Vaccine freeze-drying protective agent as well as preparation method and application thereof

Abstract

The invention relates to a vaccine freeze-drying protective agent as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The vaccine freeze-drying protective agent is prepared from the following raw materials: trehalose, mannitol, arginine, pullulan, 2-hydroxypropyl-beta-cyclodextrin, phospholipid, sodium sulfite and a protective additive, the protective additive is at least one of hydroxyl ectoine, betaine, dodecyl dimethyl betaine and polycarboxylic acid betaine. The preparation method comprises the following steps: shearing and mixing the 2-hydroxypropyl-beta-cyclodextrin and the phospholipid at a high speed, sequentially adding the other components, adjusting the pH and osmotic pressure, performing sterile filtration, and performing freeze drying. The protective agent can effectively inhibit phase separation, sugar recrystallization and interfacial adsorption in the freeze-drying process, significantly improves the activity retention rate and physical stability of vaccines in the freeze-drying and storage periods, and is suitable for preparing freeze-drying preparations of various vaccines such as viral vaccines, bacterial vaccines and recombinant protein vaccines.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a vaccine freeze-drying protectant, its preparation method, and its application. Background Technology

[0002] As preventative biological products, vaccines are frequently affected by various unstable factors during storage, transportation, and use, such as temperature fluctuations, repeated freeze-thaw cycles, drying stress, and interfacial adsorption. These factors can easily lead to decreased antigen activity and reduced potency, directly impacting immunization efficacy and vaccination safety. While freeze-drying technology can significantly improve the long-term stability of vaccines, the freeze-drying process itself involves issues such as freeze concentration, mechanical damage from ice crystals, dehydration stress, and reconstitution shock, which can also cause irreversible damage to vaccine activity. Therefore, it is usually necessary to add appropriate freeze-drying protectants to freeze-dried formulations to maintain the integrity of the vaccine structure and its biological activity.

[0003] Currently used freeze-drying protectants mainly include non-reducing sugars (such as sucrose and trehalose), excipients (such as mannitol and glycine), amino acids (such as arginine), surfactants, and buffer systems. These components alleviate the loss of activity during freeze-drying to some extent through mechanisms such as glassy solidification, water substitution, inhibition of ice crystal growth, and reduction of interfacial tension. However, existing protection systems still have the following shortcomings in practical applications: However, the existing protection system may still have the following shortcomings: (1) Insufficient physical stability: Under high temperature or humidity conditions, the glassy system of sugars is prone to phase separation or sugar recrystallization, resulting in uneven solid structure, which in turn affects the long-term stability of the active ingredients of the vaccine. (2) It is difficult to balance molding and protection: In order to obtain good freeze-dried appearance and resolubility, it is often necessary to rely on easily crystallizable components such as mannitol to provide skeletal support, and amorphous components such as trehalose to maintain glassy protection. There is a contradiction between the two in formulation design and process control, and it is difficult to optimize them in a coordinated manner. (3) Limited system adaptability: Different types of vaccines (such as viral vaccines, bacterial vaccines, recombinant protein vaccines, etc.) have significantly different sensitivities to freezing, drying, oxidation, interfacial adsorption and aggregation. Traditional protective systems based on single sugars or excipients are difficult to be universally applied, especially for vaccines that are prone to aggregation or are sensitive to interfaces.

[0004] Therefore, developing a novel freeze-drying protectant that can simultaneously inhibit freeze-drying phase separation and recrystallization, balance freeze-drying properties and reconstitution properties, and has broad vaccine compatibility is of great significance for improving the stability of vaccine formulations throughout their entire lifecycle, extending shelf life, and ensuring vaccination efficacy. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides a vaccine freeze-drying protectant, its preparation method, and its application.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A vaccine freeze-drying protectant comprises the following raw materials: trehalose, mannitol, arginine, pullulan, 2-hydroxypropyl-β-cyclodextrin, phospholipids, sodium sulfite, and a protective additive; wherein the protective additive is at least one selected from hydroxyectoine, betaine, dodecyl dimethyl betaine, and polycarboxylate betaine.

[0007] Trehalose, as a typical non-reducing sugar, can stabilize the microenvironment around antigen or virus particles during the freeze-drying stage through water substitution / vitrification, reducing conformational changes and inactivation caused by dehydration, and helping to improve the retention of activity during reconstitution after freeze-drying.

