Pig pseudorabies vaccine freeze-drying protective agent and preparation method thereof

Abstract

The invention discloses a porcine pseudorabies vaccine freeze-drying protective agent and a preparation method thereof, and belongs to the technical field of vaccine protective agents. The preservative comprises the following components with final concentration: 100-200g / L of protein hydrolysate, 80-160g / L of cane sugar, 40-80g / L of amino acid, 20-40g / L of gelatin, 20-40g / L of polyvinylpyrrolidone and 1-2g / L of pH buffer agent. The freeze-drying protective agent prepared by the invention is good in form after being freeze-dried, and a freeze-dried block mass is tidy in edge and can be completely separated from a bottle wall. Tests show that the virus content is almost not reduced within 12 months, the virus content is reduced by no more than 0.3 titer within 24 months and the virus content is reduced by no more than 0.6 titer within 36 months by using the freeze-drying protective agent prepared by the invention under the condition of 2-8 DEG C, and the virus protection time is long.

Description

Technical Field

[0001] The present invention belongs to the technical field of vaccine protectants, and particularly relates to a freeze-drying protectant for porcine pseudorabies vaccine and a preparation method thereof. Background Art

[0002] Currently, the protectants used in domestic porcine pseudorabies vaccines are mostly sucrose-milk protectants. The products using this protectant have a validity period of about 1 year, and the storage temperature is mostly below -15°C. During the storage period, the virus content shows a slow downward trend, and the protective effect on live virus is not ideal. In recent years, with the progress of technology, some new protectant formulations containing biological materials such as gelatin, protein, and amino acids have emerged. However, most of these raw materials are imported, with high prices, and some of them contain porcine-derived raw materials such as trypsin, which poses a certain risk of exogenous virus contamination for porcine vaccines. Summary of the Invention

[0003] Aiming at the above deficiencies in the prior art, the present invention provides a freeze-drying protectant for porcine pseudorabies vaccine and a preparation method thereof, which can enable the freeze-dried product to be stored under the condition of 2 - 8°C and maintain stable quality within 36 months.

[0004] To achieve the above object, the technical solution adopted by the present invention to solve its technical problems is: The object of the present invention is to provide a freeze-drying protectant for porcine pseudorabies vaccine, comprising components with the following final concentrations: Hydrolyzed protein 100 - 200 g / L, sucrose 80 - 160 g / L, amino acids 40 - 80 g / L, gelatin 20 - 40 g / L, polyvinylpyrrolidone 20 - 40 g / L, and pH buffer 1 - 2 g / L; the hydrolyzed protein includes acid-hydrolyzed casein and hydrolyzed milk protein, and the mass ratio of the two is 1.5 - 3:1 - 2.

[0005] Further, it includes components with the following final concentrations: Hydrolyzed protein 100 - 150 g / L, sucrose 100 - 160 g / L, amino acids 40 - 60 g / L, gelatin 30 - 40 g / L, polyvinylpyrrolidone 20 - 3 g / L, and pH buffer 1 - 2 g / L.

[0006] Sucrose: As a small molecule substance, sugars can make microorganisms form a uniform suspension, and at the same time have the functions of inhibiting ice crystal growth and stabilizing proteins.

[0007] Gelatin: Forms a porous grid structure, improves the re-solubility, and at the same time enhances the mechanical strength of the freeze-dried mass.

[0008] Acid-hydrolyzed casein, hydrolyzed milk protein: Provide nutritional support, regulate and maintain osmotic pressure, reduce aggregation, denaturation or inactivation of active ingredients caused by factors such as ice crystal formation and drying stress during freeze-drying, and improve stability.

[0009] Sodium glutamate, glycine: Stabilize the protein structure, act as an energy source, and improve the appearance quality of freeze-dried masses.

[0010] Polyvinylpyrrolidone: Increase the glass transition temperature, prevent ice crystal growth, reduce protein denaturation, and improve reconstitution performance.

[0011] Dipotassium hydrogen phosphate: Regulate the pH value, provide an electrolyte environment, and enhance stability.

