Heat-resistant protective agent, heat-resistant live vaccine and preparation method and application thereof

Abstract

The invention relates to the field of vaccine manufacturing and preservation, in particular to a heat-resistant protective agent, a heat-resistant live vaccine as well as a preparation method and application thereof. The invention provides a heat-resistant protective agent for a heat-resistant canine distemper and canine parvovirus bivalent live vaccine. The heat-resistant protective agent comprises the following components in percentage by mass: 3%-30% of trehalose, 5%-25% of cane sugar, 1.5%-10% of glucan, 0.5%-2% of glycine, 0.5%-2% of histidine, 1%-5% of casein hydrolysate, 0.5%-5% of gelatin and the balance of water for injection. The invention also provides a preparation method of the heat-resistant canine distemper and canine parvovirus disease bivalent live vaccine, which comprises the following steps: uniformly mixing the protective agent with a canine distemper virus solution and a canine parvovirus solution to obtain a canine distemper and canine parvovirus disease bivalent live vaccine stock solution; and carrying out gradient vacuum drying to obtain the canine distemper and canine parvovirus disease bivalent live vaccine. The canine bivalent vaccine sugar glass preparation prepared by the invention has good heat resistance, and the heat resistance effect of the canine bivalent vaccine sugar glass preparation is superior to that of a traditional freeze-dried live vaccine product.

Description

Technical Field

[0001] The present invention relates to the field of vaccine manufacturing and preservation, and particularly to heat-resistant protectants, heat-resistant live vaccines, and their preparation methods and applications. Background Art

[0002] Canine distemper and canine parvovirus disease are two viral infectious diseases that seriously endanger canids, usually causing a large number of dogs, minks and other animals to get sick, resulting in serious economic losses to the dog and mink breeding industries and pet owners. Vaccine immunization is generally considered the most direct and effective measure for the prevention and control of such viral diseases. In order to facilitate the transportation and storage of vaccines, during the preparation of traditional live vaccine products, a freeze-drying protectant is generally added first and then vacuum freeze-drying is carried out. After freeze-drying, the vaccine can be stored in a frozen environment, but the transportation of the vaccine also requires cold chain transportation support. Since cold chain transportation consumes a large amount of energy and has high technical requirements, usually the cold chain transportation fails to meet the standards, resulting in the vaccine losing cold chain protection intermittently during transportation, thus affecting the actual use effect of the vaccine. Therefore, developing a live vaccine product with heat-resistant ability, which can enable the vaccine to be stably stored at room temperature or at 2-8°C for a long time, is of great significance for reducing the cost of cold chain transportation. Summary of the Invention

[0003] In order to solve the above technical problems, the object of the present invention is to provide a heat-resistant protectant for a heat-resistant canine distemper and canine parvovirus combined live vaccine, and the heat-resistant protectant comprises, by mass percentage: 3% - 30% of trehalose, 5% - 25% of sucrose, 1.5% - 10% of dextran, 0.5% - 2% of glycine, 0.5% - 2% of histidine, 1% - 5% of hydrolyzed casein, 0.5% - 5% of gelatin, and the balance being injection water.

[0004] Further, the heat-resistant protectant comprises, by mass percentage: 15% - 30% of trehalose, 10% - 20% of sucrose, 1.5% - 3% of dextran, 0.5% - 1.5% of glycine, 0.5% - 1.5% of histidine, 1.5% - 3% of hydrolyzed casein, 1% - 2% of gelatin, and the balance being injection water.

[0005] Further, the heat-resistant protectant comprises, by mass percentage: 15% - 20% of trehalose, 20% of sucrose, 2% - 3% of dextran, 1% - 1.5% of glycine, 1.5% of histidine, 2.5% - 3% of hydrolyzed casein, 1% - 2% of gelatin, and the balance being injection water.

[0006] Further, the heat-resistant protectant comprises, by mass percentage: 15% of trehalose, 20% of sucrose, 3% of dextran, 1% of glycine, 1.5% of histidine, 3% of hydrolyzed casein, 1% of gelatin, and the balance being injection water.

[0007] The present invention also provides a method for preparing the heat-resistant protective agent, which is characterized by comprising the following steps:

[0008] (1) Dissolve trehalose, sucrose, glycine, histidine, and hydrolyzed casein in order in water for injection to obtain solution A;

[0009] (2) Dissolve gelatin and dextran in water for injection at 70-80°C, sterilize to obtain solution B;

[0010] (3) Mix solution A and solution B evenly to obtain the product.

