A vaccine heat-resistant protective agent, a preparation method and application thereof

Abstract

This invention discloses a vaccine heat protectant, its preparation method, and its application, belonging to the field of vaccine technology. The vaccine heat protectant comprises the following raw materials: polycarboxylated betaine, taurine, sugars, polysarcosine, erythritol, gelatin hydrolysate, metal ion chelating agent, and buffer solution. This invention also provides its preparation method. Compared with existing technologies, this invention significantly improves the freezing and reconstitution properties of freeze-dried cakes, shortens reconstitution time, and reduces turbidity and insoluble particles through the synergistic effect of polycarboxylated betaine, taurine, sugars, polysarcosine, gelatin hydrolysate, erythritol, and EDTA-Na2. After storage at 2-8℃, 25℃, and 37℃ and multiple freeze-thaw cycles, it is significantly superior to the traditional sucrose and mannitol system in terms of antigen content residual rate and glass transition temperature, effectively buffering the risk of inactivation caused by cold chain fluctuations.

Description

Technical Field

[0001] This invention relates to the field of vaccine technology, and in particular to a vaccine heat protectant, its preparation method, and its application. Background Technology

[0002] Vaccine heat protectants are a type of formulation system that, without altering the structure and immunogenicity of the antigen itself, enables the vaccine to remain sufficiently stable at room temperature or even higher temperatures through the rational combination of excipients and processes.

[0003] Traditional heat protectants for vaccines are mainly represented by sucrose, trehalose, mannitol, and dextran, which are used in conjunction with freeze-drying to construct a high-Tg glassy network, and this is currently the most widely used approach. However, recent studies have found that relying solely on a single sugar or a simple combination of sugar alcohols still presents problems such as increased aggregation, long reconstitution time, and increased insoluble particles under conditions of repeated freeze-thaw cycles, prolonged high-temperature acceleration, and complex antigens.

[0004] CN109157657A discloses a heat-resistant freeze-drying protectant for live infectious bursal disease (IBD) vaccine in chickens and its preparation method. This heat-resistant freeze-drying protectant is composed of the following components by weight percentage: 8%-20% skim milk powder, 3%-6% trehalose, 0.3%-1% mannitol, 0.3%-1% glycine, 0.5%-2% L-cysteine, 0.1%-0.5% sodium isoVC, 0.08%-0.4% citric acid, 2.56%-2.82% trisodium citrate, with the balance being water for injection. This heat-resistant freeze-drying protectant allows the live IBD vaccine to be stored at 2-8°C for 24 months. During the freeze-drying and storage processes, its appearance and efficacy remain largely unchanged, preventing the loss of the active ingredients over a longer period and thus reducing transportation and storage costs. CN106729734A discloses a heat-resistant freeze-drying protectant for small ruminant disease (PPR) live vaccine and its preparation method. The PPR live vaccine freeze-drying protectant is composed of the following components by weight percentage: 7.5% sucrose, 4.0% dextran, 4.0% sorbitol, 2.5% fish gelatin, 0.051% Na₂HPO₄, 0.125% NaH₂PO₄, with the balance being water for injection; it is prepared by high-temperature sterilization. This invention's PPR live vaccine freeze-drying protectant reduces the damage to viral activity caused by various physicochemical factors during freeze-drying. However, it suffers from insufficient heat resistance, poor reconstitution, and easy antigen aggregation and inactivation. Summary of the Invention

[0005] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is that the traditional sucrose and mannitol heat-resistant freeze-drying protection system has insufficient heat resistance, poor resolubility, and easy antigen aggregation and inactivation.

[0006] To achieve the above objectives, the present invention provides a vaccine heat protectant, characterized in that it comprises the following raw materials: polycarboxylic acid betaine, taurine, sugars, polysarcosine, erythritol, metal ion chelating agent, and buffer solution; Preferably, a vaccine heat protectant, using a 5-20 mM buffer solution as a solvent, comprises 0.05-0.3% (w / v) polycarboxylic acid betaine, 0.05-0.5% (w / v) taurine, 5-12% (w / v) carbohydrates, 0.05-0.5% (w / v) polysarcosine, 1-3% (w / v) erythritol, and 0.001-0.05 mM metal ion chelating agent, with a pH of 6.8-7.4; The sugar is at least one of stachyose and raffinose; more preferably, the sugar is a mixture of stachyose and raffinose in a mass ratio of (1-3):(1-3).

[0007] The metal ion chelating agent is EDTA-Na2.