[0008] Mannitol is mainly used as a molding agent and skeleton support. It is easy to crystallize to form a stable freeze-dried block structure, which improves appearance, reduces the risk of collapse, and promotes the formation of reconstitution channels, thereby improving the reconstitution speed and the compatibility with formulation processes.

[0009] Arginine, as an amino acid stabilizer and anti-aggregation component, can reduce non-specific interactions between proteins and particles, inhibit aggregation and co-aggregation during freeze-concentration and reconstitution, and also has a certain regulatory effect on the pH buffer and ionic environment of the system.

[0010] Pullulan, as a high-molecular-weight protective and thickening component, can improve the glass transition capacity and structural strength of the system, reduce the tendency of phase separation and molecular mobility during freeze-drying, and help form a more stable solid network, thereby improving the physical stability during storage.

[0011] 2-Hydroxypropyl-β-cyclodextrin has inclusion and interface regulation capabilities. It can form inclusion complexes with hydrophobic fragments to reduce interfacial adsorption and aggregation tendencies, and can synergistically improve resolution performance and tolerance to certain stresses.

[0012] Phospholipids, as surface-active / interface protection components, can form a protective layer at liquid-gas and solid-liquid interfaces, reducing the risk of adsorption, denaturation, and aggregation of vaccine components at the interface, and together with cyclodextrins and protective additives, enhance the interfacial stability of the system.

[0013] Sodium sulfite, as an antioxidant / reducing protective component, can remove dissolved oxygen in the solution or inhibit oxidation chain reactions, reducing the risk of activity reduction and quality deterioration caused by oxidation during freeze-drying and storage.

[0014] As a compatible solute / zwitterionic stabilizing component, the protective additive can enhance hydration and preferential repulsion effects, stabilize the microenvironment of antigen / virus particles, and inhibit interfacial adsorption and aggregation. Among them, polycarboxylate betaine is a zwitterionic polymer with advantages of multi-point hydration and steric hindrance stability. It does not easily migrate in the freeze-dried solid system and can synergistically inhibit phase separation / recrystallization and molecular motion with the trehalose glassy network and mannitol skeleton, thereby reducing the loss of activity and improving the potency retention rate during freeze-drying and storage.

[0015] While existing freeze-drying protection systems typically employ combinations of non-reducing sugars, forming agents, amino acids, surfactants, and buffer systems, they still face multiple stability challenges during vaccine freeze-drying and storage. For example, under high temperature / humidity conditions, glycosylated glassy systems are prone to phase separation or recrystallization, affecting activity retention. To balance freeze-dried appearance and reconstitution, it is often difficult to simultaneously satisfy the mannitol crystallization framework support and trehalose amorphous glassy protection. Furthermore, different types of vaccines exhibit significant differences in their sensitivity to stresses such as freeze concentration, ice crystal damage, drying dehydration, and reconstitution shocks, making them susceptible to inactivation pathways such as interfacial adsorption, aggregation / co-aggregation, and oxidation, leading to a decrease in potency after freeze-drying and during storage. Traditional single sugar / excipient systems have limited compatibility. Based on this, the present invention further introduces protective additives into the basic protective system of trehalose, mannitol, arginine, pullulan, 2-hydroxypropyl-β-cyclodextrin, phospholipids and sodium sulfite to solve the problem of potency reduction caused by freezing / drying stress, solid-state physical instability and interface-related inactivation during the freeze-drying process and subsequent storage.

[0016] Preferably, the vaccine freeze-drying protectant has the following mass concentrations for each component: trehalose 60-90 g / L, mannitol 25-40 g / L, arginine 3-8 g / L, pullulan 1-3 g / L, 2-hydroxypropyl-β-cyclodextrin 4-8 g / L, phospholipids 0.1-0.3 g / L, sodium sulfite 0.05-0.2 g / L, and protective additives 5-12 g / L.

