Emulsifying method for few vaccine antigens in laboratory
The vaccine antigens are emulsified through a specific procedure of a tissue grinding low-temperature homogenizer, which solves the problems of poor emulsification effect and antigen denaturation in the existing technology, achieves uniform and stable emulsification of small amounts of vaccines in the laboratory, and improves the quality of the vaccine.
Patent Information
- Application Number
- CN202510957598.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-11
- Publication Date
- 2025-10-03
AI Technical Summary
The existing technologies have problems such as poor vaccine emulsification effect and antigen denaturation, which make it difficult to meet the emulsification needs of small quantities of vaccines in the laboratory.
A tissue grinding low-temperature homogenizer was used to perform intermittent emulsification by adjusting the rotation speed to 3500-5000 rpm, the linear speed to 5-8 m/s, and the temperature to 2-8°C. The cycle was repeated 3-4 times, for a total of 5-7 runs, to avoid antigen denaturation and improve uniformity and stability.
It achieves uniform and stable emulsification of vaccine antigens, improves the stability and antigen activity of the vaccine, is suitable for small-scale vaccine preparation in the laboratory, and the equipment is simple to operate and has low cost.
Smart Images
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Abstract
Description
Technical Field
[0001] The present application relates to the technical field of vaccine emulsification, and in particular to an emulsification method for a small amount of vaccine antigens in a laboratory. Background Art
[0002] Vaccine emulsification is a key step in vaccine preparation. Its purpose is to evenly mix the antigen and adjuvant to form a stable emulsion, which plays a vital role in improving vaccine stability and immunogenicity. With the continuous development of medicine and biology, the types of vaccines are increasing, and the requirements for vaccine emulsification are becoming increasingly stringent. Good vaccine emulsification can improve vaccine quality, thereby better stimulating the body's immune response during use, providing a strong guarantee for the prevention and control of various diseases.
[0003] At present, vaccine emulsification technologies mainly include mechanical stirring, high-pressure homogenization and ultrasonic emulsification. Among them, the mechanical stirring method achieves emulsification through mechanical stirring. Due to its low cost, it is often used in situations where the emulsification effect requirements are not particularly high. However, its emulsification effect is poor and its stability is insufficient, resulting in an inability to meet the requirements of high-quality vaccine emulsification. The high-pressure homogenization method uses high pressure to force the liquid through tiny pores to achieve the purpose of emulsification. This method is widely used in some large-scale production and can guarantee the emulsification effect to a certain extent; however, its equipment is complex, the cost is high, and it is easy to cause antigen denaturation, which is not suitable for small-scale vaccine emulsification in the laboratory. The ultrasonic emulsification method uses the aeration effect of ultrasound to complete emulsification. Its operation is relatively simple and has certain conveniences for some small experiments or preliminary studies, but there is still the problem of uneven emulsification, and long-term ultrasound may also cause antigen degradation. Summary of the Invention
[0004] In order to overcome the problems of poor emulsification effect and easy antigen denaturation in existing vaccine emulsification technologies, the present application provides an emulsification method for small amounts of vaccine antigens in the laboratory.
[0005] The emulsification method for a small amount of vaccine antigen in the laboratory provided in this application adopts the following technical solution: A method for emulsifying a small amount of vaccine antigen in a laboratory, comprising the following steps: Add the mixture of vaccine antigen and adjuvant to a tissue grinding cryogenic homogenizer and emulsify according to the following program: rotation speed 3500-5000 rpm, linear speed 5-8 m / s, temperature 2-8°C; run for 30 seconds and pause for 30 seconds as one cycle, and repeat 3-4 times; The above procedure was run every 5±0.5 min for a total of 5-7 times.
[0006] The present application provides an emulsification method for a small amount of vaccine antigen in the laboratory. The emulsification method relies on a specific device, a low-temperature homogenizer, and emulsifies under a specific procedure, thereby achieving the emulsification of the vaccine antigen and obtaining a uniform and stable vaccine antigen preparation. Specifically: The present application uses a rotation speed of 3500-5000rpm and a linear speed of 5-8m / s to fully collide and mix the antigen and adjuvant, greatly improving the uniformity of the mixing of the two; by controlling the temperature in a low-temperature environment of 2-8°C, it can effectively prevent the antigen from denaturing during the emulsification process, thereby ensuring the quality of the vaccine; by running for 30s and pausing for 30s as a cycle, and repeating 3-4 times, the intermittent cycle method effectively avoids the problem of excessive heat caused by continuous friction causing a decrease in antigen activity, while also ensuring that the antigen and adjuvant have more time to adjust and fuse; by repeating the operation 5-7 times, the uniformity and stability of the entire emulsification process can be further ensured. In summary, the emulsification method provided in this application can greatly improve the uniformity of vaccine emulsification and antigen activity, and significantly enhance the stability of the vaccine while ensuring the quality of the vaccine; in addition, the emulsification method provided in this application uses a tissue grinding low-temperature homogenizer, which has relatively simple equipment operation and low cost, and is very suitable for the emulsification needs of small quantities of vaccines in the laboratory.