[0008] The buffer solution is selected from one of histidine buffer, Tris buffer, phosphate buffer, citrate buffer, and MES buffer. A further preferred embodiment of a vaccine heat protectant, using a 5-20 mM buffer solution as a solvent, comprises 0.05-0.3% (w / v) polycarboxylic acid betaine, 0.05-0.5% (w / v) taurine, 5-12% (w / v) carbohydrates, 0.05-0.5% (w / v) polysarcosine, 0.1-0.5% (w / v) gelatin hydrolysate, 1-3% (w / v) erythritol, and 0.001-0.05 mM metal ion chelating agent, with a pH of 6.8-7.4; The method for preparing the gelatin enzymatic hydrolysate is as follows: 80-120g of gelatin is added to 1L of water and allowed to stand for 30-60min. The mixture is then heated to 50-60℃ and stirred for 60-120min. The solution is passed through a 100-200 mesh sieve, and the pH of the gelatin solution is adjusted to 6.8-7.2. The solution is kept at 50-60℃, and 0.1-2wt% of enzyme preparation is added based on the weight of the gelatin. The mixture is then treated for 1-3h. After that, the solution is heated to 90-95℃ and kept at 10-20min for inactivation. After the treatment, the solution is centrifuged, the supernatant is collected, concentrated, and dried to obtain the gelatin enzymatic hydrolysate.

[0009] The enzyme preparation mentioned therein is at least one of neutral protease and alkaline protease.

[0010] The present invention also discloses a method for preparing a vaccine heat protectant.

[0011] A method for preparing a vaccine heat protectant includes the following steps: Polycarboxylated betaine, taurine, stachyose, polysarcosine, erythritol, gelatin hydrolysate, and EDTA-Na2 were added to histidine buffer and mixed evenly. The pH was adjusted, and finally the mixture was replenished with histidine buffer to obtain the vaccine heat protectant. The vaccine heat protectant and vaccine stock solution are mixed at a volume ratio of 1:1 and then freeze-dried to obtain the vaccine.

[0012] The freeze-drying process is as follows: (1) Pre-freezing stage: the temperature is reduced from 5°C to -40°C at a cooling rate of 0.5-1°C / min and held for 2-3 hours; (2) First drying stage: After the pre-freezing is completed, turn on the vacuum system and pump the chamber pressure to 80-120 mTorr. Raise the shelf temperature from -40℃ to -35℃ and keep it for 4-5 hours. Then slowly raise it to -15℃ at a rate of about 0.1-0.2℃ / min and keep it for 10-12 hours. (3) Second drying stage: Under the condition of keeping the chamber pressure no higher than 50 mTorr, the shelf temperature is raised from -15℃ to 25℃ at a heating rate of about 0.2-0.3℃ / min and maintained for 6-8h; after the freeze-drying is completed, the shelf is fully plugged under vacuum conditions.

[0013] The vaccine stock solution is one of the following: live attenuated vaccine stock solution, inactivated vaccine stock solution, viral lysate vaccine stock solution, or recombinant subunit vaccine stock solution; Polycarboxylated betaine is a typical zwitterionic polymer, with both cationic and carboxylate groups on its molecular chain. It is electrically neutral overall but highly hydrophilic. In aqueous solution, it can form a dense hydration layer around proteins or nanoparticles, weakening electrostatic attraction and hydrophobic association between particles, and inhibiting adsorption and irreversible aggregation at container and gas-liquid interfaces. Polycarboxylated betaine interacts with polysarcosine to form the outer hydrophilic protective layer. Polycarboxylated betaine focuses on strong hydration and electrical shielding, while polysarcosine provides compliant chain segments and steric hindrance, enabling the particles to maintain uniform dispersion even under concentration, freeze-drying, and heating conditions.

[0014] Taurine, as an endogenous amphoteric small molecule, primarily functions as an osmotic protectant and regulates the hydration environment. By optimizing the outer hydration layer of proteins or nanoparticles, it mitigates conformational loosening and membrane stress during heating, concentration, and freeze-drying reconstitution. Taurine, in conjunction with polycarboxylic acid betaine and polysarcosine, forms a hydration shell and flexible chain layer on the particle surface. Taurine buffers osmotic pressure and localizes dehydration in the bulk phase, ensuring particle dispersion stability even under high solids content and thermal stress conditions. Stachyose and raffinose, non-reducing oligosaccharides, form amorphous sugar glasses with high glass transition temperatures after freeze-drying. They partially replace water molecules through hydrogen bonds, fixing the spatial conformation of proteins and carriers, and reducing overall molecular mobility through vitrification, inhibiting aggregation and degradation. Due to their different molecular sizes and vitrification capabilities, stachyose and raffinose, when used in combination, form a denser and more stable mixed glassy network, providing hierarchical protection and stronger conformational stability throughout the freeze-drying-reconstitution process, resulting in a synergistic effect significantly superior to using either oligosaccharide alone.

[0015] Erythritol, a four-carbon polyol, participates in the formation of a stable backbone, improving mechanical strength; simultaneously, it interacts with stachyose and raffinose to regulate osmotic pressure and ice crystal behavior, indirectly improving the thermal stability and dispersibility of antigens and carriers. Gelatin hydrolysate forms a polypeptide buffer layer on the antigen surface, reducing interfacial denaturation and providing sacrificial antioxidant protection and inhibiting aggregation.