[0017] By introducing the aforementioned protective additives, the protective system of this invention can provide more adequate microenvironmental stabilization for antigen proteins or vaccine particles in processes such as freeze-drying concentration, ice crystal damage, dehydration and drying, and reconstitution shock. On the one hand, the protective additives, as compatible solutes / zwitterionic stabilizing components, can enhance the hydration and preferential repulsion effect of the system, reduce protein conformational changes, interfacial adsorption and aggregation tendency, and synergistically inhibit particle aggregation and oxidation-related inactivation with phospholipids, cyclodextrins and arginine. On the other hand, in particular, polycarboxylate betaine, as a zwitterionic polymer, has multi-point hydration and steric hindrance stabilization effects, and is not easily migrated in the freeze-drying solid system. It is beneficial to synergistically inhibit solid physical instability such as phase separation / recrystallization and reduce molecular mobility with the trehalose glassy network and mannitol crystallization framework, thereby improving freeze-drying and reconstitution performance, reducing activity loss during freeze-drying and storage, making the vaccine detectable at higher potency and better potency retention after freeze-drying, and improving the stability and compatibility of the formulation under temperature fluctuations and high temperature / humidity storage conditions.

[0018] The protective additive is a mixture of hydroxyethylidene and polycarboxylate betaine in a mass ratio of 1:1–5:1.

[0019] This invention employs a combination of hydroxyectoine and polycarboxylate betaine, which achieve synergistic effects through their respective independent mechanisms of action. Hydroxyectoine stabilizes the conformation of the vaccine antigen through the hydration layer, preventing structural disturbances during freeze-drying; while polycarboxylate betaine inhibits aggregation and interfacial adsorption through interfacial protection, enhancing physical stability during freeze-drying. The combined protective agent significantly improves the vaccine's activity retention rate, reconstitution rate, and stability after freeze-drying.

[0020] The phospholipids are selected from soybean lecithin or egg yolk lecithin.

[0021] The vaccine freeze-drying protectant, wherein the polycarboxylate betaine is prepared by the following method: Under an inert atmosphere, N-(3-dimethylaminopropyl)methacrylamide was reacted with tert-butyl bromoacetate in an organic solvent to give an intermediate product. The intermediate product was polymerized in the presence of a chain transfer agent and an initiator, followed by deprotection and purification to obtain polycarboxylate betaine.

[0022] The reaction mechanism of polycarboxylate betaine of this invention is as follows: The tertiary amine in the N-(3-dimethylaminopropyl)methacrylamide molecule undergoes a nucleophilic substitution reaction with tert-butyl bromoacetate to generate an intermediate with a carboxyl side chain protected by a quaternary ammonium cation and tert-butyl ester; Under the presence of a chain transfer agent and an initiator and under deoxygenation conditions, the methacryloyl double bond of the intermediate undergoes free radical polymerization to obtain a polymer with a protected carboxyl side chain; Finally, trifluoroacetic acid is added to deprotect tert-butyl ester to generate a carboxyl / carboxylate salt, which forms an amphoteric polymer with the quaternary ammonium cation as an inner salt. After purification and freeze-drying, polycarboxylate betaine is obtained.

[0023] Preferably, the preparation method of the polycarboxylate betaine is as follows: under nitrogen protection, 3-8g of N-(3-dimethylaminopropyl)methacrylamide and 40-80mL of anhydrous acetonitrile are mixed evenly, 8-11g of tert-butyl bromoacetate is added and stirring is continued for 20-60min, and the reaction is carried out at 50-65℃ for 18-36h to obtain the intermediate product; Under nitrogen protection, 0.05-0.2 g of 4-cyano-4-(phenylthiocarboxylthio)valerate, 0.01-0.03 g of benzoyl peroxide, 8-12 g of the above intermediate product and N,N-dimethylformamide were stirred until homogeneous, deoxygenated by bubbling, and reacted at 60-80℃ and 400-800 rpm for 18-36 h. Diethyl ether was added, and a white precipitate was formed. Trifluoroacetic acid was added, and the reaction was carried out at room temperature and 200-500 rpm for 1-4 h. The mixture was purified by dialysis, filtered, and freeze-dried to obtain polycarboxylate betaine.