[0012] Furthermore, the mass ratio of acid-hydrolyzed casein to hydrolyzed milk protein is 1.5:1.

[0013] Furthermore, the amino acids include glycine and sodium glutamate with a mass ratio of 0.5 - 1:0.5 - 1.

[0014] Furthermore, the mass ratio of glycine to sodium glutamate is 1:1.

[0015] Furthermore, the pH buffer is dipotassium hydrogen phosphate.

[0016] Another object of the present invention is to provide a preparation method of the above-mentioned freeze-drying protectant for porcine pseudorabies vaccine, which includes the following process: Mix each component according to the formula, dissolve in water, sterilize at 110 - 125 °C under high pressure for 20 - 30 min, and then cool to room temperature.

[0017] Furthermore, the high-pressure sterilization temperature is 116 °C, the pressure is 68 Kpa, and the sterilization time is 20 min.

[0018] Advantages of the present invention: The freeze-drying protectant prepared by the present invention has a good appearance, a good morphology after freeze-drying, the edges of the freeze-dried mass are neat, and it can be completely separated from the bottle wall. After testing, under the condition of 2 - 8 °C, the virus content hardly decreases within 12 months, the virus content decreases by no more than 0.3 titre within 24 months, and the virus content decreases by no more than 0.6 titre within 36 months, providing a long protection time for the virus.

[0019] The protectant of the present invention has low cost, is easy to prepare, and is safe and non-toxic. The raw materials in the formula can completely use the raw materials produced by domestic manufacturers, and the preparation cost is lower compared with imported raw materials. All components in the formula can be prepared by high-pressure steam sterilization, without the need for filtration sterilization, and the preparation is simple and convenient. Moreover, after injecting pigs and guinea pigs with 10 times the dosage, there are no any adverse reactions, indicating its excellent biological safety. Detailed Embodiments

[0020] The following describes the detailed embodiments of the present invention to facilitate those skilled in the art of this technology to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the detailed embodiments. For those of ordinary skill in the art of this technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions and creations using the concept of the present invention are within the scope of protection.

[0021] Example 1 A freeze-drying protectant for porcine pseudorabies vaccine is prepared as follows: (1) Weigh 140 g of sucrose, 30 g of gelatin, 60 g of acid-hydrolyzed casein, 40 g of hydrolyzed milk protein, 20 g of sodium glutamate, 20 g of glycine, 20 g of polyvinylpyrrolidone (K-30), and 1 g of dipotassium hydrogen phosphate for standby; (2) After mixing the above components evenly, add 800 mL of injection water, measure the pH value and adjust it to between 7.0 and 7.4, then make up the volume to 1000 mL with injection water, and then sterilize at 116 °C under high pressure for 20 min to obtain the freeze-drying protectant. After cooling, store it at 2 - 8 °C for no more than 30 days.

[0022] Example 2 A freeze-drying protectant for porcine pseudorabies vaccine is prepared as follows: (1) Weigh 80 g of sucrose, 20 g of gelatin, 90 g of acid-hydrolyzed casein, 60 g of hydrolyzed milk protein, 30 g of sodium glutamate, 30 g of glycine, 30 g of polyvinylpyrrolidone (K-30), and 1.5 g of dipotassium hydrogen phosphate for standby; (2) After mixing the above components evenly, add 800 mL of injection water, measure the pH value and adjust it to between 7.0 and 7.4, then make up the volume to 1000 mL with injection water, and then sterilize at 110 °C under high pressure for 25 min to obtain the freeze-drying protectant. After cooling, store it at 2 - 8 °C for no more than 30 days.

[0023] Example 3 A freeze-drying protectant for porcine pseudorabies vaccine is prepared as follows: (1) Weigh 160 g of sucrose, 40 g of gelatin, 120 g of acid-hydrolyzed casein, 80 g of hydrolyzed milk protein, 40 g of sodium glutamate, 40 g of glycine, 40 g of polyvinylpyrrolidone (K-30), and 2 g of dipotassium hydrogen phosphate for standby.