[0016] EDTA-Na2 blocks Fenton-like reaction pathways, significantly reducing the risk of protein oxidation, lipid peroxidation, and nucleic acid degradation, providing a stable chemical environment for the aforementioned physical and colloidal protection. The buffer solution maintains a pH range of 6.8-7.4 to avoid acid-base catalytic hydrolysis and conformational changes, and works with EDTA-Na2 to slow down thermally accelerated chemical reactions.

[0017] The beneficial effects of this invention are: Compared with existing technologies, this invention significantly improves the forming and reconstitution properties of freeze-dried cakes, shortens reconstitution time, and reduces turbidity and insoluble particles through the synergistic effect of polycarboxylated betaine, taurine, sugars, polysarcosine, gelatin hydrolysate, erythritol, and EDTA-Na2. After storage at 2-8℃, 25℃, and 37℃ and multiple freeze-thaw cycles, it is significantly superior to the traditional sucrose and mannitol system in terms of antigen content residual rate and glass transition temperature, effectively buffering the risk of inactivation caused by cold chain fluctuations. Detailed Implementation

[0018] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this invention.

[0019] The preparation method of gelatin enzymatic hydrolysate in the embodiment is as follows: 100g of gelatin is added to 1L of water and left to stand for 30min, heated to 55℃ and stirred for 60min, passed through a 100-mesh sieve, the pH of the gelatin solution is adjusted to 6.8, and the temperature is maintained at 55℃. 1wt% enzyme preparation is added according to the weight of gelatin, and the treatment is continued for 3h. Then, the temperature is heated to 95℃ and maintained for 15min to inactivate the enzyme. After the treatment, the solution is centrifuged, the supernatant is collected, concentrated, and dried to obtain gelatin enzymatic hydrolysate. The enzyme preparation is a neutral protease (enzyme activity of 50000U / g, food grade, Shandong Longket Enzyme Preparation Co., Ltd.).

[0020] Polycarboxylated betaine, product number: RC-6336, purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0021] Polysarcosine, molecular weight 5kDa, was purchased from Xiamen Xinke Biomedical Technology Co., Ltd.

[0022] A commercially available live porcine pseudorabies vaccine (PRV) is prepared by passage of an attenuated strain of PRV into a cell culture. The example used an attenuated live vaccine with the Bartha-K61 strain as the seed virus.

[0023] Other raw materials not mentioned are all common raw materials. The above content is only for the purpose of illustrating the present invention and should not be construed as a strict limitation of the present invention. Those skilled in the art can directly purchase the same / similar raw materials from the market or prepare them themselves.

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1 A vaccine heat protectant is prepared by mixing 0.15% (w / v) polycarboxybetaine, 0.20% (w / v) taurine, 8.0% (w / v) stachyose, 0.20% (w / v) polysarcosine, 2.0% (w / v) erythritol, 0.2% (w / v) gelatin hydrolysate, and 0.01 mM EDTA-Na2 in 10 mM histidine buffer, adjusting the pH to 6.9, and finally adding histidine buffer to a final volume of 1 mL to obtain the vaccine heat protectant. The vaccine heat protectant and the vaccine stock solution are mixed at a volume ratio of 1:1 and then freeze-dried to obtain the vaccine; the vaccine stock solution is a live porcine pseudorabies vaccine.

[0026] The freeze-drying process is as follows: (1) Pre-freezing stage: the temperature is reduced from 5℃ to -40℃ at a cooling rate of 0.5℃ / min and held for 3 hours; (2) First drying stage: After the pre-freezing is completed, the vacuum system is turned on and the chamber pressure is pumped to 80 mTorr. The shelf temperature is raised from -40℃ to -35℃ and maintained for 4 hours. Then, it is slowly raised to -15℃ at a rate of about 0.2℃ / min and maintained for 12 hours. (3) Second drying stage: Under the condition of keeping the chamber pressure no higher than 50 mTorr, the shelf temperature is raised from -15℃ to 25℃ at a heating rate of about 0.3℃ / min and maintained for 6 hours; after the freeze drying is completed, the shelf is fully plugged under vacuum conditions.

[0027] Example 2 A vaccine heat protectant is prepared by mixing 0.15% (w / v) polycarboxybetaine, 0.20% (w / v) taurine, 8.0% (w / v) stachyose, 0.20% (w / v) polysarcosine, 2.0% (w / v) erythritol, 0.2% (w / v) gelatin hydrolysate, and 0.01 mM EDTA-Na2 in 10 mM phosphate buffer, adjusting the pH to 6.9, and finally adding phosphate buffer to a final volume of 1 mL to obtain the vaccine heat protectant. The vaccine heat protectant and the vaccine stock solution are mixed at a volume ratio of 1:1 and then freeze-dried to obtain the vaccine; the vaccine stock solution is a live porcine pseudorabies vaccine.

