Vaccine antigen preserving fluid as well as preparation method and application thereof

Abstract

The invention discloses a vaccine antigen preserving fluid as well as a preparation method and application thereof, and belongs to the technical field of biological products. The vaccine antigen preserving fluid is prepared from the following raw materials in parts by weight: 10 to 30 parts of cane sugar, 5 to 20 parts of trehalose, 5 to 20 parts of dextran, 5 to 15 parts of lucid ganoderma extract, 5 to 15 parts of radix astragali seu hedysari extract and the balance of phosphoric acid buffer solution based on 1 * 10 < 3 > parts. The invention further discloses a preparation method and application of the vaccine antigen preserving fluid. The vaccine antigen preserving fluid prepared by the invention can solve the problems that the preservation time of vaccine antigens is short, the required preservation temperature is low, and the activity of the vaccine antigens is reduced along with the increase of time in the preservation process.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological products, and in particular relates to a vaccine antigen preservation solution and a preparation method and application thereof. Background Art

[0002] Vaccines are one of the important achievements of modern medicine. They play an extremely important role in maintaining human and animal health and maintaining public health security, especially in preventing and controlling the occurrence and spread of infectious diseases. Vaccination is also the most economical and effective method for preventing and controlling infectious diseases. At present, most vaccines are viral vaccines and subunit vaccines. Viral vaccines mainly include inactivated vaccines and attenuated live vaccines, and subunit vaccines are mainly recombinant protein vaccines. In the production process of vaccines, the preparation and preservation of antigens are one of the most critical links of vaccines. Generally, in the production process of vaccines, the prepared antigens cannot be immediately used for subsequent production processes. It is usually necessary to test the antigens for sterility, content and other items. Only after passing the test can they be put into the subsequent production process. Especially for multi-linked and multivalent vaccines, multiple antigens need to be prepared and tested before the vaccine can be prepared in batches. Therefore, in most cases, the antigens often need to be stored for a period of time after preparation.

[0003] The activity of antigens directly determines the product quality and stability of vaccines, and the activity of antigens is affected by many factors such as temperature, pH value, storage conditions, etc. Therefore, the preservation of antigens is a huge challenge for vaccine production. In particular, with the continuous improvement of the modernization of vaccine production companies, such as the extensive use of bioreactors, the large-scale production of antigens can greatly reduce production costs, but this is accompanied by challenges brought about by the preservation of antigens.

[0004] At present, after the antigen is prepared, most of them are directly stored in refrigerated or frozen conditions to minimize the reduction of antigen activity. Some antigens are extremely sensitive to heat and must be stored at -70℃ ultra-low temperature to maintain their activity stability. However, the equipment and energy costs required for ultra-low temperature conditions are increased, which greatly increases the manufacturing cost of the vaccine production process.

[0005] Therefore, how to maintain the activity of antigens under freezing and other storage conditions and extend their shelf life is of great significance for vaccine production in terms of reducing production costs, facilitating production arrangements, etc., while also maximizing the effectiveness and safety of vaccines. Summary of the invention

[0006] The object of the present invention is to provide a vaccine antigen preservation solution, a preparation method and an application thereof. The vaccine antigen preservation solution can solve the problems of short preservation time of vaccine antigen, low required preservation temperature and reduction of its activity over time during preservation.

[0007] The technical solution adopted to achieve the above object is to provide a vaccine antigen preservation solution. Based on 1*10 3 parts of the vaccine antigen preservation solution, it includes the following raw materials in parts by weight: 10 - 30 parts of sucrose, 5 - 20 parts of trehalose, 5 - 20 parts of dextran, 5 - 15 parts of Ganoderma lucidum extract, and 5 - 15 parts of Astragalus membranaceus extract, with the balance being phosphate buffer solution.

