Inactivation method for improving recovery rate of enterovirus antigen
By using TritionX-100, arginine and lysine mixed protective agents in the preparation of enterovirus vaccines, the problem of low antigen recovery rate caused by formaldehyde inactivation was solved, and the antigen recovery rate and vaccine quality were significantly improved.
Patent Information
- Application Number
- CN202510319779.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-18
- Publication Date
- 2025-06-20
AI Technical Summary
The formaldehyde inactivation method results in low antigen recovery rate during the preparation of enterovirus vaccine, affecting the yield and cost efficiency of the vaccine.
The protective agent composed of TritionX-100, arginine and lysine was used to add the purified enterovirus solution for incubation, and then the formaldehyde solution was added to inactivate it. This method significantly improved the antigen recovery rate.
It significantly improves the recovery rate of enterovirus antigens, reduces the residual amount of TritonX-100 and formaldehyde, meets the requirements of the National Pharmacopeia for impurity residues, and improves the product quality and immunity effect of the vaccine.
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Abstract
Description
Technical Field
[0001] The present application relates to the technical field of viral antigens, and in particular to an inactivation method for improving the recovery rate of enterovirus antigens. Background Art
[0002] Formaldehyde is the most commonly used virus inactivator and is usually used to inactivate viruses in biological products. Its inactivation mechanism is: on the one hand, it acts on the amino-containing nucleotide bases of the virus, and denatures the nucleic acid by combining with the amino-containing bases such as adenine, guanine, and cytosine; on the other hand, it acts on the virus capsid protein and combines with the amino group of the protein to form a hydroxymethyl derivative. As the action time increases, the hydroxymethyl derivative can react with the amide to cross-link the microorganism, causing the microorganism to lose its toxicity and reproductive ability, but still maintain good immunity. However, it is easy to cause protein cross-linking or virus particle aggregation, and the antigenicity of the pathogen protein is severely damaged. This process is often accompanied by antigen loss, resulting in a low antigen recovery rate, which affects the yield and cost efficiency of the vaccine.
[0003] At present, in the process of preparing enteric vaccines, especially the production of enterovirus 71 vaccines, the formaldehyde inactivation method is usually used to inactivate the vaccine: the purified virus liquid is sterile filtered (or coarsely filtered), and then 200μg of formaldehyde solution is added to inactivate for 72 hours to obtain antigens with stable protein structure and high immunogenicity for the preparation of the final vaccine product. However, formaldehyde inactivation is often accompanied by protein cross-linking or viral particle aggregation, resulting in low antigen recovery rate, affecting the yield and cost efficiency of the vaccine. Summary of the invention
[0004] In order to solve the above technical problems, the present application provides an inactivation method for improving the recovery rate of enterovirus antigens.
[0005] The present application provides an inactivation method for improving the recovery rate of enterovirus antigens, which specifically comprises the following steps: Add a protective agent with a final concentration of 50-250 μg / ml to the purified enterovirus solution; after fully mixing, stir and incubate the mixture; The protective agent is composed of TritionX-100, arginine, and lysine mixed in a mass ratio of 140-160:20-50:4-16; The incubation temperature is 25-40°C and the incubation time is 4-12h; Then, 0.8-1.2% formaldehyde solution is added to make the final concentration 180-220 μg / ml, and the solution is placed at 35-39° C. and inactivated for 68-76 hours to obtain the inactivated vaccine; finally, the inactivated solution is filtered through a 300KDa membrane to remove TritionX-100 and formaldehyde in the sample.
[0006] In this application, a protective agent composed of Triton X-100, arginine, and lysine with a mass ratio of 140-160:20-50:4-16 is added to the purified enterovirus solution, incubated for a certain period of time, and then inactivated with 1% formaldehyde solution. The antigen recovery rate of the inactivated solution obtained after washing and filtration is greatly improved, and the residual amounts of Triton X-100 and formaldehyde are less.
[0007] Triton X-100 in the protective agent is a non-ionic surfactant. Triton X-100 contains a hydrophilic polyoxyethylene chain and a hydrophobic aromatic group, and can form a stable micelle structure at a certain concentration, surrounding the virus in the form of a physical barrier to prevent direct contact and aggregation between viruses, thereby maintaining the suspended state of the virus and also protecting the antigen from loss caused by shear force during stirring, and then improving the antigen recovery rate. Arginine and lysine both carry positive charges and can interact with the negative charges on the virus surface, thereby stabilizing the virus particles and preventing their aggregation or precipitation during inactivation, ensuring that more virus particles remain independent and intact, which is beneficial for subsequent recovery and purification. In addition, this combination of the protective agent can also reduce the damage of formaldehyde to antigenicity, so that the inactivated virus can still maintain high antigenicity, which is crucial for the recovery and utilization of antigens in the vaccine R & D and production process. During the experiment, the applicant found that the protective agent composed of Triton X-100, arginine, and lysine mixed in a specific mass ratio significantly improved the recovery rate during the virus inactivation process through the synergistic effects in multiple aspects such as stabilizing virus particles.