[0028] The freeze-drying process is as follows: (1) Pre-freezing stage: the temperature is reduced from 5℃ to -40℃ at a cooling rate of 0.5℃ / min and held for 3 hours; (2) First drying stage: After the pre-freezing is completed, the vacuum system is turned on and the chamber pressure is pumped to 80 mTorr. The shelf temperature is raised from -40℃ to -35℃ and maintained for 4 hours. Then, it is slowly raised to -15℃ at a rate of about 0.2℃ / min and maintained for 12 hours. (3) Second drying stage: Under the condition of keeping the chamber pressure no higher than 50 mTorr, the shelf temperature is raised from -15℃ to 25℃ at a heating rate of about 0.3℃ / min and maintained for 6 hours; after the freeze drying is completed, the shelf is fully plugged under vacuum conditions.

[0029] Example 3 A vaccine heat protectant is prepared by mixing 0.15% (w / v) polycarboxybetaine, 0.20% (w / v) taurine, 8.0% (w / v) raffinose, 0.20% (w / v) polysarcosine, 2.0% (w / v) erythritol, 0.2% (w / v) gelatin hydrolysate, and 0.01 mM EDTA-Na2 in 10 mM histidine buffer, adjusting the pH to 6.9, and finally adding histidine buffer to a final volume of 1 mL to obtain the vaccine heat protectant. The vaccine heat protectant and the vaccine stock solution are mixed at a volume ratio of 1:1 and then freeze-dried to obtain the vaccine; the vaccine stock solution is a live porcine pseudorabies vaccine.

[0030] The freeze-drying process is as follows: (1) Pre-freezing stage: the temperature is reduced from 5℃ to -40℃ at a cooling rate of 0.5℃ / min and held for 3 hours; (2) First drying stage: After the pre-freezing is completed, the vacuum system is turned on and the chamber pressure is pumped to 80 mTorr. The shelf temperature is raised from -40℃ to -35℃ and maintained for 4 hours. Then, it is slowly raised to -15℃ at a rate of about 0.2℃ / min and maintained for 12 hours. (3) Second drying stage: Under the condition of keeping the chamber pressure no higher than 50 mTorr, the shelf temperature is raised from -15℃ to 25℃ at a heating rate of about 0.3℃ / min and maintained for 6 hours; after the freeze drying is completed, the shelf is fully plugged under vacuum conditions.

[0031] Example 4 A vaccine heat protectant is prepared by mixing 0.15% (w / v) polycarboxybetaine, 0.20% (w / v) taurine, 4.0% (w / v) stachyose, 4.0% (w / v) raffinose, 0.20% (w / v) polysarcosine, 2.0% (w / v) erythritol, 0.2% (w / v) gelatin hydrolysate, and 0.01 mM EDTA-Na2 in 10 mM histidine buffer, adjusting the pH to 6.9, and finally adding histidine buffer to a final volume of 1 mL to obtain the vaccine heat protectant. The vaccine heat protectant and the vaccine stock solution are mixed at a volume ratio of 1:1 and then freeze-dried to obtain the vaccine; the vaccine stock solution is a live porcine pseudorabies vaccine.

[0032] The freeze-drying process is as follows: (1) Pre-freezing stage: the temperature is reduced from 5℃ to -40℃ at a cooling rate of 0.5℃ / min and held for 3 hours; (2) First drying stage: After the pre-freezing is completed, the vacuum system is turned on and the chamber pressure is pumped to 80 mTorr. The shelf temperature is raised from -40℃ to -35℃ and maintained for 4 hours. Then, it is slowly raised to -15℃ at a rate of about 0.2℃ / min and maintained for 12 hours. (3) Second drying stage: Under the condition of keeping the chamber pressure no higher than 50 mTorr, the shelf temperature is raised from -15℃ to 25℃ at a heating rate of about 0.3℃ / min and maintained for 6 hours; after the freeze drying is completed, the shelf is fully plugged under vacuum conditions.

[0033] Comparative Example 1 A vaccine heat protectant is prepared by mixing 0.20% (w / v) taurine, 8.0% (w / v) stachyose, 2.0% (w / v) erythritol, 0.2% (w / v) gelatin hydrolysate, and 0.01 mM EDTA-Na2 in 10 mM histidine buffer, adjusting the pH to 6.9, and finally adding histidine buffer to a final volume of 1 mL. The vaccine heat protectant and the vaccine stock solution are mixed at a volume ratio of 1:1 and then freeze-dried to obtain the vaccine; the vaccine stock solution is a live porcine pseudorabies vaccine.

[0034] The freeze-drying process is as follows: (1) Pre-freezing stage: the temperature is reduced from 5℃ to -40℃ at a cooling rate of 0.5℃ / min and held for 3 hours; (2) First drying stage: After the pre-freezing is completed, the vacuum system is turned on and the chamber pressure is pumped to 80 mTorr. The shelf temperature is raised from -40℃ to -35℃ and maintained for 4 hours. Then, it is slowly raised to -15℃ at a rate of about 0.2℃ / min and maintained for 12 hours. (3) Second drying stage: Under the condition of keeping the chamber pressure no higher than 50 mTorr, the shelf temperature is raised from -15℃ to 25℃ at a heating rate of about 0.3℃ / min and maintained for 6 hours; after the freeze drying is completed, the shelf is fully plugged under vacuum conditions.

