Process method for removing mycoplasma polluted by influenza vaccine virus seed in serum-free suspension MDCK cell culture
Through methods such as high-speed centrifugation, monolayer cell culture and cholesterol-free serum-free maintenance medium culture, the problem of mycoplasma contamination of influenza vaccines has been solved, efficient removal of contamination has been achieved, and the purity and safety of the vaccine have been improved.
Patent Information
- Application Number
- CN202310882440.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-18
- Publication Date
- 2025-01-21
AI Technical Summary
The prior art is difficult to effectively remove mycoplasma contamination in influenza vaccine poisoning species, and traditional methods have problems such as incomplete pollution and complex operation.
A comprehensive method of high-speed centrifugation, monolayer cell culture, virus adsorption, buffer washing, cholesterol-free serum-free maintenance solution culture and pH adjustment was repeated to remove mycoplasma contamination.
It achieves efficient removal of mycoplasma contamination, improves the purity and safety of vaccine strains, and meets Chinese Pharmacopoeia standards.
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Figure CN119331831A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a method for removing mycoplasma contaminated by influenza vaccine strains, which is used for the method for removing mycoplasma contaminated by viruses such as influenza vaccine strains, and belongs to the field of biotechnology. Specifically, it relates to a complex multi-step mycoplasma removal process such as high-speed centrifugation and cell culture, which mainly includes high-speed centrifugation at 10,000-15,000 rpm, adding virus adsorption for 1-2 hours during the virus culture process of monolayer cells, discarding the culture medium, washing with buffer or cholesterol-free and serum-free cell culture medium for 3-5 times, and the virus harvesting liquid is centrifuged again at 10,000-15,000 rpm, and then removed once by virus adsorption method and pH 6.9-7.0 cholesterol-free and serum-free maintenance medium culture method, and the PCR test result is negative, which is an effective method for removing mycoplasma contamination. Background Art
[0002] The replacement of chicken embryos for influenza vaccine production by mammalian cell culture has become a trend in the research and development of influenza vaccines. The cell culture method has the advantages of easy scale-up and controllable quality, is not limited by the interruption of chicken embryo supply, and can quickly adapt to the market. Therefore, the innovative development of cell influenza vaccine preparation technology solves the current difficulties of chicken embryo technology and reaches the international advanced level.
[0003] Removal of mycoplasma contamination from influenza virus means applying various physical and chemical methods to remove non-viral impurities such as mycoplasma without damaging or inactivating the virus, and optimizing the screening of high-purity and high-virus titer strains. Vaccine strains are a prerequisite for the development of high-quality vaccines, so strains without any exogenous contamination are required. The size of mycoplasma is 0.1-0.3um, which can pass through the filter, often causing trouble for cell culture. The requirements for mycoplasma culture are higher than those for general bacteria. In addition to basic nutrients, 10-20% human or animal serum must be added to provide the cholesterol required by mycoplasma. The optimum pH is between 7.8 and 8.0, and death occurs below 7.0. Mycoplasma contamination of cell culture (especially passage cells) is a global problem.
[0004] After mycoplasma contaminates cells and toxic species, it is necessary to remove mycoplasma. Common methods include antibiotic treatment, antiserum treatment, antibiotic plus antiserum and complement, mycoplasma remover and other methods. This process requires a large amount of reagents and is more likely to bring about re-contamination, so it is not applicable to remove mycoplasma contaminated by toxic species. The centrifugation and monolayer cell culture methods are used to remove mycoplasma contamination. The conditions are mild, repeatable, and easy to use. It is an effective method for removing mycoplasma contamination from vaccine strains. Therefore, in the vaccine preparation process, a set of mature and mild mycoplasma removal technologies are needed to make the vaccine strains meet the Chinese Pharmacopoeia standards.
[0005] The molecular weight of viruses ranges from tens of thousands to millions, and the molecular weight of mycoplasma is 4*10 8 -1*10 9 , much larger than the virus. Centrifugation of the virus requires about 30,000 revolutions, so centrifugation at 1,000-15,000 revolutions can partially remove some mycoplasmas.
[0006] The virus infects the monolayer cells by adsorption. The virus electrostatically and specifically binds to the cell surface receptors, but mycoplasma does not have this function. The purpose of partially removing mycoplasma is achieved by flushing with buffer or culture medium.
