microscope observation

[0084] The freeze-dried foot-and-mouth disease O virus-like particles are reconstituted, and the foot-and-mouth disease O virus-like particles before and after freeze-drying are observed by electron microscope. The results show that the morphology of the foot-and-mouth disease O virus-like particles before and after freeze-drying is basically consistent.

[0085] The results show that the morphology of the foot-and-mouth disease O virus-like particles does not change significantly after freeze-drying, and the morphology of the virus-like particles after freeze-drying remains basically consistent with that before freeze-drying. (Results are shown in Figure 1 and Figure 2 )

[0086] (4) Particle size analysis

[0087] The freeze-dried foot-and-mouth disease O virus-like particles are reconstituted, and the foot-and-mouth disease O virus-like particles before and after freeze-drying are analyzed by dynamic light scattering instrument. The results show that the particle size of the foot-and-mouth disease O virus-like particles before and after freeze-drying is 20-30 nm. The results show that the particle size of the foot-and-mouth disease O virus-like particles does not change during freeze-drying, and the particle size of the virus-like particles after freeze-drying remains basically consistent with that before freeze-drying.

[0088] (Results see Figure 4 )

[0089] (5) Analysis of assembly rate of virus-like particles

[0090] The virus-like particles of foot-and-mouth disease virus type O after lyophilization were reconstituted, and sucrose gradient centrifugation was used to analyze the assembly rate of the virus-like particles before and after lyophilization. The results showed that the assembly rate of the virus-like particles of foot-and-mouth disease virus type O before and after lyophilization was basically consistent. The results showed that the assembly rate of the virus-like particles of foot-and-mouth disease virus type O did not change significantly during lyophilization, and the assembly rate of the virus-like particles after lyophilization was basically consistent with that before lyophilization. (Results see Figure 5 )

[0091] (6) Evaluation of immune effect

[0092] The virus-like particles of foot-and-mouth disease virus type O after lyophilization were reconstituted to prepare virus-like particle vaccines, and the same batch of virus-like particles of foot-and-mouth disease virus type O without lyophilization treatment were diluted to the same concentration. The vaccines were prepared according to the preparation process of the virus-like particle vaccine of foot-and-mouth disease virus type O, and healthy pigs were immunized. Blood samples were collected at 28 days after immunization to separate sera, and the antibody titers were determined by liquid-phase blocking ELISA.

[0093] As shown in Table 1, the antibody level produced after immunization of piglets with the vaccine prepared from the virus-like particles of foot-and-mouth disease virus type O after lyophilization was higher than that before lyophilization. The results showed that due to the immunopotentiating effect of the galacto-oligosaccharide contained in the lyoprotectant, the immune effect of the virus-like particles of foot-and-mouth disease virus type O after lyophilization was better than that before lyophilization.

[0094] Table 1 Evaluation results of immune effect of virus-like particle vaccine of foot-and-mouth disease virus type O before and after lyophilization

[0095]

[0096] 3. Preservation period test of virus-like particles after lyophilization

[0097] The virus-like particle vaccine after lyophilization was stored at -15°C for 24 months, and sampling was performed for moisture determination, sterility test, electron microscope observation, particle size analysis, assembly rate analysis and immune effect evaluation to determine the preservation period of the virus-like particles after lyophilization.

[0098] (1) Moisture determination

[0099] Four bottles of the freeze-dried FMDV O type virus-like particles after 24 months of storage were taken out, and the moisture content was determined according to the current appendix of Chinese Veterinary Pharmacopoeia. The results of the moisture content of the four bottles were 2.5%, 2.4%, 2.5% and 2.5% respectively. The results showed that there was no significant difference between the moisture content of the freeze-dried FMDV O type virus-like particles after 24 months of storage at -15°C and that before storage.

[0100] (2) Sterility test

[0101] Five bottles of the freeze-dried FMDV O type virus-like particles after 24 months of storage were taken out, and the sterility test was performed according to the current appendix of Chinese Veterinary Pharmacopoeia. The results of the sterility test were 5 / 5. The results showed that there was no significant difference between the sterility test results of the freeze-dried FMDV O type virus-like particles after 24 months of storage at -15°C and that before storage.