[0035] Comparative Example 2 A vaccine heat protectant is prepared by mixing 0.15% (w / v) polycarboxybetaine, 8.0% (w / v) stachyose, 0.20% (w / v) polysarcosine, 2.0% (w / v) erythritol, 0.2% (w / v) gelatin hydrolysate, and 0.01 mM EDTA-Na2 in 10 mM histidine buffer, adjusting the pH to 6.9, and finally adding histidine buffer to bring the volume to 1 mL to obtain the vaccine heat protectant. The vaccine heat protectant and the vaccine stock solution are mixed at a volume ratio of 1:1 and then freeze-dried to obtain the vaccine; the vaccine stock solution is a live porcine pseudorabies vaccine.

[0036] The freeze-drying process is as follows: (1) Pre-freezing stage: the temperature is reduced from 5℃ to -40℃ at a cooling rate of 0.5℃ / min and held for 3 hours; (2) First drying stage: After the pre-freezing is completed, the vacuum system is turned on and the chamber pressure is pumped to 80 mTorr. The shelf temperature is raised from -40℃ to -35℃ and maintained for 4 hours. Then, it is slowly raised to -15℃ at a rate of about 0.2℃ / min and maintained for 12 hours. (3) Second drying stage: Under the condition of keeping the chamber pressure no higher than 50 mTorr, the shelf temperature is raised from -15℃ to 25℃ at a heating rate of about 0.3℃ / min and maintained for 6 hours; after the freeze drying is completed, the shelf is fully plugged under vacuum conditions.

[0037] Comparative Example 3 A vaccine heat protectant is prepared by mixing 0.15% (w / v) polycarboxybetaine, 0.20% (w / v) taurine, 8.0% (w / v) stachyose, 0.20% (w / v) polysarcosine, 0.2% (w / v) gelatin hydrolysate, and 0.01 mM EDTA-Na2 in 10 mM histidine buffer, adjusting the pH to 6.9, and finally adding histidine buffer to bring the volume to 1 mL to obtain the vaccine heat protectant. The vaccine heat protectant and the vaccine stock solution are mixed at a volume ratio of 1:1 and then freeze-dried to obtain the vaccine; the vaccine stock solution is a live porcine pseudorabies vaccine.

[0038] The freeze-drying process is as follows: (1) Pre-freezing stage: the temperature is reduced from 5℃ to -40℃ at a cooling rate of 0.5℃ / min and held for 3 hours; (2) First drying stage: After the pre-freezing is completed, the vacuum system is turned on and the chamber pressure is pumped to 80 mTorr. The shelf temperature is raised from -40℃ to -35℃ and maintained for 4 hours. Then, it is slowly raised to -15℃ at a rate of about 0.2℃ / min and maintained for 12 hours. (3) Second drying stage: Under the condition of keeping the chamber pressure no higher than 50 mTorr, the shelf temperature is raised from -15℃ to 25℃ at a heating rate of about 0.3℃ / min and maintained for 6 hours; after the freeze drying is completed, the shelf is fully plugged under vacuum conditions.

[0039] Comparative Example 4 A vaccine heat protectant is prepared by mixing 0.15% (w / v) polycarboxybetaine, 0.20% (w / v) taurine, 8.0% (w / v) stachyose, 0.20% (w / v) polysarcosine, 2.0% (w / v) erythritol, and 0.2% (w / v) gelatin hydrolysate in 10 mM histidine buffer, adjusting the pH to 6.9, and finally adding histidine buffer to a final volume of 1 mL to obtain the vaccine heat protectant. The vaccine heat protectant and the vaccine stock solution are mixed at a volume ratio of 1:1 and then freeze-dried to obtain the vaccine; the vaccine stock solution is a live porcine pseudorabies vaccine.

[0040] The freeze-drying process is as follows: (1) Pre-freezing stage: the temperature is reduced from 5℃ to -40℃ at a cooling rate of 0.5℃ / min and held for 3 hours; (2) First drying stage: After the pre-freezing is completed, the vacuum system is turned on and the chamber pressure is pumped to 80 mTorr. The shelf temperature is raised from -40℃ to -35℃ and maintained for 4 hours. Then, it is slowly raised to -15℃ at a rate of about 0.2℃ / min and maintained for 12 hours. (3) Second drying stage: Under the condition of keeping the chamber pressure no higher than 50 mTorr, the shelf temperature is raised from -15℃ to 25℃ at a heating rate of about 0.3℃ / min and maintained for 6 hours; after the freeze drying is completed, the shelf is fully plugged under vacuum conditions.