[0024] (2) After mixing the above components evenly, add 800 mL of injection water, measure the pH value and adjust it to between 7.0 and 7.4, then make up the volume to 1000 mL with injection water, and then sterilize it at 125 °C under high pressure for 30 min to obtain the lyoprotectant. After cooling, store it at 2 - 8 °C for no more than 30 days.

[0025] Comparative Example 1 Weigh 100 g of skim milk and 50 g of sucrose respectively, add them to 800 mL of injection water, measure the pH value and adjust it to between 7.0 and 7.4, make up the volume to 1000 mL with injection water, sterilize it at 116 °C under high pressure for 20 min, and store it at 2 - 8 °C for no more than 30 days after cooling.

[0026] Test Example 1 Toxicity Test of the Protectant 1. Purchase 20 piglets at 4 - 6 weeks old, randomly divide them into 4 groups with 5 piglets in each group. Inject 10 mL of the lyoprotectant prepared in Examples 1 - 3 and Comparative Example 1 into the 4 groups of piglets through intramuscular injection in the neck, and continuously observe for 21 days with no adverse reactions.

[0027] 2. Purchase another 20 healthy guinea pigs, randomly divide them into 4 groups with 5 guinea pigs in each group. Inject 2 mL of the lyoprotectant prepared in Examples 1 - 3 and Comparative Example 1 into the 4 groups of guinea pigs through intraperitoneal injection, and continuously observe for 21 days with no adverse reactions.

[0028] Test Example 2 Live Freeze - Dried Porcine Pseudorabies Vaccine 1. The virus solution of porcine pseudorabies (SA215 strain) used in the present invention is provided by Animal Husbandry Bio - engineering Co., Ltd., and its virus content is 10 8.5 TCID 50 / mL. Mix the live porcine pseudorabies vaccine with the protectants prepared in Examples 1 - 3 and Comparative Example 1 in a ratio of 1:1 and sub - pack them at 2 mL per vial. After sub - packing, place them in a freeze - dryer. Carry out freeze - drying according to the following procedure: Drop to below - 45 °C within 2 hours and maintain for 4 hours; Evacuate the air and keep the vacuum degree within 15 Pa, heat up to - 6 °C at a rate of 0.5 °C / h, and maintain at - 6 °C until the water line disappears; Heat up to 25 °C at a rate of 4 °C / h and maintain at 25 °C for 1.5 hours; Take out and crimp the cap.

[0029] 2. Detect the freeze - dried vaccine samples prepared with the protectants in Examples 1 - 3 and Comparative Example 1. The specific process is as follows: 1) Appearance: All are spongy and loose lumps, easy to separate from the vial wall, and dissolve rapidly after adding the diluent.

[0030] 2) Sterility test: Conduct the test according to the method in the appendix of the current "Chinese Veterinary Pharmacopoeia", and no bacteria grow.

[0031] 3) Mycoplasma test: The test was carried out according to the method in the appendix of the current "Chinese Veterinary Pharmacopoeia", and no Mycoplasma growth was found.

[0032] 4) Vacuum degree test: The test was carried out according to the method in the appendix of the current "Chinese Veterinary Pharmacopoeia", and purple glow appeared in all cases.

[0033] 5) Residual moisture determination: The test was carried out according to the method in the appendix of the current "Chinese Veterinary Pharmacopoeia", and the residual moisture contents were as follows: Example 1: 1.8%, Example 2: 1.5%, Example 3: 1.5%, Comparative Example 1: 1.6%.

[0034] 6) Safety test: The freeze-dried porcine pseudorabies live vaccine prepared with the cryoprotectants of Examples 1-3 and Comparative Example 1 was used, and then it was respectively injected into healthy piglets at 21 days old with negative porcine pseudorabies virus antibodies through the neck muscle. 5 piglets were injected with each freeze-dried porcine pseudorabies live vaccine. Each piglet was injected with 1 bottle of the freeze-dried porcine pseudorabies live vaccine (diluted with 2 mL of phosphate buffer solution and then injected through the neck muscle), and observed continuously for 21 days. No adverse reactions were found, indicating that the vaccine samples freeze-dried with the cryoprotectants prepared in the examples of the present invention have good safety for piglets.