[0007] The optimum pH for mycoplasma is between 7.8 and 8.0, and it will die if the pH is lower than 7.0. By adjusting the pH to 6.9-7.0 through cell virus culture method, a certain mycoplasma inactivation effect can be achieved.
[0008] The requirements for mycoplasma culture are higher than those for general bacteria. In addition to basic nutrients, 10-20% human or animal serum must be added to provide the cholesterol required by mycoplasma. Therefore, using cholesterol-free serum-free medium for viral cell culture can reduce the replication and proliferation of mycoplasma.
[0009] Literature reports that after mycoplasma contaminates cells and virus strains, the commonly used methods for removing mycoplasma include antibiotic treatment, antiserum treatment, antibiotic plus antiserum and complement, mycoplasma remover and other methods, and the removal is not thorough. Therefore, it is difficult to solve the mycoplasma contamination problem using traditional mycoplasma removal technology. Therefore, it is necessary to conduct innovative research on the mycoplasma contamination removal technology for vaccine virus strains.
[0010] No reports were found on mycoplasma removal processes that combined centrifugation, cell culture adsorption, cholesterol-free serum-free culture medium, and pH 6.9-7.0. In short, the mycoplasma removal process of this paper was not found to be consistent with those reported in the literature. Summary of the invention
[0011] The present invention overcomes the technical defects of traditional vaccine virus seed removal of mycoplasma, and provides a set of innovative and complex vaccine virus seed mycoplasma removal methods, which have good removal effect, good repeatability, mild conditions, and are easy to operate. Even if there is no contaminating mycoplasma, this operation process can also prevent mycoplasma contamination. The vaccine virus seed contaminated with mycoplasma is centrifuged at 1000-15000 revolutions, inoculated with a monolayer of cells for adsorption at 37°C for 1-2 hours, rinsed 3-5 times with a buffer or cholesterol-free and serum-free cell culture fluid, and then cultured with a cholesterol-free and serum-free maintenance fluid at a pH of 6.9-7.0 at 34°C for 48-120 hours for harvesting, and the above operation is repeated once again to achieve the purpose of mycoplasma removal.
[0012] The purpose of the present invention is achieved through the following technical solutions and steps.
[0013] Centrifugation The influenza vaccine virus strain contaminated with mycoplasma is centrifuged at 10,000-15,000 rpm in a high-speed centrifuge to precipitate some of the mycoplasma and harvest the vaccine virus culture supernatant.
[0014] Cell culture The supernatant of the vaccine virus was centrifuged and inoculated into MDCK monolayer cells. The cells were adsorbed at 37°C for 1-2 hours, and then rinsed 3-5 times with buffer or cholesterol-free and serum-free cell culture medium. The cholesterol-free and serum-free maintenance medium was replaced with a pH of 6.9-7.0. The vaccine virus seed liquid was harvested after culturing at 34°C for 48-72 hours.
[0015] Centrifuge again The influenza vaccine virus harvested liquid was centrifuged at 10,000-15,000 rpm in a high-speed centrifuge to precipitate some of the mycoplasmas and harvest the virus supernatant.
[0016] Second cell culture The supernatant of influenza vaccine virus seed centrifugation is inoculated into MDCK monolayer cells, adsorbed at 37°C for 1-2 hours, rinsed 3-5 times with buffer or cholesterol-free and serum-free cell culture medium, replaced with cholesterol-free and serum-free maintenance medium with a pH of 6.9-7.0, and cultured at 34°C for 48-72 hours to harvest the vaccine virus seed liquid.
[0017] In the above-mentioned method for removing mycoplasma, influenza A and B viruses are preferred;
[0018] In the above method for removing mycoplasma, a high-speed centrifuge is preferred, and the centrifugal force parameter is 10000-15000 rpm;
[0019] In the above-mentioned method for removing mycoplasma, preferably, the virus is inoculated into a monolayer of cells, adsorbed at 37°C for 1-2 hours, and washed 3-5 times with a buffer or a cholesterol-free and serum-free cell culture medium;
[0020] In the above-mentioned method for removing mycoplasma, a cholesterol-free and serum-free maintenance medium is preferred;
[0021] In the above method for removing mycoplasma, the pH of the cell virus maintenance solution is preferably 6.9-7.0;
[0022] After collection in each step, samples are taken for verification of various indicators. Some of the vaccine viruses may include: H1N1 influenza virus, H3N2 influenza virus, H5N1 influenza virus, By type influenza B virus, Bv type influenza B virus, etc. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1The invention relates to a process flow chart for removing mycoplasma contamination from the virus strain of influenza vaccine cultured in serum-free suspension MDCK cells. The virus strain of influenza vaccine cultured in serum-free suspension MDCK cells is centrifuged at 10,000-15,000 rpm, infects MDCK suspension cells, adsorbs cells at 37° C. for 1-2 hours, discards the virus solution, rinses with buffer or cholesterol-free and serum-free culture solution, adds cholesterol-free serum-free maintenance solution for culture, harvests the virus culture supernatant at 33-34° C., pH 6.9-7.0, and then centrifuges at 10,000-15,000 rpm again, infects again, harvests the virus culture supernatant, and samples for various tests.