[0041] Comparative Example 5 A vaccine heat protectant is prepared by mixing 5% (w / v) sucrose and 3% (w / v) mannitol in 10mM histidine buffer, adjusting the pH to 6.9, and finally adding histidine buffer to make up to 1mL. The vaccine heat protectant and the vaccine stock solution are mixed at a volume ratio of 1:1 and then freeze-dried to obtain the vaccine; the vaccine stock solution is a live porcine pseudorabies vaccine.

[0042] The freeze-drying process is as follows: (1) Pre-freezing stage: the temperature is reduced from 5℃ to -40℃ at a cooling rate of 0.5℃ / min and held for 3 hours; (2) First drying stage: After the pre-freezing is completed, the vacuum system is turned on and the chamber pressure is pumped to 80 mTorr. The shelf temperature is raised from -40℃ to -35℃ and maintained for 4 hours. Then, it is slowly raised to -15℃ at a rate of about 0.2℃ / min and maintained for 12 hours. (3) Second drying stage: Under the condition of keeping the chamber pressure no higher than 50 mTorr, the shelf temperature is raised from -15℃ to 25℃ at a heating rate of about 0.3℃ / min and maintained for 6 hours; after the freeze drying is completed, the shelf is fully plugged under vacuum conditions.

[0043] Comparative Example 6 A vaccine heat protectant is prepared by mixing 0.20% (w / v) taurine, 8.0% (w / v) stachyose, 0.20% (w / v) polysarcosine, 2.0% (w / v) erythritol, 0.2% (w / v) gelatin hydrolysate, and 0.01 mM EDTA-Na2 in 10 mM histidine buffer, adjusting the pH to 6.9, and finally adding histidine buffer to bring the volume to 1 mL. The vaccine heat protectant and the vaccine stock solution are mixed at a volume ratio of 1:1 and then freeze-dried to obtain the vaccine; the vaccine stock solution is a live porcine pseudorabies vaccine.

[0044] The freeze-drying process is as follows: (1) Pre-freezing stage: the temperature is reduced from 5℃ to -40℃ at a cooling rate of 0.5℃ / min and held for 3 hours; (2) First drying stage: After the pre-freezing is completed, the vacuum system is turned on and the chamber pressure is pumped to 80 mTorr. The shelf temperature is raised from -40℃ to -35℃ and maintained for 4 hours. Then, it is slowly raised to -15℃ at a rate of about 0.2℃ / min and maintained for 12 hours. (3) Second drying stage: Under the condition of keeping the chamber pressure no higher than 50 mTorr, the shelf temperature is raised from -15℃ to 25℃ at a heating rate of about 0.3℃ / min and maintained for 6 hours; after the freeze drying is completed, the shelf is fully plugged under vacuum conditions.

[0045] Comparative Example 7 A vaccine heat protectant is prepared by mixing 0.15% (w / v) polycarboxybetaine, 0.20% (w / v) taurine, 8.0% (w / v) stachyose, 2.0% (w / v) erythritol, 0.2% (w / v) gelatin hydrolysate, and 0.01 mM EDTA-Na2 in 10 mM histidine buffer, adjusting the pH to 6.9, and finally adding histidine buffer to bring the volume to 1 mL to obtain the vaccine heat protectant. The vaccine heat protectant and the vaccine stock solution are mixed at a volume ratio of 1:1 and then freeze-dried to obtain the vaccine; the vaccine stock solution is a live porcine pseudorabies vaccine.

[0046] The freeze-drying process is as follows: (1) Pre-freezing stage: the temperature is reduced from 5℃ to -40℃ at a cooling rate of 0.5℃ / min and held for 3 hours; (2) First drying stage: After the pre-freezing is completed, the vacuum system is turned on and the chamber pressure is pumped to 80 mTorr. The shelf temperature is raised from -40℃ to -35℃ and maintained for 4 hours. Then, it is slowly raised to -15℃ at a rate of about 0.2℃ / min and maintained for 12 hours. (3) Second drying stage: Under the condition of keeping the chamber pressure no higher than 50 mTorr, the shelf temperature is raised from -15℃ to 25℃ at a heating rate of about 0.3℃ / min and maintained for 6 hours; after the freeze drying is completed, the shelf is fully plugged under vacuum conditions.

[0047] Comparative Example 8 A vaccine heat protectant is prepared by mixing 0.15% (w / v) polycarboxybetaine, 0.20% (w / v) taurine, 8.0% (w / v) stachyose, 0.20% (w / v) polysarcosine, 2.0% (w / v) erythritol, and 0.01 mM EDTA-Na2 in 10 mM histidine buffer, adjusting the pH to 6.9, and finally adding histidine buffer to bring the volume to 1 mL to obtain the vaccine heat protectant. The vaccine heat protectant and the vaccine stock solution are mixed at a volume ratio of 1:1 and then freeze-dried to obtain the vaccine; the vaccine stock solution is a live porcine pseudorabies vaccine.