[0008] Preferably, the protective agent is composed of Triton X-100, arginine, and lysine mixed in a mass ratio of 145-155:30-40:8-12.
[0009] Preferably, the protective agent is composed of Triton X-100, arginine, and lysine mixed in a mass ratio of 147-152:32-37:9-11.
[0010] In a specific embodiment, in the protective agent, the mass ratio of Triton X-100, arginine, and lysine can be 140:20:4, 140:30:4, 140:32:4, 140:35:4, 140:37:4, 140:40:4, 145:20:4, 145:30:4, 145:32:4, 145:35:4, 145:37:4, 145:40:4, 150:20:4, 150:30:4, 150:32:4, 150:35:4, 150:37:4, 150:40:4, 155:20:4, 155:30:4, 155:32:4, 155:35:4, 155:37:4, 155:40:4, 160:20:4, 160:30:4, 160:32:4, 160:35:4, 160:37:4, 160:40:4, 140:20:8, 140:30:8, 140:32:8, 140:35:8, 140:37:8, 140:40:8, 145:20:9, 145:30:9, 145:32:9, 145:35:9, 145:37:9, 145:40:9, 150:20:10, 150:30:10, 150:32:10, 150:35:10, 150:37:10, 150:40:10, 155:20:11, 155:30:11, 155:32:11, 155:35:11, 155:37:11, 155:40:11, 160:20:12, 160:30:12, 160:32:12, 160:35:12, 160:37:12, 160:40:12, 160:20:16, 160:30:16, 160:32:16, 160:35:16, 160:37:16, 160:40:16.
[0011] Through experiments, it is known that the protective agent composed of Triton X-100, arginine, and lysine in the above mass ratio can further improve the recovery rate of virus antigen in the inactivating solution.
[0012] Preferably, the final concentration of the protective agent is 100 - 230 μg / ml.
[0013] Preferably, the final concentration of the protective agent is 150 - 200 μg / ml.
[0014] In a specific embodiment, the final concentration of the protective agent can be 50 μg / ml, 100 μg / ml, 120 μg / ml, 150 μg / ml, 180 μg / ml, 200 μg / ml, 230 μg / ml, 250 μg / ml.
[0015] Through experiments, it is known that by controlling the final concentration of the protective agent within the above range in this application, the recovery rate of virus antigen in the inactivated solution can be further improved.
[0016] Preferably, the incubation temperature is 35 - 38 °C and the incubation time is 4 - 12 h.
[0017] Preferably, the incubation temperature is 35 - 38 °C and the incubation time is 5 - 8 h.
[0018] In a specific embodiment, the incubation temperature can be 25 °C, 30 °C, 35 °C, 37 °C, 38 °C, 40 °C.
[0019] In a specific embodiment, the incubation time can be 4 h, 5 h, 6 h, 8 h, 10 h, 12 h.
[0020] Through experiments, it is known that by controlling the incubation temperature within the above range in this application, the recovery rate of virus antigen in the inactivated solution can be further improved.
[0021] Preferably, the final concentration of the formaldehyde solution is 190 - 210 μg / ml.
[0022] In a specific embodiment, the final concentration of the formaldehyde solution can be 180 μg / ml, 190 μg / ml, 200 μg / ml, 210 μg / ml, 220 μg / ml.
[0023] Preferably, the inactivation time is 70 - 74 h.
[0024] Preferably, the enterovirus is EV71 virus.
[0025] In summary, the technical solution of this application has the following effects: This application provides a novel antigen inactivation method for enterovirus. Compared with the traditional inactivation process, a protective agent composed of a mixture of TritionX-100, arginine, and lysine is added and incubated, and the antigen recovery rate after inactivation is greatly improved.
[0026] For the inactivated vaccine finally obtained in this application, 300KDa membrane washing and filtration are carried out to remove TritionX-100 and formaldehyde, so that their residual amounts are lower than the detection standard values, meeting the requirements of the fourth part of the "National Pharmacopoeia" for the residual amount of impurities, and effectively avoiding the stress effects of TritionX-100 and formaldehyde on the body.
[0027] Using the preparation method of the inactivated vaccine in this application is more conducive to maintaining the integrity of the virus, enhancing the immune effect, and improving the product quality. Detailed implementation methods
[0028] The present application will be further described in detail below in combination with examples, comparative examples, and performance detection tests. These examples should not be construed as limiting the scope claimed by the present application. Examples
[0029] Examples 1 - 5 Examples 1 - 5 respectively provide an inactivation method for improving the recovery rate of enterovirus antigen.