[0024] Figure 2 Electrophoresis diagram of mycoplasma PCR test results before and after treatment of each mycoplasma positive virus strain The first five bands are the positive results of PCR electrophoresis bands of five samples of influenza virus H1N1, influenza virus H3N2, influenza virus H5N1, influenza virus By type B, and influenza virus B type B. The two negative results are the PCR electrophoresis band results of H5N1 influenza virus and influenza virus By type B after treatment. The last two positive results are one positive control for other samples and the other for the standard positive control.
[0025] Figure 3 Electrophoresis diagram of mycoplasma PCR test results after treatment of each mycoplasma-positive virus strain The first three bands are the negative results of mycoplasma detection after processing the three samples of H1N1, H3N2, and B (Bv), and the last clear band is the positive control sample. DETAILED DESCRIPTION
[0026] Embodiment 1:
[0027] This embodiment provides a complex multi-step vaccine virus liquid mycoplasma removal method, which includes the following steps:
[0028] Normal virus culture: H1N1 type: Influenza virus infectious IVR-215 (H1N1) 45620E4 / D7 / E1 virus infected serum-free suspension cultured MDCK cells, the infection parameters are cell density of 3.5*10 6 cells / ml, cell viability 98.7%, TPCK trypsin concentration 2μg / ml, pH 7.4, culture on a shaker at 90 rpm, temperature 34°C, and harvest after 48 hours.
[0029] The vaccine virus harvest liquid contaminated with mycoplasma is centrifuged at 10,000-15,000 rpm in a high-speed centrifuge to precipitate some of the mycoplasma and harvest the virus supernatant.
[0030] The supernatant of the vaccine virus seed centrifuged was inoculated into MDCK monolayer cells, adsorbed at 37°C for 1-2 hours, rinsed 3-5 times with 0.01M PBS or CDMDCK Production Medium (serum-free) cell culture medium, replaced with cholesterol-free CD MDCK Production Medium (serum-free) maintenance medium, TPCK trypsin concentration of 2μg / ml, maintenance medium pH of 6.9-7.0, and cultured at 34°C for 48-72 hours to harvest the vaccine virus seed liquid.
[0031] Centrifuge again The vaccine virus harvest fluid was centrifuged at 10,000-15,000 rpm in a high-speed centrifuge to precipitate some of the mycoplasmas and harvest the virus supernatant.
[0032] Second cell culture The supernatant of influenza vaccine virus seed centrifugation was inoculated into MDCK monolayer cells, adsorbed at 37°C for 1-2 hours, rinsed 3-5 times with 0.01M PBS or cholesterol-free CD MDCK Production Medium (serum-free) cell culture medium, replaced with cholesterol-free CD MDCK Production Medium (serum-free) maintenance medium, TPCK trypsin concentration of 2 μg / ml, maintenance liquid base pH of 6.9-7.0, and cultured at 34°C for 48-72 hours to harvest the vaccine virus seed liquid. Detection indicators at each step step Mycoplasma removal rate% Hemagglutination titer Centrifugation 80.7 512 Cell culture 95.2 1024 Centrifuge again 99.8 512 Second cell culture 100 1024 After the key two-step method, all test indicators met the requirements of the experimental design plan.
[0033] Embodiment 2:
[0034] This embodiment provides a complex multi-step method for removing mycoplasma from influenza vaccine virus fluid, which includes the following steps:
[0035] Normal virus seed propagation: Infected with Influenza virus infectious (H3N2) type virus with MOI of 0.01 and infection parameters of shake flask cell density of 3.5*10 6 cells / ml, cell survival rate 98.7%, TPCK trypsin concentration 2μg / ml, pH 7.4, shaker speed 90 rpm, culture temperature 34℃, harvest time 48 hours.