[0008] The beneficial effects of the present invention adopting the above technical solution are as follows: In the vaccine antigen preservation solution of the present invention, sucrose serves as the main cryoprotectant, forming a high-viscosity glassy matrix during the freezing process to inhibit ice crystal growth. Its hydroxyl groups fix water molecules through a hydrogen bond network, reducing the exposure of protein hydrophobic regions. Trehalose has super water-holding capacity, can form a stable amorphous state to prevent protein thermal denaturation, synergistically reduces the phase transition temperature with sucrose, enhances the glassy state formation ability, and has good safety. Dextran binds to water molecules, maintains the hydration layer of proteins, enhances the thermal stability of vaccine antigens, and can also prevent vaccine antigens from aggregating and precipitating at high temperatures through steric hindrance effects, reducing the structural damage caused by molecular collisions. The main extract in Astragalus membranaceus extract is Astragalus polysaccharide, and the main component in Ganoderma lucidum extract is Ganoderma polysaccharide. Astragalus polysaccharide and Ganoderma polysaccharide produce a synergistic effect through hydrogen bonding and electrostatic interactions to jointly stabilize the protein structure and lipid structure of vaccine antigens, prevent vaccine antigens from denaturing, and also have the effects of reducing the oxidative damage of vaccine antigens, inhibiting the activities of nucleases and proteases, preventing vaccine antigen degradation, preventing microbial contamination, and maintaining the environmental pH and ionic strength. The two interact with each other to form an oxidation-reduction relay, achieving a three-dimensional block of free radical chain reactions, thereby playing a role in stabilizing vaccine antigens and extending the preservation time. In addition, Astragalus polysaccharide and Ganoderma polysaccharide also have significant immunomodulatory effects, can enhance the immune resistance of the body, and can improve the immune use effect of subsequent vaccines. At the same time, Astragalus membranaceus extract and Ganoderma lucidum extract can also increase the protein denaturation temperature, and cooperate with sugars to effectively extend the half-life of antigen thermal denaturation. The phosphate buffer solution ensures the stability of the solution ionic strength through dynamic regulation, prevents the change of protein charge distribution, and can play a role in maintaining the pH of vaccine antigens and preventing vaccine antigens from denaturing.

[0009] Preferably, based on 1*10 3 parts of the vaccine antigen preservation solution, it includes the following raw materials in parts by weight: 25 parts of sucrose, 10 parts of trehalose, 10 parts of dextran, 10 parts of Ganoderma lucidum extract, and 10 parts of Astragalus membranaceus extract, with the balance being phosphate buffer solution.

[0010] More preferably, the pH of the phosphate buffer solution is 7 to 7.5; based on 1*10 3 parts of the phosphate buffer solution, it comprises the following raw materials in parts by weight: 80 to 85 parts of sodium chloride, 1 to 2 parts of potassium chloride, 14 to 15 parts of disodium hydrogen phosphate, and 2 to 3 parts of potassium dihydrogen phosphate, and the balance is water.

[0011] More preferably, the pH of the phosphate buffer solution is 7.2; based on 1*10 3 parts of the phosphate buffer solution, it comprises the following raw materials in parts by weight: 80 parts of sodium chloride, 2 parts of potassium chloride, 14.4 parts of disodium hydrogen phosphate, and 2.4 parts of potassium dihydrogen phosphate, and the balance is water.

[0012] The present invention also provides a method for preparing the vaccine antigen preservation solution, which comprises the following steps: Dissolve sucrose, trehalose, dextran, Ganoderma lucidum extract and Astragalus membranaceus extract in the phosphate buffer solution, and obtain it after filtering through a membrane.

[0013] Preferably, the pore size of the membrane is 0.22 μm.

[0014] The present invention also provides the application of the vaccine antigen preservation solution in the preservation of vaccine antigen.

[0015] More preferably, the application of the vaccine antigen preservation solution in the preservation of vaccine antigen comprises the following steps: Mix the vaccine antigen and the vaccine antigen preservation solution according to a volume ratio of 8 to 9:1 to 2, and store it under the condition of -15 to 37 °C.

[0016] More preferably, the volume ratio of the vaccine antigen to the vaccine antigen preservation solution is 9:1.