[0030] The differences between the above - mentioned examples are as follows: the compositions of the protectants are different, as shown in Table 1 specifically.
[0031] The specific steps of the inactivation method for improving the recovery rate of enterovirus antigen in the above - mentioned examples are as follows: Add a protectant with a final concentration of 150 μg / ml to the purified enterovirus; after thorough mixing, stir and incubate the mixture; the incubation temperature is 37 °C, and the incubation time is 6 h; Then add a 1.0% formaldehyde solution to make its final concentration 200 μg / ml, and place it at 37 °C for inactivation for 72 h to obtain the inactivated vaccine; finally, wash and filter the inactivated solution through a 300 KDa membrane to remove impurities and formaldehyde in the sample.
[0032] Table 1 Compositions of protectants in Examples 1 - 5 Example Composition of the protective agent 1 Composed of Trition X-100, arginine, and lysine mixed in a mass ratio of 150:35:10 2 Composed of Trition X-100, arginine, and lysine mixed in a mass ratio of 145:40:8 3 Composed of Trition X-100, arginine, and lysine mixed in a mass ratio of 155:30:12 4 Composed of Trition X-100, arginine, and lysine mixed in a mass ratio of 140:50:4 5 Composed of Trition X-100, arginine, and lysine mixed in a mass ratio of 160:20:16 Examples 6 - 10 Examples 6 - 10 respectively provide an inactivation method for improving the recovery rate of enterovirus antigen.
[0033] The specific differences between the above - mentioned examples and Example 1 are as follows: the final concentrations of the protectants are different, as shown below.
[0034] In Example 6: the final concentration of the protectant is 50 μg / ml.
[0035] In Example 7: the final concentration of the protectant is 100 μg / ml.
[0036] In Example 8: the final concentration of the protectant is 200 μg / ml.
[0037] In Example 9: the final concentration of the protectant is 230 μg / ml.
[0038] In Example 10: the final concentration of the protectant is 250 μg / ml.
[0039] Other process parameters in the above - mentioned examples are the same as those in Example 1.
[0040] Examples 11 - 18 Examples 11 - 18 respectively provide an inactivation method for improving the recovery rate of enterovirus antigen.
[0041] The differences between the above examples and Example 1 are specifically as follows: the incubation conditions are different, as shown in Table 2 specifically.
[0042] Table 2 Incubation conditions in Examples 11 - 18 Other process parameters in the above examples are the same as those in Example 1.
[0043] Examples 19 - 22 Examples 19 - 22 respectively provide an inactivation method for improving the recovery rate of enterovirus antigen.
[0044] The differences between the above examples and Example 1 are specifically as follows: the final concentration of formaldehyde solution is different, as shown below.
[0045] In Example 19: the final concentration of formaldehyde solution is 180 μg / ml.
[0046] In Example 20: the final concentration of formaldehyde solution is 190 μg / ml.
[0047] In Example 21: the final concentration of formaldehyde solution is 210 μg / ml.
[0048] In Example 22: the final concentration of formaldehyde solution is 220 μg / ml.
[0049] Other process parameters in the above examples are the same as those in Example 1.
[0050] Comparative Examples Comparative Examples 1 - 3 Comparative Examples 1 - 3 respectively provide an inactivation method for the recovery rate of enterovirus antigen.
[0051] The differences between the above comparative examples and Example 1 are specifically as follows.
[0052] In Comparative Example 1: the protective agent is TritionX - 100.
[0053] In Comparative Example 2: the protective agent is composed of TritionX - 100, arginine, and glycine mixed in a mass ratio of 150:35:10.
[0054] In Comparative Example 3: the protective agent is composed of TritionX - 100, arginine, and lysine mixed in a mass ratio of 170:16:18.
[0055] Other process parameters in the above comparative examples are the same as those in Example 1.
[0056] Comparative Example 4 Comparative Example 4 provides a method for inactivating enterovirus antigen recovery rate.
[0057] No protective agent was added in this comparative example; the specific method was as follows: 1% formaldehyde solution was added to the purified enterovirus to make its final concentration 200 μg / ml, and it was inactivated at 37°C for 72 h to obtain the inactivated vaccine; finally, the inactivated solution was washed and filtered through a 300KDa membrane to remove formaldehyde in the sample.
[0058] Performance detection test Detection of virus antigen content: According to the enzyme-linked immunosorbent assay (ELISA), the antigen content in the inactivated solutions obtained in the examples and comparative examples was detected, and the antigen content increase rate of the examples and comparative examples relative to Comparative Example 4 was calculated.