[0024] The preparation method of the vaccine freeze-drying protectant includes the following steps: (1) Mix 2-hydroxypropyl-β-cyclodextrin with phospholipids and subject to high-speed shearing to obtain mixture A; (2) Add trehalose, pullulan, sodium sulfite, mannitol and arginine to mixture A and stir to mix; (3) Add protective additives and continue stirring; (4) Adjust the pH to 6.0-7.8, adjust the osmotic pressure to 400-600 mosm / kg, make up the volume, perform sterile filtration, freeze dry, and obtain the vaccine freeze-dried protectant.

[0025] Preferably, the high-speed shearing process in step (1) has a rotation speed of 8000-12000 rpm and a time of 2-5 min; the stirring speed in steps (2) and (3) is 400-800 rpm.

[0026] The application of the vaccine freeze-drying protectant in the preparation of vaccine freeze-dried formulations.

[0027] The vaccine includes at least one of a viral vaccine, a bacterial vaccine, or a recombinant protein vaccine.

[0028] The beneficial effects of this invention are: This invention provides a vaccine freeze-drying protectant, its preparation method, and its application. The vaccine freeze-drying protectant of this invention introduces protective additives into a basic system consisting of trehalose, mannitol, arginine, pullulan, 2-hydroxypropyl-β-cyclodextrin, phospholipids, and sodium sulfite. In particular, the combination of hydroxyectoine and polycarboxylate betaine significantly improves the stability of freeze-dried vaccines without increasing process complexity. Under the same freeze-drying conditions, it effectively reduces potency loss during freeze-drying and storage, resulting in better titer retention after freeze-drying and storage at 4°C for 28 days. Simultaneously, it significantly improves the appearance and reconstitution performance of the freeze-dried vaccine, resulting in faster reconstitution, clearer reconstituted solutions with less precipitation, less shrinkage of the freeze-dried block, and a more reasonable osmotic pressure after reconstitution. This balances stability, potency retention, and compatibility with the formulation process, improving the reliability of vaccine preparation storage, transportation, and use. Detailed Implementation

[0029] The invention will now be described in further detail with reference to specific embodiments, but it should not be construed as limiting the scope of the invention to the following embodiments.

[0030] The raw materials described in this application are partially described; all other raw materials not described are commercially available. Hydroxyectoine is also known as 1,4,5,6-tetrahydro-2-methyl-5-hydroxy-4-pyrimidinecarboxylic acid, CAS: 165542-15-4.

[0031] Example 1 A vaccine freeze-dried protective agent is composed of the following raw materials at the following mass concentrations: 80 g / L trehalose, 35 g / L mannitol, 5 g / L arginine, 2 g / L pullulan, 6 g / L 2-hydroxypropyl-β-cyclodextrin, 0.15 g / L soybean lecithin, 0.1 g / L sodium sulfite, and 8 g / L protective additive.

[0032] The protective additive is a mixture of hydroxyethylidene and polycarboxylate betaine in a mass ratio of 3:2.

[0033] The preparation method of the polycarboxylate betaine is as follows: Under nitrogen protection, 5g of N-(3-dimethylaminopropyl)methacrylamide and 50mL of anhydrous acetonitrile are mixed evenly. Under the conditions of room temperature and stirring at 600rpm, 9.5g of tert-butyl bromoacetate is added and stirring is continued for 30min. The mixture is reacted at 55℃ for 24h. After cooling to room temperature, 500mL of diethyl ether is added, stirred, and allowed to stand. A white precipitate is precipitated, filtered, washed, and dried to obtain the intermediate product. Under nitrogen protection, 0.1 g of 4-cyano-4-(phenylthiocarboxylthio)valerate, 0.015 g of benzoyl peroxide, 10 g of the above intermediate product and N,N-dimethylformamide were stirred until homogeneous. The mixture was deoxygenated by bubbling under nitrogen / argon for 60 min and reacted at 70 °C and 600 rpm for 24 h. After the reaction was completed, the mixture was cooled with liquid nitrogen, and 500 mL of diethyl ether was added. After stirring and standing, a white precipitate was formed. Trifluoroacetic acid was added, and the mixture was reacted at room temperature and 300 rpm for 2 h. The mixture was purified by dialysis. The dialysis solution was filtered through a 0.22 μm filter and freeze-dried to obtain polycarboxylate betaine.