[0017] More preferably, the vaccine antigen is an RNA virus antigen, a DNA virus antigen or a recombinant protein antigen.

[0018] More preferably, the vaccine antigen is a porcine epidemic diarrhea virus antigen solution, a porcine parvovirus antigen solution or a prokaryotically expressed recombinant duck circovirus Cap nanoparticle protein.

[0019] The present invention has the following beneficial effects: The vaccine antigen preservation solution of the present invention can support the vaccine antigen and protect it from damage caused by environmental stresses such as freezing, drying and heat stress; the vaccine antigen preservation solution can effectively extend the preservation time of the vaccine antigen, maintain the activity of the vaccine antigen, and at the same time has a significant immune regulation effect to improve the immune use effect of subsequent vaccines, and has a wide application prospect. Specific Embodiments

[0020] The features and properties of the present invention are further described in detail below in conjunction with examples. The Ganoderma lucidum extract and the Astragalus membranaceus extract are both from Xi'an Tianyi Biotechnology Co., Ltd.

[0021] Example 1 A vaccine antigen preservation solution, comprising the following raw materials in parts by weight: 25 g of sucrose, 10 g of trehalose, 10 g of dextran, 10 g of Ganoderma lucidum extract, and 10 g of Astragalus membranaceus extract, made up to 1 L with phosphate buffer solution; Among them, the phosphate buffer solution comprises the following raw materials in parts by weight: 80 g of sodium chloride, 2 g of potassium chloride, 14.4 g of disodium hydrogen phosphate, and 2.4 g of potassium dihydrogen phosphate, made up to 1 L with injection water; The phosphate buffer solution is prepared through the following steps: Weigh sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate, dissolve them in 800 mL of injection water, adjust the pH to 7.2 after complete dissolution, and then make up to 1 L.

[0022] This example also provides a preparation method for the above vaccine antigen preservation solution, comprising the following steps: Dissolve sucrose, trehalose, dextran, Ganoderma lucidum extract, and Astragalus membranaceus extract in 800 mL of phosphate buffer solution, make up to 1 L after complete dissolution, and obtain the product after passing through a filter membrane with a pore size of 0.22 μm.

[0023] Example 2 A vaccine antigen preservation solution, comprising the following raw materials in parts by weight: 10 g of sucrose, 5 g of trehalose, 5 g of dextran, 5 g of Ganoderma lucidum extract, and 5 g of Astragalus membranaceus extract, made up to 1 L with phosphate buffer solution; Among them, the phosphate buffer solution comprises the following raw materials in parts by weight: 80 g of sodium chloride, 2 g of potassium chloride, 14.4 g of disodium hydrogen phosphate, and 2.4 g of potassium dihydrogen phosphate, made up to 1 L with injection water; The phosphate buffer solution is prepared through the following steps: Weigh sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate, dissolve them in 800 mL of injection water, adjust the pH to 7.2 after complete dissolution, and then make up to 1 L.

[0024] This example also provides a preparation method for the above vaccine antigen preservation solution, comprising the following steps: Dissolve sucrose, trehalose, dextran, Ganoderma lucidum extract, and Astragalus membranaceus extract in 800 mL of phosphate buffer solution, make up to 1 L after complete dissolution, and obtain the product after passing through a filter membrane with a pore size of 0.22 μm.

[0025] Example 3 A vaccine antigen preservation solution, comprising the following raw materials in parts by weight: 30 g of sucrose, 20 g of trehalose, 20 g of dextran, 15 g of Ganoderma lucidum extract, and 15 g of Astragalus membranaceus extract, made up to 1 L with phosphate buffer solution; Among them, the phosphate buffer solution comprises the following raw materials in parts by weight: 80 g of sodium chloride, 2 g of potassium chloride, 14.4 g of disodium hydrogen phosphate, and 2.4 g of potassium dihydrogen phosphate, and is made up to 1 L with water for injection; The phosphate buffer solution is prepared through the following steps: Weigh sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate, dissolve them in 800 mL of water for injection, adjust the pH to 7.2 after complete dissolution, and then make up to 1 L.