[0011] The present invention also provides a method for preparing a heat-resistant canine distemper and canine parvovirus combined live vaccine. Mix the protective agent, canine distemper virus venom, and canine parvovirus venom evenly according to the ratio of (1-3):1:1 to obtain the stock solution of the canine distemper and canine parvovirus combined live vaccine, and obtain the live vaccine of the canine distemper and canine parvovirus combined live vaccine after gradient vacuum drying.

[0012] Further, the foam drying procedure is as follows: Set the shelf temperature at 15-25°C, set the cold trap temperature at -80°C. After the sample is put into the chamber, maintain the equilibrium temperature for 1-2 hours, and then start vacuum for foam drying. The initial vacuum is 50000 Pa, set the vacuum degree every 3 minutes to slowly reduce the vacuum degree to 200-500 Pa. After the sample starts to foam, maintain for 1.5-3 h, adjust the vacuum degree to 15 Pa, maintain for 10-20 hours, then pump to the ultimate vacuum, maintain for 5-15 hours to end drying, and press the plug out of the chamber under vacuum to obtain the product.

[0013] Further, the ratio of the protective agent, canine distemper virus venom, and canine parvovirus venom is 2:1:1.

[0014] The present invention also provides the application of the heat-resistant protective agent in the high-temperature preservation of the canine distemper and canine parvovirus combined live vaccine.

[0015] Further, the temperature is 10-40°C.

[0016] The beneficial effects of the present invention compared with the prior art are as follows:

[0017] The canine combined live vaccine of the present invention is a solid sugar glass vaccine in which the antigen is embedded in a non-reducing sugar with a relatively high viscosity and gradually solidifies as the water is lost. Since the non-reducing sugar is very stable, the sugar glass encapsulating the vaccine will not participate in chemical reactions or protein reactions, and has good heat resistance and stable preservation effect. Through experiments, the sugar glass preparation of the canine combined vaccine prepared by the present invention has good heat resistance, and its heat resistance effect is better than that of traditional freeze-dried live vaccine products. Specific embodiments

[0018] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0019] Embodiment 1: Formulation and preparation method of the protective agent

[0020] It is divided into two parts, liquid A and liquid B.

[0021] Preparation of liquid A: Take trehalose, sucrose, glycine, histidine, and hydrolyzed casein respectively according to the concentrations required by the formula in Table 1. Dissolve the above components in injection water in sequence, shake well after full dissolution, filter and sterilize with a 0.22μm filter membrane, and store at 2 - 8°C for later use.

[0022] Preparation of liquid B: Take hydrolyzed gelatin according to the formula in Table 1. After dextran is fully dissolved with injection water at 70 - 80°C, sterilize it at 121°C under high pressure for 30 minutes for later use.

[0023] During use, mix liquid A and liquid B evenly and adjust the pH of the formula to be between 7.2 and 7.4, then the sugar glass preparation protective agent for the combined live vaccine against canine distemper and canine parvovirus can be obtained.

[0024] The component ratios of the present invention include but are not limited to the component ratios described in the following table, aiming to provide a protective agent for the sugar glass preparation of the combined live vaccine against canine distemper and canine parvovirus for the embodiments.

[0025] Table 1 Formulation of the protective agent for the sugar glass preparation of the combined live vaccine against canine distemper and canine parvovirus

[0026]

[0027] In this embodiment, "%" when describing the component content of the heat-resistant protective agent represents mass-volume percentage, with the unit of g / 100ml. The protective agents prepared according to different components shown in Table 1 are respectively named Protective Agent 1, Protective Agent 2, Protective Agent 3, and Protective Agent 4.

[0028] Embodiment 2: Preparation of the vaccine stock solution

[0029] Mix the above protective agent with canine distemper virus venom and canine parvovirus venom evenly at a ratio of 2:1:1 to obtain the combined live vaccine stock solution against canine distemper and canine parvovirus. At the same time, mix the above protective agent with porcine pseudorabies virus venom evenly at a ratio of 1:1 to obtain the porcine pseudorabies virus live vaccine stock solution. Dispense it into 7ml vials at 1ml / vial and perform foam drying according to the following procedure:

[0030] Set the temperature of the plate layer to 15 - 25°C, set the temperature of the cold trap to -80°C. After the sample is put into the box, maintain the equilibrium temperature for 1 - 2 hours and then start the vacuum for foam drying. The initial vacuum is 50000 Pa. Set the vacuum degree every 3 minutes to slowly reduce the vacuum degree to 200 - 500 Pa. After the sample starts to foam, maintain it for 1.5 - 3 hours, adjust the vacuum degree to 15 Pa, maintain it for 10 - 20 hours and then pump to the ultimate vacuum, maintain it for 5 - 15 hours to end the drying. Press the plug out of the box under vacuum.