[0034] The preparation method of the vaccine freeze-drying protectant is as follows: 2-hydroxypropyl-β-cyclodextrin and soybean lecithin are mixed and sheared at 10,000 rpm for 3 min to obtain mixture A; trehalose, pullulan, sodium sulfite, mannitol and arginine are added to mixture A, and the mixture is stirred at 600 rpm for 15 min; protective additives are added and stirring is continued for 20 min; the pH is adjusted to between 6.0 and 7.8 with 0.1 M phosphate buffer solution, and the osmotic pressure is measured to be within the range of 400 mosm. After volume adjustment, the solution is aseptically filtered through a 0.22 μm filter membrane. The aseptically filtered solution is pre-frozen at -40℃ for 6 h, vacuum dried at -20℃ for 24 h, and finally dried at room temperature for 8 h to obtain the vaccine freeze-drying protectant.

[0035] Example 2 The invention is basically the same as the example, except that the protective additive is hydroxyectoin.

[0036] Example 3 The invention is basically the same as the example, except that the protective additive is polycarboxylate betaine.

[0037] The preparation method of the polycarboxylate betaine is the same as that in Example 1.

[0038] Example 4 A vaccine freeze-dried protective agent is composed of the following raw materials at the following mass concentrations: 80 g / L trehalose, 35 g / L mannitol, 5 g / L arginine, 2 g / L pullulan, 6 g / L 2-hydroxypropyl-β-cyclodextrin, 0.15 g / L soybean lecithin, 0.1 g / L sodium sulfite, and 8 g / L protective additive.

[0039] The protective additive is betaine.

[0040] The preparation method of the vaccine freeze-drying protectant is as follows: 2-hydroxypropyl-β-cyclodextrin and soybean lecithin are mixed and sheared at 10,000 rpm for 3 min to obtain mixture A; trehalose, pullulan, sodium sulfite, mannitol and arginine are added to mixture A, and the mixture is stirred at 600 rpm for 15 min; protective additives are added and stirring is continued for 20 min; the pH is adjusted to between 6.0 and 7.8 with 0.1 M phosphate buffer solution, and the osmotic pressure is measured to be within the range of 400 mosm. After volume adjustment, the solution is aseptically filtered through a 0.22 μm filter membrane. The aseptically filtered solution is pre-frozen at -40℃ for 6 h, vacuum dried at -20℃ for 24 h, and finally dried at room temperature for 8 h to obtain the vaccine freeze-drying protectant.

[0041] Example 5 A vaccine freeze-dried protective agent is composed of the following raw materials at the following mass concentrations: 80 g / L trehalose, 35 g / L mannitol, 5 g / L arginine, 2 g / L pullulan, 6 g / L 2-hydroxypropyl-β-cyclodextrin, 0.15 g / L soybean lecithin, 0.1 g / L sodium sulfite, and 8 g / L protective additive.

[0042] The protective additive is dodecyl dimethyl betaine.

[0043] The preparation method of the vaccine freeze-drying protectant is as follows: 2-hydroxypropyl-β-cyclodextrin and soybean lecithin are mixed and sheared at 10,000 rpm for 3 min to obtain mixture A; trehalose, pullulan, sodium sulfite, mannitol and arginine are added to mixture A, and the mixture is stirred at 600 rpm for 15 min; protective additives are added and stirring is continued for 20 min; the pH is adjusted to between 6.0 and 7.8 with 0.1 M phosphate buffer solution, and the osmotic pressure is measured to be within the range of 400 mosm. After volume adjustment, the solution is aseptically filtered through a 0.22 μm filter membrane. The aseptically filtered solution is pre-frozen at -40℃ for 6 h, vacuum dried at -20℃ for 24 h, and finally dried at room temperature for 8 h to obtain the vaccine freeze-drying protectant.

[0044] Example 6 A vaccine freeze-dried protective agent is composed of the following raw materials at the following mass concentrations: 80 g / L trehalose, 35 g / L mannitol, 5 g / L arginine, 2 g / L pullulan, 6 g / L 2-hydroxypropyl-β-cyclodextrin, 0.15 g / L soybean lecithin, 0.1 g / L sodium sulfite, and 8 g / L protective additive.

[0045] The protective additive is a mixture of hydroxyectoin and dodecyl dimethyl betaine in a mass ratio of 3:2.