[0026] This example also provides a method for preparing the above vaccine antigen preservation solution, which includes the following steps: Dissolve sucrose, trehalose, dextran, Ganoderma lucidum extract, and Astragalus membranaceus extract in 800 mL of phosphate buffer solution, make up to 1 L after complete dissolution, and obtain the product after passing through a filter membrane with a pore size of 0.22 μm.

[0027] Comparative Example 1 A vaccine antigen preservation solution, which includes the following raw materials in parts by weight: 25 g of sucrose, 10 g of trehalose, 10 g of Ganoderma lucidum extract, and 10 g of Astragalus membranaceus extract, and is made up to 1 L with phosphate buffer solution; Among them, the phosphate buffer solution comprises the following raw materials in parts by weight: 80 g of sodium chloride, 2 g of potassium chloride, 14.4 g of disodium hydrogen phosphate, and 2.4 g of potassium dihydrogen phosphate, and is made up to 1 L with water for injection; The phosphate buffer solution is prepared through the following steps: Weigh sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate, dissolve them in 800 mL of water for injection, adjust the pH to 7.2 after complete dissolution, and then make up to 1 L.

[0028] This comparative example also provides a method for preparing the above vaccine antigen preservation solution, which includes the following steps: Dissolve sucrose, trehalose, Ganoderma lucidum extract, and Astragalus membranaceus extract in 800 mL of phosphate buffer solution, make up to 1 L after complete dissolution, and obtain the product after passing through a filter membrane with a pore size of 0.22 μm.

[0029] Comparative Example 2 A vaccine antigen preservation solution, which includes the following raw materials in parts by weight: 80 g of sodium chloride, 2 g of potassium chloride, 14.4 g of disodium hydrogen phosphate, and 2.4 g of potassium dihydrogen phosphate, and is made up to 1 L with water for injection.

[0030] This comparative example also provides a method for preparing the above vaccine antigen preservation solution, which includes the following steps: Weigh sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate, dissolve them in 800 mL of water for injection, adjust the pH to 7.2 after complete dissolution, and then make up to 1 L.

[0031] Comparative Example 3 A vaccine antigen preservation solution, comprising the following raw materials in parts by weight: 25 g of sucrose, 10 g of trehalose, and 10 g of dextran, made up to 1 L with phosphate buffer solution; Among them, the phosphate buffer solution comprises the following raw materials in parts by weight: 80 g of sodium chloride, 2 g of potassium chloride, 14.4 g of disodium hydrogen phosphate, and 2.4 g of potassium dihydrogen phosphate, made up to 1 L with injection water; The phosphate buffer solution is prepared through the following steps: Weigh sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate, dissolve them in 800 mL of injection water, adjust the pH to 7.2 after complete dissolution, and then make up to 1 L.

[0032] This comparative example also provides a method for preparing the above vaccine antigen preservation solution, comprising the following steps: Dissolve sucrose, trehalose, and dextran in 800 mL of phosphate buffer solution, make up to 1 L after complete dissolution, and obtain the product after passing through a filter membrane with a pore size of 0.22 μm.

[0033] Comparative Example 4 A vaccine antigen preservation solution, comprising the following raw materials in parts by weight: 25 g of sucrose, 10 g of trehalose, 10 g of dextran, and 10 g of Ganoderma lucidum extract, made up to 1 L with phosphate buffer solution; Among them, the phosphate buffer solution comprises the following raw materials in parts by weight: 80 g of sodium chloride, 2 g of potassium chloride, 14.4 g of disodium hydrogen phosphate, and 2.4 g of potassium dihydrogen phosphate, made up to 1 L with injection water; The phosphate buffer solution is prepared through the following steps: Weigh sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate, dissolve them in 800 mL of injection water, adjust the pH to 7.2 after complete dissolution, and then make up to 1 L.