[0102] (3) Electron microscope observation

[0103] The freeze-dried FMDV O type virus-like particles after 24 months of storage were reconstituted and observed under an electron microscope. The results showed that there was no significant difference between the electron microscope observation results of the freeze-dried FMDV O type virus-like particles after 24 months of storage at -15°C and that before storage. (Results are shown in Figure 3 )

[0104] (4) Particle size analysis

[0105] The freeze-dried FMDV O type virus-like particles after 24 months of storage were reconstituted and analyzed for particle size. The results showed that there was no significant difference between the particle size analysis results of the freeze-dried FMDV O type virus-like particles after 24 months of storage at -15°C and that before storage. (Results are shown in Figure 4 )

[0106] (5) Virus-like particle assembly rate analysis

[0107] The freeze-dried FMDV O type virus-like particles after 24 months of storage were reconstituted and analyzed for assembly rate. The results showed that there was no significant difference between the assembly rate of the freeze-dried FMDV O type virus-like particles after 24 months of storage at -15°C and that before storage. (Results are shown in Figure 5 )

[0108] (6) Evaluation of immune effect

[0109] The freeze-dried FMDV O type virus-like particles after 24 months of storage were reconstituted and prepared into vaccines according to the preparation process of FMDV O type virus-like particle vaccine. Healthy pigs were immunized to evaluate the immune effect. The results showed that the antibody level produced by the animals after immunization with the freeze-dried FMDV O type virus-like particles after 24 months of storage at -15°C was slightly lower than that before storage, but there was no significant difference.

[0110] Table 2 Immune effect of the freeze-dried FMDV O type virus-like particles after 24 months of storage

[0111]

[0112] The freeze-dried foot-and-mouth disease virus-like particles of type O are stored at -15℃ for 24 months, and the characteristics and immunization effects thereof do not change significantly. The above storage period test results show that the storage period of the freeze-dried foot-and-mouth disease virus-like particles of type O under the condition of -15℃ is at least 21 months or more.

Claims

1. A freeze-drying protectant for a swine foot-and-mouth disease virus type O virus-like particle vaccine, characterized in that, It is prepared by mixing A liquid and B liquid in a volume ratio of 1:1~3:1; The A liquid is prepared from components with the following final concentrations: 0.01~0.5mol / L basic solution I, 0.5~2mmol / L basic solution II, 0.01~0.1% polysorbate 80, 5~20% sucrose, and 1~5% tryptone; the basic solution I is Tris solution with a pH value of 8.0; the basic solution II is EDTA solution with a pH value of 8.0; The B liquid is prepared from components with the following final concentrations: 0.1~5% trehalose, 0.1~5% oligogalactose, and 0.1~2% PEG6000.

2. The freeze-dried protectant for swine foot-and-mouth disease virus O type virus-like particle vaccine according to claim 1, characterized by, The volume ratio of A liquid to B liquid is 2:

1.

3. The freeze-dried protectant for swine foot-and-mouth disease virus O type virus-like particle vaccine according to claim 1 or 2, characterized in that, The A liquid is prepared from components with the following final concentrations: 0.01mol / L basic solution I, 1mmol / L basic solution II, 0.05% polysorbate 80, 10% sucrose, and 2.5% tryptone.

4. The freeze-dried protectant for swine foot-and-mouth disease virus O type virus-like particle vaccine according to claim 1 or 2, characterized in that, The B liquid is prepared from components with the following final concentrations: 2% trehalose, 2% oligogalactose, and 1% PEG6000.

5. A process for the preparation of a lyoprotectant for a swine foot-and-mouth disease virus type O virus-like particle vaccine according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: (1) preparing basic solution I and basic solution II; (2) mixing and dissolving the components of A liquid according to the formula, and sterilizing at 100~150℃ and 80~100KPa; (3) mixing and dissolving the components of B liquid according to the formula, and then filtering and sterilizing; (4) after the A liquid is cooled to room temperature, mixing the A liquid and the B liquid in a volume ratio of 1:1~3:

1.

6. The production method according to claim 5, wherein The sterilization temperature of A liquid is 116℃, and the pressure is 100KPa.

Images