[0007] In some embodiments, the program may be run 5-6 times or 6-7 times.
[0008] In a specific embodiment, the program can be run 5 times, 6 times or 7 times.
[0009] In some embodiments, the rotation speed may be 3500-4000 rpm, 3500-4500 rpm, 4000-4500 rpm, 4000-5000 rpm, or 4500-5000 rpm.
[0010] In a specific embodiment, the rotation speed may also be 3500 rpm, 4000 rpm, 4500 rpm or 5000 rpm.
[0011] In some embodiments, the line speed may be 5-6 m / s, 5-7 m / s, 6-7 m / s, 6-8 m / s, or 7-8 m / s.
[0012] In a specific embodiment, the linear speed may also be 5 m / s, 6 m / s, 7 m / s or 8 m / s.
[0013] In some embodiments, the temperature may be 2-4°C, 2-6°C, 4-6°C, 4-8°C, or 6-8°C.
[0014] In a specific embodiment, the temperature may also be 2°C, 4°C, 6°C or 8°C.
[0015] Optionally, the adjuvant is selected from Freund's complete adjuvant, Freund's incomplete adjuvant or white oil.
[0016] Optionally, when the adjuvant is Freund's complete adjuvant or Freund's incomplete adjuvant, the volume ratio of the vaccine antigen to the adjuvant is 1:(1-1.2).
[0017] Optionally, when the adjuvant is white oil, the volume ratio of the vaccine antigen to the adjuvant is 1:(1.5-3).
[0018] Optionally, when the adjuvant is white oil, an emulsifier and a preservative need to be added to the mixture.
[0019] Optionally, the emulsifier is Siben 80 and the preservative is thimerosal.
[0020] Optionally, the rotation speed is 4000-4500 rpm, the linear speed is 6-7 m / s, and the temperature is 2-6°C.
[0021] The rotation speed, line speed, and temperature during the emulsification process of this application affect the emulsification uniformity and antigenic activity of the vaccine. The inventors of this application have found through experimental research that further controlling the rotation speed, line speed, and temperature during the emulsification process within the above ranges results in better emulsification uniformity and stability of the vaccine, as well as better vaccine activity.
[0022] Optionally, the model of the tissue grinding low-temperature homogenizer is Tgrinder H24R.
[0023] In summary, this application has the following beneficial effects: 1. The present application provides a method for emulsifying vaccine antigens, which relies on a tissue grinding and low-temperature homogenizer for emulsification; the present application adjusts the parameters such as the rotation speed, linear speed, and temperature in the homogenizer within a specific range, thereby achieving emulsification of the vaccine antigen while avoiding antigen denaturation, thereby obtaining a vaccine antigen preparation with good stability and high antigen activity.
[0024] 2. This application further controls the rotation speed in the emulsification process within the range of 4000-4500 rpm, the linear speed within the range of 6-7 m / s, and the temperature within the range of 2-6°C. The obtained vaccine has better emulsification uniformity and stability, and the vaccine activity is better. DETAILED DESCRIPTION
[0025] The present application provides an emulsification method for a small amount of vaccine antigen in the laboratory, comprising the following steps: adding a mixture of the vaccine antigen and the adjuvant into a tissue grinding low-temperature homogenizer, and emulsifying according to the following procedure: rotation speed 3500-5000 rpm, linear speed 5-8 m / s, temperature 2-8°C; running for 30 seconds and pausing for 30 seconds as one cycle, and repeating 3-4 times; the above procedure is run once every 5±0.5 minutes, and is run 5-7 times in total.
[0026] Wherein, the adjuvant is selected from Freund's complete adjuvant, Freund's incomplete adjuvant or white oil; when the adjuvant is Freund's complete adjuvant or Freund's incomplete adjuvant, the volume ratio of the vaccine antigen to the adjuvant is 1:(1-1.2); when the adjuvant is white oil, the volume ratio of the vaccine antigen to the adjuvant is 1:(1.5-3).
[0027] The model of the tissue grinding low-temperature homogenizer used in the examples of the present application is Tgrinder H24R; the vaccine antigen is a recombinant Cap protein solution of porcine circovirus type 2, which is produced by the company; the product number of Freund's complete adjuvant is F5881, purchased from Sigma; the product number of Freund's incomplete adjuvant is F5506, purchased from Sigma; white oil was purchased from ExxonMobil Chemical Business Co., Ltd. of the United States; the raw materials, reagents, solvents, etc. used in this application can all be obtained commercially.
[0028] The present application is further described in detail below with reference to embodiments and performance testing.