[0034] This comparative example also provides a method for preparing the above vaccine antigen preservation solution, comprising the following steps: Dissolve sucrose, trehalose, dextran, and Ganoderma lucidum extract in 800 mL of phosphate buffer solution, make up to 1 L after complete dissolution, and obtain the product after passing through a filter membrane with a pore size of 0.22 μm.

[0035] Comparative Example 5 A vaccine antigen preservation solution, comprising the following raw materials in parts by weight: 25 g of sucrose, 10 g of trehalose, 10 g of dextran, and 10 g of Astragalus membranaceus extract, made up to 1 L with phosphate buffer solution; Among them, the phosphate buffer solution comprises the following raw materials in parts by weight: 80 g of sodium chloride, 2 g of potassium chloride, 14.4 g of disodium hydrogen phosphate, and 2.4 g of potassium dihydrogen phosphate, made up to 1 L with injection water; The phosphate buffer solution is prepared through the following steps: Weigh sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate, dissolve them in 800 mL of water for injection. After complete dissolution, adjust the pH to 7.2, and then make up the volume to 1 L.

[0036] This comparative example also provides a method for preparing the above vaccine antigen preservation solution, which includes the following steps: Dissolve sucrose, trehalose, dextran and astragalus extract in 800 mL of phosphate buffer solution. After complete dissolution, make up the volume to 1 L, and then filter through a membrane with a pore size of 0.22 μm to obtain the solution.

[0037] Experimental Example 1 Preservation Experiment of RNA Virus Mix the porcine epidemic diarrhea virus (PEDV) virus antigen solution (content is 10 8.1 TCID 50 / mL) with the vaccine antigen preservation solutions prepared in Example 1, Example 2, and Comparative Examples 1-5 according to a volume ratio of 9:1. Place the mixed virus solutions under the conditions of 37 °C, 2-8 °C and -15 °C for preservation respectively, and sample at different times after preservation to measure the virus content. The results are shown in Tables 1-3.

[0038] Table 1 Detection Results of Virus Content of PEDV after Preservation at 37 °C for Different Times

[0039] Table 2 Detection Results of Virus Content of PEDV after Preservation at 2-8 °C for Different Times

[0040] Table 3 Detection Results of Virus Content of PEDV after Preservation at -15 °C for Different Times

[0041] The results in Tables 1-3 show that, compared with the comparative examples, the vaccine antigen preservation solutions prepared in Example 1 and Example 2 of the present invention can significantly increase the virus content of the RNA virus (PEDV) after preservation at 37 °C, 2-8 °C and -15 °C for different times, effectively maintain the activity of the RNA virus during preservation, and thus play a role in prolonging the virus preservation time.

[0042] Experimental Example 2 Preservation Experiment of DNA Virus Mix the porcine parvovirus (PPV) virus antigen solution (content is 10 7.8 TCID 50 / mL) with the vaccine antigen preservation solutions prepared in Example 1, Example 2, and Comparative Examples 1-5 according to a volume ratio of 9:1. Place the mixed virus solutions under the conditions of 37 °C, 2-8 °C, -15 °C for preservation respectively, and sample at different times after preservation to measure the virus content. The results are shown in Tables 4-6.

[0043] Table 4 Detection results of virus content after PPV was stored at 37°C for different times

[0044] Table 5 Detection results of virus content after PPV was stored at 2-8°C for different times

[0045] Table 6 Detection results of virus content after PPV was stored at -15°C for different times

[0046] It can be seen from the results of Tables 4-6 that, compared with the comparative examples, the vaccine antigen preservation solutions prepared in Examples 1-2 of the present invention can significantly increase the virus content of DNA virus (PPV) after being stored at 37°C, 2-8°C and -15°C for different times, effectively maintain the activity of DNA virus during storage, and thus play a role in extending the virus storage time.