[0035] 3. Virus content determination It was diluted to 1 mL / bottle with serum-free DMEM nutrient solution (restored to the virus liquid volume before freeze-drying), 100 μL was taken for 10-fold serial dilution, and then 10 3 、10 4 、10 5 、10 6 、10 7 、10 8 dilutions were inoculated into Vero cells. 6 wells were inoculated for each dilution, and 0.1 μL was inoculated into each well. It was cultured in an environment of 37 °C and 5% CO2 for 72 hours, and the cytopathic effects of each dilution were observed. The TCID 50 was calculated according to the Reed-Muench method, and the results are shown in Table 1.

[0036] As shown in Table 1, the virus content only decreased by 0.1-0.2 lg before and after freeze-drying with the cryoprotectant prepared by the present invention, while it decreased by 0.7 lg in the comparative example.

[0037] 4. Stability investigation The freeze-dried vaccines of Examples 1, 2, 3 and Comparative Example 1 were taken and stored at 2-8 °C. 3 samples were taken every 12 months and the virus content was determined according to the above virus content detection method. The results were averaged, and the test lasted for 3 years. The results are shown in Table 1.

[0038] Table 1 Virus content determination results before and after freeze-drying and after 36 months of storage for each group

[0039] As shown in Table 1, the samples freeze-dried with the cryoprotectant prepared by the present invention are placed at 2-8°C. After being stored for 12 months, the virus content hardly decreases. After being stored for 24 months, it only decreases by 0.2-0.3 lg. After being stored for 36 months, it only decreases by 0.5-0.6 lg, showing excellent stability. While the sucrose milk cryoprotectant (comparative example) used for comparison decreases by 1.6, 2.4, and 2.8 lg respectively after 12, 24, and 36 months, further indicating that the cryoprotectant prepared by the present invention has more excellent protective performance on the activity of pseudorabies virus in the environment of 2-8°C.

[0040] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the examples, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A freeze-drying protective agent for porcine pseudorabies vaccine, characterized in that, Comprising components with the following final concentrations: Protein hydrolysate 100 - 200 g / L, sucrose 80 - 160 g / L, amino acids 40 - 80 g / L, gelatin 20 - 40 g / L, polyvinylpyrrolidone 20 - 40 g / L, and pH buffer 1 - 2 g / L; the protein hydrolysate includes acid-hydrolyzed casein and hydrolyzed milk protein, and the mass ratio of the two is 1.5 - 3:1 - 2.

2. The freeze-drying protective agent for porcine pseudorabies vaccine according to claim 1, wherein Comprising components with the following final concentrations: Protein hydrolysate 100 - 150 g / L, sucrose 100 - 160 g / L, amino acids 40 - 60 g / L, gelatin 30 - 40 g / L, polyvinylpyrrolidone 20 - 30 g / L, and pH buffer 1 - 2 g / L.

3. The freeze-drying protective agent for porcine pseudorabies vaccine according to claim 1, characterized in that, The mass ratio of acid-hydrolyzed casein to hydrolyzed milk protein is 1.5:

1.

4. The freeze-drying protective agent for porcine pseudorabies vaccine according to claim 1 or 2, characterized in that, The amino acids include glycine and sodium glutamate with a mass ratio of 0.5 - 1:0.5 - 1.

5. The freeze-drying protective agent for porcine pseudorabies vaccine according to claim 4, wherein The mass ratio of glycine to sodium glutamate is 1:

1.

6. The lyophilization protectant for porcine pseudorabies vaccine according to claim 1 or 2, characterized in that, The pH buffer is dipotassium hydrogen phosphate.

7. A preparation method of the freeze-drying protective agent for porcine pseudorabies vaccine according to any one of claims 1 to 6, characterized in that, Comprising the following process: Mix each component according to the formula, dissolve in water, sterilize at 110 - 125 °C under high pressure for 20 - 30 min, and then cool to room temperature.

8. The preparation method according to claim 7, wherein The high-pressure sterilization temperature is 116 °C, and the sterilization time is 20 min.

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