[0031] Example 3: Detection of virus content

[0032] The sugar glass preparations prepared in groups 1 - 4 in Example 1 and the canine distemper and canine parvovirus combined live vaccine were subjected to foam drying, and the virus content before and after drying was detected. Another traditional freeze-dried canine combined live vaccine product was set as group 5 as the test control. The preparation method is as follows: Weigh 15% sucrose, 3% gelatin, 3.5% enzymolyzed casein, 0.25% dipotassium hydrogen phosphate, 0.105% potassium dihydrogen phosphate, 0.055% potassium hydroxide respectively, and the rest is injection water. After autoclaving at 116°C for 30 minutes, it is reserved for use. The prepared protective agent, canine distemper virus venom, and canine parvovirus venom are mixed evenly in a ratio of 1:1:1 and then freeze-dried.

[0033] The object of virus content detection before drying is the original vaccine preparation solution, and the object of detection after drying is the sugar glass preparation prepared by reconstituting with the culture medium to the volume of the original vaccine preparation solution. The detection results are shown in Table 2 below.

[0034] Table 2 Detection results of virus content of canine distemper and canine parvovirus combined live vaccine

[0035]

[0036] From the detection results in Table 2 above, it can be seen that among the 4 formulations in groups 1, 2, 3, and 4, after foam drying, the group with the largest decrease in CDV virus content is group 3, with a decrease of 0.4 Lg TCID 50 / ml, and the group with the smallest decrease is group 2, with a decrease in CDV virus content of 0.15 Lg TCID 50 / ml; the group with the largest decrease in CPV virus content is group 1, with a decrease of 0.25 Lg TCID 50 / ml, and the group with the smallest decrease is group 3, with no decrease in virus content. Although the protective agents in each group are different, the foam drying process of the virus has fully achieved a good protection purpose.

[0037] The live vaccine of porcine pseudorabies virus was separately foam-dried with 1 to 4 groups of sugar glass preparations formulated in Example 1, and the virus content before and after drying was detected. Additionally, a traditional freeze-dried live vaccine of porcine pseudorabies virus was set as the 5th group as a test control. The preparation method is as follows: Weigh 15% sucrose, 3% gelatin, 3.5% enzymatically hydrolyzed casein, 0.25% dipotassium hydrogen phosphate, 0.105% potassium dihydrogen phosphate, and 0.055% potassium hydroxide respectively, with the rest being injection water. After the prepared protective agent and the porcine pseudorabies virus venom were mixed evenly in a ratio of 1:2, they were freeze-dried.

[0038] The detection object before drying was the original solution of the prepared vaccine preparation, and the detection object after drying was the sugar glass preparation prepared and reconstituted with a culture medium to the volume of the original vaccine preparation. The detection results are shown in Table 3 below.

[0039] Table 3 Detection Results of Virus Content in Sugar Glass Preparation of Live Vaccine of Porcine Pseudorabies Virus

[0040]

[0041] From the detection results in Table 3 above, it can be seen that the virus content in the sugar glass preparation of the live vaccine of porcine pseudorabies virus after drying decreased significantly, and the virus content decreased to 0.92 - 1.02 Lg TCID 50 / ml, which was higher than the freeze damage of the 5th group with the traditional formula. The protective effect of this formula on the live vaccine of porcine pseudorabies virus was significantly lower than its protective effect on the live vaccines of canine distemper and canine parvovirus. Thus, it can be seen that this formula may be more suitable for the combined live vaccine of canine distemper and canine parvovirus, rather than for other virus vaccines.

[0042] Example 4: Detection of Virus Content in Aging Resistance Experiment

[0043] The samples of the sugar glass preparations of the combined live vaccine of canine distemper and canine parvovirus prepared in Example 1 - 4 after foam drying were placed in an incubator at 37°C for the aging resistance experiment. At the same time, the 5th group of freeze-dried combined live vaccine for dogs was taken as the experimental control and placed in the incubator at 37°C for the aging resistance experiment synchronously. Each group was placed for 7 days, and the virus content was detected after 14 days. The results are shown in Table 4:

[0044] Table 4 Results of Aging Resistance Experiment of Sugar Glass Preparation of Combined Live Vaccine of Canine Distemper and Canine Parvovirus

[0045]

[0046] In addition, from the detection results in Table 4, it can be seen that after storing at 37°C for 14 days for aging resistance in groups 1, 2, 3, and 4, the CDV virus content decreased the most in the 4th group, by 0.5 LgTCID 50 / ml, and the least in the 2nd group, only decreasing by 0.15 Lg TCID 50 / ml. The group with the most significant decrease in the CPV virus content was Group 3, with a decrease of 0.65 Lg TCID 50 / ml, and the group with the least decrease was Group 1, with a decrease of 0.15 Lg TCID 50 / ml.