[0047] Experimental Example 3 Preservation experiment of recombinant protein The recombinant duck circovirus Cap nanoparticle protein (rCap) (particle assembly rate of 73%) expressed prokaryotically was mixed with the vaccine antigen preservation solutions prepared in Examples 1 and 2 and Comparative Examples 1-5 at a volume ratio of 9:1, and the mixed virus solutions were stored at 37°C, 2-8°C and -15°C respectively, and samples were taken at different times after storage for determination of the particle assembly rate. The results are shown in Tables 7-9.

[0048] Table 7 Detection results of particle assembly rate after rCap was stored at 37°C for different times

[0049] Table 8 Detection results of particle assembly rate after rCap was stored at 2-8°C for different times

[0050] Table 9 Detection results of particle assembly rate after rCap was stored at -15°C for different times

[0051] The results of Tables 7-9 show that, compared with the comparative examples, the vaccine antigen preservation solutions prepared in Examples 1-2 of the present invention can help to stabilize the stability of the recombinant nanoparticle protein at 37°C, 2-8°C and -15°C, and thus extend the storage time of the recombinant nanoparticle protein.

[0052] The present invention has been described according to the above embodiments. It should be understood that the above embodiments do not limit the present invention in any form. Any technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the scope of the present invention.

Claims

1. A vaccine antigen preservation solution, characterized in that, Based on 1 * 10 3 parts of vaccine antigen preservation solution, it comprises the following raw materials in parts by weight: 10 - 30 parts of sucrose, 5 - 20 parts of trehalose, 5 - 20 parts of dextran, 5 - 15 parts of Ganoderma lucidum extract, and 5 - 15 parts of Astragalus membranaceus extract, with the balance being phosphate buffer solution.

2. The vaccine antigen preservation solution according to claim 1, characterized in that, Based on 1 * 10 3 parts of the vaccine antigen preservation solution, it comprises the following raw materials in parts by weight: 25 parts of sucrose, 10 parts of trehalose, 10 parts of dextran, 10 parts of Ganoderma lucidum extract, and 10 parts of Astragalus membranaceus extract, with the balance being phosphate buffer solution.

3. The vaccine antigen preservation solution according to claim 1 or 2, characterized in that, The pH of the phosphate buffer solution is 7 to 7.5; based on 1*10 3 parts of the phosphate buffer solution, it comprises the following raw materials in parts by weight: 80 to 85 parts of sodium chloride, 1 to 2 parts of potassium chloride, 14 to 15 parts of disodium hydrogen phosphate, and 2 to 3 parts of potassium dihydrogen phosphate, with the balance being water.

4. The vaccine antigen preservation solution according to claim 3, characterized in that, The pH of the phosphate buffer solution is 7.2; based on 1*10 3 parts of the phosphate buffer solution, it comprises the following raw materials in parts by weight: 80 parts of sodium chloride, 2 parts of potassium chloride, 14.4 parts of disodium hydrogen phosphate, and 2.4 parts of potassium dihydrogen phosphate, and the balance is water.

5. The preparation method of the vaccine antigen preservation solution according to any one of claims 1 to 4, characterized in that, It includes the following steps: Dissolve sucrose, trehalose, dextran, Ganoderma lucidum extract and Astragalus membranaceus extract in phosphate buffer solution, and obtain the solution after filtering through a membrane.

6. The preparation method of the vaccine antigen preservation solution according to claim 5, characterized in that, The pore size of the membrane is 0.22 μm.

7. Use of the vaccine antigen preservation solution according to any one of claims 1 to 4 in the preservation of vaccine antigens.

8. The application according to claim 7, wherein It includes the following steps: Mix the vaccine antigen and the vaccine antigen preservation solution according to a volume ratio of 8-9:1-2, and store it at -15-37°C.

9. The application according to claim 8, wherein The volume ratio of the vaccine antigen to the vaccine antigen preservation solution is 9:

1.

10. The application according to claim 8, characterized in that, The vaccine antigen is an RNA virus antigen, a DNA virus antigen or a recombinant protein antigen.

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