[0029] Example 1 Example 1 provides a method for emulsifying a small amount of vaccine antigen in the laboratory, comprising the following steps: The vaccine antigen and Freund's complete adjuvant were mixed in a volume ratio of 1:1. The mixture was added to a tissue grinding low-temperature homogenizer and emulsified according to the following procedure: rotation speed of 4500 rpm, linear speed of 7 m / s, temperature of 4°C; one cycle was 30 s and a pause of 30 s, and the cycle was repeated 3 times; the above procedure was repeated every 5 minutes for a total of 5 times.
[0030] Example 2 Example 2 provides an emulsification method for small amounts of vaccine antigens in the laboratory.
[0031] The difference between the above embodiment and embodiment 1 is that the program is run 6 times.
[0032] Example 3 Example 3 provides an emulsification method for small amounts of vaccine antigens in the laboratory.
[0033] The difference between the above embodiment and embodiment 1 is that the program is run 7 times.
[0034] Examples 4-12 Examples 4-12 each provide an emulsification method for a small amount of vaccine antigen in the laboratory.
[0035] The difference between the above embodiment and embodiment 2 is that the rotation speed, linear speed and temperature in the program are shown in Table 1 below.
[0036] Table 1 Rotational speed, linear speed and temperature in the procedures of Example 2 and Examples 4-12 Example 13 Example 13 provides a method for emulsifying a small amount of vaccine antigen in the laboratory, comprising the following steps: The vaccine antigen and Freund's incomplete adjuvant were mixed in a volume ratio of 1:1. The mixture was added to a tissue grinding low-temperature homogenizer and emulsified according to the following procedure: rotation speed of 4500 rpm, linear speed of 7 m / s, temperature of 4°C; one cycle was 30 s and a pause of 30 s, and the cycle was repeated 3 times; the above procedure was repeated every 5 minutes for a total of 6 times.
[0037] Example 14 Example 14 provides a method for emulsifying a small amount of vaccine antigen in the laboratory, comprising the following steps: White oil and Siben-80 were mixed in a volume ratio of 95:5 to obtain an oil phase; thimerosal was added to the vaccine antigen at a final concentration of 0.01 wt% to obtain an aqueous phase; the aqueous phase and the oil phase were mixed in a volume ratio of 1:2, and the mixture was added to a tissue grinding low-temperature homogenizer and emulsified according to the following procedure: rotation speed 4500 rpm, linear speed 7 m / s, temperature 4°C; one cycle was run for 30 seconds and paused for 30 seconds, and the cycle was repeated 3 times; the above procedure was repeated once every 5 minutes for a total of 6 times.
[0038] Comparative Example 1 Comparative Example 1 provides an emulsification method for a small amount of vaccine antigen in the laboratory.
[0039] The difference between the comparative example and Example 2 is that the program is run 3 times.
[0040] Comparative Example 2 Comparative Example 2 provides an emulsification method for a small amount of vaccine antigen in the laboratory.
[0041] The difference between the comparative example and Example 2 is that the rotation speed in the procedure is 3000 rpm, the linear speed is 10 m / s, and the temperature is 4°C.
[0042] Comparative Example 3 Comparative Example 3 provides an emulsification method for a small amount of vaccine antigen in the laboratory.
[0043] The difference between the comparative example and Example 2 is that the rotation speed in the procedure is 6000 rpm, the linear speed is 3 m / s, and the temperature is 4°C.
[0044] Comparative Example 4 Comparative Example 4 provides an emulsification method for a small amount of vaccine antigen in the laboratory.
[0045] The difference between the comparative example and Example 2 is that the temperature in the procedure is 25°C.
[0046] Comparative Example 5 Comparative Example 5 provides an emulsification method for a small amount of vaccine antigen in the laboratory, comprising the following steps: The vaccine antigen and Freund's complete adjuvant were mixed in a volume ratio of 1:1. The mixture was added to a tissue grinding low-temperature homogenizer and emulsified according to the following procedure: rotation speed of 4500 rpm, linear speed of 7 m / s, temperature of 4°C; one cycle was 60 s and a pause of 60 s, and the cycle was repeated twice; the above procedure was repeated every 5 minutes for a total of 5 times.
[0047] Performance testing The stability and activity of the emulsified vaccine antigens of Examples 1-14 and Comparative Examples 1-5 were tested, and the results are shown in Table 2 below.
[0048] (1) Stability: Samples of the vaccine were placed in a 37°C constant temperature box and taken out every day to observe their physical and chemical properties until stratification or demulsification occurred. The time for stratification or demulsification of the vaccine was calculated to characterize the stability and emulsification effect of the vaccine.
[0049] Note: The longer it takes for the vaccine to stratify or break, the better the stability of the vaccine and the better the emulsification effect.