[0047] Example 5: Detection of Residual Moisture Content

[0048] The samples of the foam-dried sugar glass preparations of the canine distemper and canine parvovirus bivalent live vaccines prepared in each example were tested for residual moisture content according to the method of the Chinese Veterinary Pharmacopoeia. The results all met the requirements of the Veterinary Pharmacopoeia standards, as shown in Table 5:

[0049] Table 5 Detection Results of Residual Moisture Content of Sugar Glass Preparations of Canine Distemper and Canine Parvovirus Bivalent Live Vaccines

[0050]

[0051] The above results indicate that the four groups of sugar glass preparations of canine bivalent virus components prepared in this example all have good heat resistance, and their heat resistance effect is better than that of traditional freeze-dried live vaccine products.

Claims

1. A heat-resistant protective agent for a heat-resistant canine distemper and canine parvovirus dual live vaccine, characterized in that: The heat-resistant protective agent comprises, by mass percentage, trehalose 3%-30%, sucrose 5%-25%, dextran 1.5%-10%, glycine 0.5%-2%, histidine 0.5%-2%, hydrolyzed casein 1%-5%, gelatin 0.5%-5%, and the rest is water for injection.

2. The heat-resistant protective agent according to claim 1, characterized in that The heat-resistant protective agent comprises, by mass percentage, 15% to 30% trehalose, 10% to 20% sucrose, 1.5% to 3% dextran, 0.5% to 1.5% glycine, 0.5% to 1.5% histidine, 1.5% to 3% hydrolyzed casein, 1% to 2% gelatin, and the rest is water for injection.

3. The heat-resistant protective agent according to claim 1, characterized in that The heat-resistant protective agent comprises, by mass percentage, 15% to 20% trehalose, 20% sucrose, 2% to 3% dextran, 1% to 1.5% glycine, 1.5% histidine, 2.5% to 3% hydrolyzed casein, 1% to 2% gelatin, and the rest is water for injection.

4. The heat-resistant protective agent according to claim 1, characterized in that The heat-resistant protective agent comprises, by mass percentage, 15% trehalose, 20% sucrose, 3% dextran, 1% glycine, 1.5% histidine, 3% hydrolyzed casein, 1% gelatin, and the rest is water for injection.

5. The method for preparing a heat-resistant protective agent according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) dissolving trehalose, sucrose, glycine, histidine, and hydrolyzed casein in water for injection in order to obtain solution A; (2) Dissolve gelatin and dextran in 70-80° C. water for injection and sterilize to obtain solution B; (3) Mix solution A and solution B evenly.

6. A method for preparing a heat-resistant canine distemper and canine parvovirus disease dual live vaccine, characterized in that: The protective agent according to any one of claims 1 to 4 is mixed with canine distemper virus venom, canine parvovirus venom, The canine distemper and canine parvovirus disease dual live vaccine stock solution is obtained after uniform mixing in a ratio of (1-3):1:1, and the canine distemper and canine parvovirus disease dual live vaccine is obtained after gradient vacuum drying.

7. The method for preparing a heat-resistant live vaccine according to claim 6, characterized in that: The drying procedure is: set the plate temperature at 15-25°C, set the cold trap temperature at -80°C, maintain the equilibrium temperature for 1-2 hours after the sample enters the box, then turn on the vacuum for foam drying, the initial vacuum is 50000Pa, set the vacuum degree every 3 minutes to slowly drop to 200-500Pa, maintain the sample for 1.5-3h after it starts foaming, adjust the vacuum degree to 15Pa, maintain it for 10-20 hours, then draw the ultimate vacuum, maintain it for 5-15 hours to end the drying, and press the plug out of the box under vacuum.

8. The method for preparing a heat-resistant live vaccine according to claim 6, characterized in that: The ratio of the protective agent to canine distemper virus venom and canine parvovirus venom is 2:1:

1.

9. Use of the heat-resistant protective agent according to claim 1 in the high-temperature storage of canine distemper and canine parvovirus disease combined live vaccines.

10. The use according to claim 9, characterized in that The temperature is 10-40°C.

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