[0048] The freeze-drying process is as follows: (1) Pre-freezing stage: the temperature is reduced from 5℃ to -40℃ at a cooling rate of 0.5℃ / min and held for 3 hours; (2) First drying stage: After the pre-freezing is completed, the vacuum system is turned on and the chamber pressure is pumped to 80 mTorr. The shelf temperature is raised from -40℃ to -35℃ and maintained for 4 hours. Then, it is slowly raised to -15℃ at a rate of about 0.2℃ / min and maintained for 12 hours. (3) Second drying stage: Under the condition of keeping the chamber pressure no higher than 50 mTorr, the shelf temperature is raised from -15℃ to 25℃ at a heating rate of about 0.3℃ / min and maintained for 6 hours; after the freeze drying is completed, the shelf is fully plugged under vacuum conditions.

[0049] Test Example 1 The appearance and reconstitution properties of the lyophilized formulations were tested using the following method: Each group of lyophilized vaccines was sealed and placed overnight at 2-8℃ for equilibration. Then, an equal volume of water for injection was added at room temperature. The mixture was gently inverted and mixed without vigorous shaking or sonication. The time required for the lyophilized cake to completely disintegrate and for the solution to become clear was recorded by visual observation. At the same time, the absorbance of the solution was measured at 600 nm as a turbidity indicator, and the presence of visible particles or flocculent precipitates was observed under a standard optical microscope.

[0050] Accelerated thermal stability test: The freeze-dried vaccine products of each group were stored in a sealed container away from light at 2-8℃, 25℃, and 37℃, and samples were taken at four time points: 0d, 7d, 14d, and 28d. At each time point, the freeze-dried preparation was reconstituted using the method described above, and the soluble antigen content was determined using an antigen quantification method suitable for this vaccine (such as ELISA based on antigen-specific antibodies). The value measured at 0d was taken as 100%, and the antigen residual rate at each time point was calculated.

[0051] Physicochemical stability testing: Residual moisture content and glass transition temperature were measured for both the experimental and comparative lyophilized formulations. Residual moisture content was determined by coulometric Karl Fischer titration, and glass transition temperature was determined by differential scanning calorimetry.

[0052] Freeze-thaw stability test: After reconstituted, the freeze-dried vaccines of the examples and comparative examples were placed between -20°C and 25°C for multiple freeze-thaw cycles. Each cycle was defined as being frozen at -20°C for no less than 12 hours and then completely thawed at room temperature. Samples were taken after the 0th, 3rd and 5th cycles to detect the antigen content and observe the appearance of the solution.

[0053] Table 1. Results of tests on the appearance and reconstitution properties of freeze-dried cakes. Table 2. Test results of antigen content residual rate under conditions of 2-8℃ and 25℃. Table 3. Results of antigen content residual rate test under accelerated conditions at 37℃ Table 4 Results of Physicochemical Property Tests Table 5. Results of Freeze-Thaw Stability Test As can be seen from Tables 1-5 above, when stachyose and raffinose are used in Example 4, the intermolecular hydrogen bond network is more compact and complementary, thus increasing the glass transition temperature (Tg) and reducing residual moisture. This provides a favorable physical basis for the antigen to maintain conformational stability under high temperature or freeze-thaw stress. In contrast, using stachyose or raffinose respectively often leads to a decrease in Tg and an increase in migration rate, which in turn manifests as a decrease in antigen activity and an increase in turbidity after reconstitution.

[0054] Table 1 shows that, based on the appearance and resolubility of the freeze-dried cakes, the freeze-dried cakes of Examples 1 and 4 were full, without collapse or obvious cracks, and had short resolubility with almost no particles. A common feature of these two groups is the use of histidine buffer, and the simultaneous presence of polycarboxylated betaine, taurine, stachyose, raffinose, polysarcosine, erythritol, gelatin hydrolysate, and EDTA. Stachyose, raffinose, and erythritol construct a dense, high-Tg glassy phase, while polycarboxylated betaine, polysarcosine, and gelatin hydrolysate together form a three-dimensional framework and colloidal protective layer. This buffers the shrinkage stress during freezing and drying, and inhibits protein denaturation and aggregation at the interface, resulting in a macroscopically intact cake structure, rapid resolubility, and clear solution.

[0055] Compared to Example 1, Example 2 only replaced the histidine buffer with a phosphate buffer. The lyophilized cake showed a slight central depression, and the reconstitution time and turbidity increased slightly. Example 3 used raffinose instead of stachyose in the sugar composition, and similarly showed a slight depression and slightly more fine cracks. This indicates that, with other components remaining the same, the ionic strength of the buffer system and the composition of stachyose and raffinose affect glass phase formation and drying shrinkage behavior. A higher phosphate ionic strength may interfere with ice crystal and glass phase structure, while stachyose or raffinose systems are more prone to localized phase separation than composite systems.