[0046] The preparation method of the vaccine freeze-drying protectant is as follows: 2-hydroxypropyl-β-cyclodextrin and soybean lecithin are mixed and sheared at 10,000 rpm for 3 min to obtain mixture A; trehalose, pullulan, sodium sulfite, mannitol and arginine are added to mixture A, and the mixture is stirred at 600 rpm for 15 min; protective additives are added and stirring is continued for 20 min; the pH is adjusted to between 6.0 and 7.8 with 0.1 M phosphate buffer solution, and the osmotic pressure is measured to be within the range of 400 mosm. After volume adjustment, the solution is aseptically filtered through a 0.22 μm filter membrane. The aseptically filtered solution is pre-frozen at -40℃ for 6 h, vacuum dried at -20℃ for 24 h, and finally dried at room temperature for 8 h to obtain the vaccine freeze-drying protectant.

[0047] Example 7 It is basically the same as Example 3, except that the protective additive is polycarboxylate betaine.

[0048] The preparation method of the polycarboxylate betaine is as follows: Under nitrogen protection, 5g of dimethylaminoethyl methacrylate and 50mL of anhydrous acetonitrile are mixed evenly. Under the conditions of room temperature and stirring at 600rpm, 9.5g of tert-butyl chloroacetate is added and stirring is continued for 30min. The mixture is then reacted at 55℃ for 24h. After cooling to room temperature, 500mL of diethyl ether is added, stirred, and allowed to stand. A white precipitate is precipitated, filtered, washed, and dried to obtain the intermediate product. Under nitrogen protection, 0.1 g of 4-cyano-4-(phenylthiocarboxylthio)valerate, 0.015 g of benzoyl peroxide, 10 g of the above intermediate product and N,N-dimethylformamide were stirred until homogeneous. The mixture was deoxygenated by bubbling under nitrogen / argon for 60 min and reacted at 70 °C and 600 rpm for 24 h. After the reaction was completed, the mixture was cooled with liquid nitrogen, and 500 mL of diethyl ether was added. After stirring and standing, a white precipitate was formed. Trifluoroacetic acid was added, and the mixture was reacted at room temperature and 300 rpm for 2 h. The mixture was purified by dialysis. The dialysis solution was filtered through a 0.22 μm filter and freeze-dried to obtain polycarboxylate betaine.

[0049] Comparative Example 1 A freeze-dried vaccine protectant is composed of the following raw materials at the following mass concentrations: 80 g / L trehalose, 35 g / L mannitol, 5 g / L arginine, 2 g / L pullulan, 6 g / L 2-hydroxypropyl-β-cyclodextrin, 0.15 g / L soybean lecithin, and 0.1 g / L sodium sulfite.

[0050] The preparation method of the vaccine freeze-drying protectant is as follows: 2-hydroxypropyl-β-cyclodextrin and soybean lecithin are mixed and sheared at 10,000 rpm for 3 min to obtain mixture A; trehalose, pullulan, sodium sulfite, mannitol and arginine are added to mixture A and stirred at 600 rpm for 15 min; the pH is adjusted to between 6.0 and 7.8 with 0.1M phosphate buffer solution, and the osmotic pressure is measured to be within the range of 400 mosm. After volume adjustment, the solution is aseptically filtered through a 0.22 μm filter membrane. The aseptically filtered solution is pre-frozen at -40℃ for 6 h, vacuum dried at -20℃ for 24 h, and finally dried at room temperature for 8 h to obtain the vaccine freeze-drying protectant.

[0051] Comparative Example 2 The method is basically the same as in Example 1, except that the step-by-step freeze-drying method is not used in the preparation of the vaccine freeze-drying protectant: 2-hydroxypropyl-β-cyclodextrin and soybean lecithin are mixed and sheared at 10,000 rpm for 3 min to obtain mixture A; trehalose, pullulan, sodium sulfite, mannitol and arginine are added to mixture A and stirred at 600 rpm for 15 min; protective additives are added and stirring is continued for 20 min; the pH is adjusted to between 6.0 and 7.8 with 0.1 M phosphate buffer solution, the osmotic pressure is measured to be within the range of 400 mosm, and after volume adjustment, the solution is aseptically filtered through a 0.22 μm filter membrane. The aseptically filtered solution is stored at 4 °C to obtain the vaccine freeze-drying protectant.