[0050] (2) Antibody content: The activity of the emulsified vaccine was characterized by animal experiments. The specific process is as follows: Experimental animals: A total of 60 2-month-old piglets were divided into 20 groups (experimental groups 1-14, control groups 1-5, and a blank control group), with 3 piglets in each group. Experimental process: Piglets were immunized with each porcine circovirus vaccine, each group corresponding to one vaccine. The blank control group did not receive any immunization treatment. The immunization dose was 2 ml / head. A second immunization was performed at the same dose 14 days later. Blood was collected 21 days after the second immunization to separate serum. The serum was diluted and the porcine circovirus 2-dCap-ELISA antibody detection kit produced by Beijing Jinnuo Biotech Co., Ltd. was used to measure the serum dilutions diluted to different gradients. The maximum dilution with a positive serum test result was used as the antibody titer. The results are shown in Table 2 below.
[0051] Table 2 Stability and activity test results of vaccine antigens obtained in Examples 1-14 and Comparative Examples 1-5 According to the test results in Table 2, the porcine circovirus vaccine was emulsified using the vaccine antigen emulsification method provided in Examples 1-14 of the present application. After the accelerated aging test, the stable time of the vaccine was 26-34 days, and the titer of the vaccine antigen was 1:128 to 1:1024; while the porcine circovirus vaccine was emulsified using the vaccine antigen emulsification method provided in Comparative Examples 1-5. After the accelerated aging test, the stable time of the vaccine was only 19-31 days, and the titer of the vaccine antigen was only 1:64 to 1:128. Therefore, it is shown that the vaccine antigen emulsification method provided in the present application can obtain vaccine antigens with good stability and good antigen activity.
[0052] Further comparison of the test results of Example 2 and Examples 4-12 shows that in Example 4, the rotation speed is controlled at 3500 rpm, and the stability time of the obtained vaccine is only 28 days; in Example 6, the rotation speed is controlled at 5000 rpm, and the titer of the obtained vaccine antigen is 1:128; in Example 7, the linear speed is controlled at 5 m / s, and the stability time of the obtained vaccine is only 27 days; in Example 9, the linear speed is controlled at 8 m / s, and the titer of the obtained vaccine antigen is 1:128; in Example 12, the temperature is controlled at 8°C, and the titer of the obtained vaccine antigen is 1:128; and in Examples 2, 5, 8, and 10-11, the rotation speed is controlled at 4000-4500 rpm, the linear speed is controlled at 6-7 m / s, and the temperature is controlled at 2-6°C, and the stability time of the obtained vaccine is 31-34 days, and the titer of the vaccine antigen is 1:256~1:024. It is explained that the present application further controls the rotation speed in the emulsification process within the range of 4000-4500 rpm, the linear speed within the range of 6-7 m / s, and the temperature within the range of 2-6°C, which has a better emulsification effect on the vaccine antigen, and the obtained vaccine antigen has better stability and better antigen activity.
[0053] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.
Claims
1. A method for emulsifying a small amount of vaccine antigen in the laboratory, characterized in that: The following steps are involved: Add the mixture of vaccine antigen and adjuvant to a tissue grinding cryogenic homogenizer and emulsify according to the following program: rotation speed 3500-5000 rpm, linear speed 5-8 m / s, temperature 2-8°C; run for 30 seconds and pause for 30 seconds as one cycle, and repeat 3-4 times; The above procedure was run every 5±0.5 min for a total of 5-7 times.
2. The emulsification method for a small amount of vaccine antigen in the laboratory according to claim 1, characterized in that: The adjuvant is selected from Freund's complete adjuvant, Freund's incomplete adjuvant or white oil.
3. The emulsification method for a small amount of vaccine antigen in the laboratory according to claim 2, characterized in that: When the adjuvant is Freund's complete adjuvant or Freund's incomplete adjuvant, the volume ratio of the vaccine antigen to the adjuvant is 1:(1-1.2).
4. The emulsification method for a small amount of vaccine antigen in the laboratory according to claim 2, characterized in that: When the adjuvant is white oil, the volume ratio of the vaccine antigen to the adjuvant is 1:(1.5-3).
5. The emulsification method for a small amount of vaccine antigen in a laboratory according to claim 4, characterized in that: When the adjuvant is white oil, an emulsifier and a preservative need to be added to the mixture.
6. The emulsification method for a small amount of vaccine antigen in the laboratory according to claim 1, characterized in that: The emulsifier is Siben 80 and the preservative is thimerosal.
7. The emulsification method for a small amount of vaccine antigen in a laboratory according to claim 1, characterized in that: The rotation speed is 4000-4500 rpm, the linear speed is 6-7 m / s, and the temperature is 2-6°C.
8. The emulsification method for a small amount of vaccine antigen in a laboratory according to claim 1, characterized in that: The model of the tissue grinding low-temperature homogenizer is Tgrinder H24R.