[0059] Detection of TritonX-100 residue: Using the virus antigen obtained in the examples and comparative examples as the detection object, the residue of TritonX-100 in the sample to be detected was detected according to the detection method specified in the Chinese Pharmacopoeia 2020 Edition.
[0060] Detection of formaldehyde residue: Using the virus antigen obtained in the examples and comparative examples as the detection object, the formaldehyde residue in the sample to be detected was detected by the acetylacetone colorimetric method.
[0061] Detection results: As shown in Table 3.
[0062] Table 3 Performance detection results of virus inactivated solutions in Examples 1-22 and Comparative Examples 1-4 Combined with Table 3, by comparing the detection results of the examples and comparative examples, it can be seen that in this application, a protective agent composed of a mixture of TritionX-100, arginine, and lysine with a mass ratio of 140-160:20-50:4-16 was added to the purified enterovirus solution, incubated for a certain time, and then formaldehyde solution was added for inactivation. The antigen recovery rate in the obtained inactivated solution was greatly improved, and the residues of TritonX-100 and formaldehyde were less.
[0063] In Comparative Example 1, only TritionX-100 was used as the protective agent. In Comparative Example 2, a protective agent composed of TritionX-100, arginine, and glycine with a mass ratio of 150:35:10 was used. In Comparative Example 3, a protective agent composed of TritionX-100, arginine, and lysine with a mass ratio of 170:16:18 was used, and the increase rate of antigen recovery rate in the inactivated solution was less.
[0064] After comparing the detection results of Examples 1-5, the present application selects to further control the protective agent to be composed of a mixture of TritionX-100, arginine, and lysine with a mass ratio of 145-155:30-40:8-12, which can further improve the antigen recovery rate in the inactivated solution.
[0065] After comparing the detection results of Examples 6-10, the present application selects to control the final concentration of the protective agent to 100-230 μg / ml, which can further improve the antigen recovery rate in the inactivated solution.
[0066] After comparing the detection results of Examples 11-18, after adding the protective agent, the present application selects to control the incubation temperature to 35-38 °C and the incubation time to 5-8 h, which can further improve the antigen recovery rate in the inactivated solution.
[0067] After comparing the detection results of Examples 19-22, after adding the protective agent, the present application selects to control the final concentration of the formaldehyde solution to 190-220 μg / ml, which can further improve the antigen recovery rate in the inactivated solution.
[0068] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.
Claims
1. A method for inactivating enterovirus antigens to improve the recovery rate, characterized in that: The specific steps include: Add a protective agent with a final concentration of 50-250 μg / ml to the purified enterovirus solution; after fully mixing, stir and incubate the mixture; The protective agent is composed of TritionX-100, arginine, and lysine mixed in a mass ratio of 140-160:20-50:4-16; The incubation temperature is 25-40°C and the incubation time is 4-12h; Then, 0.8-1.2% formaldehyde solution is added to make the final concentration 180-220 μg / ml, and the mixture is placed at a temperature of 35-39° C. and inactivated for 68-76 hours to obtain the inactivated vaccine; finally, the inactivated solution is filtered through a 300KDa membrane to remove TritionX-100 and formaldehyde in the sample.
2. The inactivation method for improving the recovery rate of enterovirus antigens according to claim 1, characterized in that: The protective agent is composed of TritionX-100, arginine and lysine mixed in a mass ratio of 145-155:30-40:8-12.
3. The inactivation method for improving the recovery rate of enterovirus antigens according to claim 2, characterized in that: The protective agent is composed of TritionX-100, arginine and lysine mixed in a mass ratio of 147-152:32-37:9-11.
4. The inactivation method for improving the recovery rate of enterovirus antigens according to claim 1, characterized in that: The final concentration of the protective agent is 100-230 μg / ml.
5. The inactivation method for improving the recovery rate of enterovirus antigens according to claim 4, characterized in that: The final concentration of the protective agent is 150-200 μg / ml.
6. The inactivation method for improving the recovery rate of enterovirus antigens according to claim 1, characterized in that: The incubation temperature is 35-38° C., and the incubation time is 4-12 h.
7. The inactivation method for improving the recovery rate of enterovirus antigens according to claim 6, characterized in that: The incubation temperature is 35-38° C., and the incubation time is 5-8 h.
8. The inactivation method for improving the recovery rate of enterovirus antigens according to claim 1, characterized in that: The final concentration of the formaldehyde solution is 190-210 μg / ml.
9. The inactivation method for improving the recovery rate of enterovirus antigens according to claim 1, characterized in that: The inactivation time is 70-74h.
10. The inactivation method for improving the recovery rate of enterovirus antigens according to claim 1, characterized in that: The enterovirus is EV71 virus.