[0052] Test Example 1 Using human embryonic lung diploid cells as the culture medium, varicella virus harvested material was prepared. Appropriate amounts of the lyophilization protectant prepared in Examples 1-7 and Comparative Examples 1-2 were directly mixed with the varicella virus harvested material, with a weight ratio of lyophilization protectant to varicella virus harvested material of 5:1. The mixture was gently vortexed until homogeneous. The mixture was then freeze-thawed at -70°C or digested with EDTA. The harvested virus was centrifuged at 1500 rpm for 15 min, and the seed virus was obtained by reconstitution with the lyophilization protectant to collect the precipitate.

[0053] The harvested virus samples were dispensed into 2mL vials at a volume of 0.58mL, partially stoppered, and placed in a freeze-drying chamber for freeze-drying.

[0054] The obtained vaccine was pre-frozen at -40℃ for 6 hours, vacuum dried at -20℃ for 24 hours, and finally dried at room temperature for 8 hours to obtain the freeze-dried vaccine.

[0055] The titers of the vaccines before and after freeze-drying were determined, and the stability of the freeze-dried vaccines at 4°C was also tested. Each group was tested 5 times, and the average value results are shown in Table 1.

[0056] Table 1. Results of titer performance test

[0057] Test Example 2 The above-mentioned freeze-dried vaccine was reconstituted, and 0.5 mL of water for injection was added. The reconstitution time and osmotic pressure were recorded. Four groups were tested for each group, and the average value was taken and rounded to the nearest integer. The results are shown in Table 2.

[0058] Table 2. Results of reconstitution properties, lyophilized appearance, and osmotic pressure test after reconstitution.

[0059] The results above show that the vaccine freeze-drying protectant prepared by this invention can improve the activity retention rate and physical stability of the vaccine during freeze-drying and storage. Specific analysis is as follows: In Example 1, the combination of hydroxyethylidene and polycarboxylate betaine resulted in the best titer retention at 0 and 28 days after freeze-drying, the shortest reconstitution time, clear reconstitution without visible precipitation, and normal freeze-dried appearance. This indicates that the combined protective additive can significantly improve the potency retention and storage stability after freeze-drying while ensuring freeze-drying and reconstitution performance. Compared to Example 2, which uses only hydroxyethylidene, and Example 3, which uses only polycarboxylate betaine, Comparative Example 1, which does not contain protective additives, can reduce titer loss during freeze-drying and storage, shorten reconstitution time, and improve appearance. However, its titer retention and reconstitution performance are still generally weaker than those of Example 1. In Comparative Example 2, with the same protective agent formulation as Example 1, the lack of a matching step-by-step freeze-drying process resulted in decreased titer retention after freeze-drying, prolonged reconstitution time, a small amount of precipitation, and shrinkage of the freeze-dried block. This is because the technical effect of the present invention comes from the synergistic effect of the protective agent formulation and the step-by-step freeze-drying process. Without the corresponding process, the freeze-drying molding properties, reconstitution, and activity stability are easily reduced due to increased stress from freezing concentration and dehydration.

[0060] Comparative Example 1, without protective additives, showed the most significant drop in titer after freeze-drying, the longest reconstitution time, and the appearance of particulate precipitation, accompanied by appearance defects such as shrinkage of the freeze-dried block. This indicates that relying solely on the traditional sugar / forming agent / amino acid / surfactant system is insufficient to adequately suppress the activity loss and physical instability caused by freeze-drying and storage. Although Example 4, using betaine, and Example 5, using dodecyl dimethyl betaine, added the same amount of protective additives, their titer retention, reconstitution, and freeze-dried appearance were inferior to the example systems, showing that the comprehensive protective ability of this type of additive for this system is limited. Example 6, using a combination of hydroxyectoine and dodecyl dimethyl betaine, showed some improvement compared to Example 5, but still fell short of Example 1. The combination of hydroxyectoine and dodecyl dimethyl betaine failed to achieve a synergistic effect. Although the titer retention and reconstitution performance improved after the combination, the effect was still lower than using hydroxyectoine alone. Small molecule betaine has limited role in the combination, and high dosages may cause problems such as increased osmotic pressure and freeze-concentration, affecting freeze-drying stability. Example 7 and Example 3 are both polycarboxylate betaine systems, but due to the different preparation routes of the polycarboxylate betaine used, the titer retention and reconstitution clarity of Example 7 are lower than those of Example 3, indicating that the structural differences of polycarboxylate betaine can also affect the protective effect.