[0056] Comparative Example 1 lacked polycarboxylated betaine and polysarcosine, resulting in insufficient skeletal components. Its freeze-dried cake exhibited edge shrinkage, increased surface cracks, significantly prolonged reconstitution time, and increased turbidity. Comparative Example 2 lacked taurine; Comparative Example 3 lacked erythritol; Comparative Example 4 lacked EDTA; Comparative Example 6 lacked polycarboxylated betaine; Comparative Example 7 lacked polysarcosine; and Comparative Example 8 lacked gelatin hydrolysate. All of these examples showed varying degrees of collapse, shrinkage, increased cracking, and increased turbidity and particle count after reconstitution. Comparative Example 5 used only the traditional combination of sucrose and mannitol. While its freeze-dried cake had a relatively acceptable overall shape, it exhibited edge collapse, the longest reconstitution time, the highest turbidity, and significant sedimentation at the bottom. This aligns with the characteristics of sucrose and mannitol, where mannitol easily crystallizes, resulting in severe phase separation and a fragile structure, making reconstitution difficult.

[0057] As shown in Table 2-4, the difference between Example 1 and Example 2 lies only in the buffer, which is histidine and phosphate, respectively. Example 1 showed a slightly higher antigen residue rate and Tg at 25°C and 37°C, indicating that the histidine buffer with lower ionic strength is more conducive to forming a homogeneous glass phase with high Tg and low internal stress, resulting in less disturbance to the antigen conformation. In contrast, the phosphate buffer system may promote partial degradation or aggregation under high-temperature conditions through its higher ionic strength and the participation of phosphate groups in the reaction.

[0058] Table 5 shows that after 5 freeze-thaw cycles, the antigen residue rate in Examples 1-4 remained at approximately 85%-93%, and the solutions were mainly clear with only a few bubbles or fine particles. In contrast, the antigen residue rates in Comparative Examples 2 and 5 decreased to 78.6% and 74.4%, respectively, with significantly turbid solutions and sediment at the bottom of the bottles. This indicates that the combination of oligosaccharides and erythritol helps control ice crystal morphology and absorb freeze-thaw stress, while the presence of taurine, polycarboxylic acid betaine, polysarcosine, and gelatin hydrolysate effectively alleviates osmotic pressure fluctuations, interfacial stress, and particle aggregation during the freeze-thaw process. The absence of these components significantly increases structural damage and aggregation under freeze-thaw stress, leading to a rapid decline in antigen activity.

[0059] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A vaccine heat-stable protective agent, characterized by: It includes the following raw materials: polycarboxylated betaine, taurine, sugars, polysarcosine, erythritol, metal ion chelating agents, and buffer solutions.

2. The vaccine thermostable protective agent of claim 1, wherein: The solvent is a 5-20 mM buffer solution containing 0.05-0.3% (w / v) polycarboxybetaine, 0.05-0.5% (w / v) taurine, 5-12% (w / v) carbohydrates, 0.05-0.5% (w / v) polysarcosine, 1-3% (w / v) erythritol, and 0.001-0.05 mM metal ion chelating agent.

3. The vaccine heat protectant as described in claim 1, characterized in that: The sugar is at least one of stachyose and raffinose.

4. The vaccine heat protectant as described in claim 1, characterized in that: The metal ion chelating agent is disodium ethylenediaminetetraacetate.

5. The vaccine heat protectant as described in claim 1, characterized in that: The buffer solution is selected from one or more of histidine buffer, Tris buffer, phosphate buffer, citrate buffer, and MES buffer.

6. The vaccine heat protectant as described in claim 1, characterized in that: It also includes gelatin hydrolysate.

7. A method for preparing a vaccine heat protectant as described in any one of claims 1-6, characterized in that: Polycarboxylated betaine, taurine, stachyose, polysarcosine, erythritol, gelatin hydrolysate, and EDTA-Na2 were added to histidine buffer and mixed thoroughly. The pH was adjusted, and finally the mixture was replenished with histidine buffer to obtain the vaccine heat protectant.

8. The application of a vaccine heat protectant as described in any one of claims 1-6, characterized in that: The heat-resistant protective agent for the vaccine is mixed with the vaccine stock solution and then freeze-dried to obtain a freeze-dried vaccine.

9. The application of the vaccine heat protectant as described in claim 8, characterized in that: The freeze-drying process is as follows: (1) Pre-freezing stage: the temperature is reduced from 5℃ to -40℃ at a cooling rate of 0.5-1℃ / min and maintained for 2-3 hours; (2) First drying stage: After the pre-freezing is completed, turn on the vacuum system and pump the chamber pressure to 80-120 mTorr. Raise the shelf temperature from -40℃ to -35℃ and keep it for 4-5 hours. Then slowly raise it to -15℃ at a rate of about 0.1-0.2℃ / min and keep it for 10-12 hours. (3) Second drying stage: Under the condition of keeping the chamber pressure no higher than 50 mTorr, the shelf temperature is raised from -15℃ to 25℃ at a heating rate of about 0.2-0.3℃ / min and maintained for 6-8h; after the freeze-drying is completed, the shelf is fully plugged under vacuum conditions.

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