Claims

1. A vaccine freeze-drying protectant, characterized in that, It includes the following raw materials: trehalose, mannitol, arginine, pullulan, 2-hydroxypropyl-β-cyclodextrin, phospholipids, sodium sulfite, and protective additives; the protective additives are at least one of hydroxyectoine, betaine, dodecyl dimethyl betaine, and polycarboxylate betaine.

2. The vaccine freeze-drying protectant according to claim 1, characterized in that, The mass concentrations of each component are as follows: trehalose 60-90 g / L, mannitol 25-40 g / L, arginine 3-8 g / L, pullulan 1-3 g / L, 2-hydroxypropyl-β-cyclodextrin 4-8 g / L, phospholipids 0.1-0.3 g / L, sodium sulfite 0.05-0.2 g / L, and protective additives 5-12 g / L.

3. The vaccine freeze-drying protectant according to claim 2, characterized in that, The protective additive is a mixture of hydroxyethylidene and polycarboxylate betaine in a mass ratio of 1:1 to 5:

1.

4. The vaccine freeze-drying protectant according to any one of claims 1-3, characterized in that, The phospholipids are selected from soybean lecithin or egg yolk lecithin.

5. The vaccine freeze-drying protectant according to any one of claims 1-3, characterized in that, The polycarboxylate betaine is prepared by the following method: Under an inert atmosphere, N-(3-dimethylaminopropyl)methacrylamide was reacted with tert-butyl bromoacetate in an organic solvent to give an intermediate product. The intermediate product was polymerized in the presence of a chain transfer agent and an initiator, followed by deprotection and purification to obtain polycarboxylate betaine.

6. The vaccine freeze-drying protectant according to claim 5, characterized in that, The preparation method of the polycarboxylate betaine is as follows: Under nitrogen protection, 3-8g of N-(3-dimethylaminopropyl)methacrylamide and 40-80mL of anhydrous acetonitrile are mixed evenly, 8-11g of tert-butyl bromoacetate is added and stirring is continued for 20-60min, and the reaction is carried out at 50-65℃ for 18-36h to obtain the intermediate product. Under nitrogen protection, 0.05-0.2 g of 4-cyano-4-(phenylthiocarboxylthio)valerate, 0.01-0.03 g of benzoyl peroxide, 8-12 g of the above intermediate product and N,N-dimethylformamide were stirred until homogeneous, deoxygenated by bubbling, and reacted at 60-80℃ and 400-800 rpm for 18-36 h. Diethyl ether was added, and a white precipitate was formed. Trifluoroacetic acid was added, and the reaction was carried out at room temperature and 200-500 rpm for 1-4 h. The mixture was purified by dialysis, filtered, and freeze-dried to obtain polycarboxylate betaine.

7. A method for preparing a vaccine freeze-drying protectant as described in any one of claims 1-6, characterized in that, Includes the following steps: (1) Mix 2-hydroxypropyl-β-cyclodextrin with phospholipids and subject to high-speed shearing to obtain mixture A; (2) Add trehalose, pullulan, sodium sulfite, mannitol and arginine to mixture A and stir to mix; (3) Add protective additives and continue stirring; (4) Adjust the pH to 6.0-7.8, adjust the osmotic pressure to 400-600 mosm / kg, make up the volume, perform sterile filtration, freeze dry, and obtain the vaccine freeze-dried protectant.

8. The preparation method according to claim 7, characterized in that, The high-speed shearing process in step (1) is performed at a speed of 8000-12000 rpm for 2-5 min; the stirring speed in steps (2) and (3) is 400-800 rpm.

9. The use of a vaccine freeze-drying protectant as described in any one of claims 1-6 in the preparation of a vaccine freeze-dried formulation.

10. The application according to claim 9, characterized in that, The vaccine includes at least one of a viral vaccine, a bacterial vaccine, or a recombinant protein vaccine.

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