[0036] The vaccine virus harvest fluid contaminated with mycoplasma is centrifuged at 10,000-15,000 rpm in a high-speed centrifuge to precipitate some of the mycoplasma and harvest the virus supernatant.
[0037] The supernatant of the vaccine virus seed centrifugation is inoculated into MDCK monolayer cells, adsorbed at 37°C for 1-2 hours, rinsed 3-5 times with 0.01M PBS or CD MDCK Production Medium (serum-free) cell culture medium, replaced with cholesterol-free CD MDCK Production Medium (serum-free) maintenance medium, TPCK trypsin concentration of 2μg / ml, maintenance medium pH of 6.9-7.0, and cultured at 34°C for 48-72 hours to harvest the vaccine virus seed liquid.
[0038] Centrifuge again The vaccine virus harvest fluid was centrifuged at 10,000-15,000 rpm in a high-speed centrifuge to precipitate some of the mycoplasmas and harvest the virus supernatant.
[0039] Second cell culture The supernatant of influenza vaccine virus seed centrifugation was inoculated into MDCK monolayer cells, adsorbed at 37°C for 1-2 hours, rinsed 3-5 times with 0.01M PBS or cholesterol-free CD MDCK Production Medium (serum-free) cell culture medium, replaced with cholesterol-free CD MDCK Production Medium (serum-free) maintenance medium, the TPCK trypsin concentration was 2 μg / ml, the pH of the maintenance medium was 6.9-7.0, and the vaccine virus seed liquid was harvested after culturing at 34°C for 48-72 hours. Detection indicators at each step step Mycoplasma removal rate% Hemagglutination titer Centrifugation 85.3 512 Cell culture 96.6 1024 Centrifuge again 99.9 512 Second cell culture 100 1024
[0040] Embodiment 3
[0041] This embodiment provides a complex multi-step method for removing mycoplasma from influenza vaccine virus fluid, which includes the following steps: Normal virus passage: serum-free suspension MDCK cells were cultured in shake flasks and infected with Influenza virusinfectious BX-85C, Bv type virus at an MOI of 0.001. The infection parameters were a cell density of 4.2*10 6 cells / ml, cell survival rate 98.5%, TPCK trypsin concentration 2.0μg / ml, pH 7.4, shaking flask rotation number 90 rpm, culture temperature 34°C, and harvest time 48 hours.
[0042] The vaccine virus harvest liquid contaminated with mycoplasma is centrifuged at 10,000-15,000 rpm in a high-speed centrifuge to precipitate some of the mycoplasma and harvest the virus supernatant.
[0043] The supernatant of the vaccine virus seed centrifugation is inoculated into MDCK monolayer cells, adsorbed at 37°C for 1-2 hours, rinsed 3-5 times with 0.01M PBS or CD MDCK Production Medium (serum-free) cell culture medium, replaced with cholesterol-free CD MDCK Production Medium (serum-free) maintenance medium, TPCK trypsin concentration of 2μg / ml, maintenance medium pH of 6.9-7.0, and cultured at 34°C for 48-72 hours to harvest the vaccine virus seed liquid.
[0044] Centrifuge again The vaccine virus harvest fluid was centrifuged at 10,000-15,000 rpm in a high-speed centrifuge to precipitate some of the mycoplasmas and harvest the virus supernatant.
[0045] Second cell culture The supernatant of the vaccine virus seed centrifugation was inoculated into MDCK monolayer cells, adsorbed at 37°C for 1-2 hours, rinsed 3-5 times with 0.01M PBS or cholesterol-free CD MDCK Production Medium (serum-free) cell culture medium, replaced with cholesterol-free CD MDCK Production Medium (serum-free) maintenance medium, the TPCK trypsin concentration was 2 μg / ml, the pH of the maintenance medium was 6.9-7.0, and the vaccine virus seed liquid was harvested after culturing at 34°C for 48-72 hours. Detection indicators at each step step Mycoplasma removal rate% Hemagglutination titer Centrifugation 86.1 1024 Cell culture 96.8 2048 Centrifuge again 99.9 1024 Second cell culture 100 2048
[0046] Embodiment 4
[0047] This embodiment provides a complex multi-step method for removing mycoplasma from influenza vaccine virus fluid, which includes the following steps:
[0048] Normal virus passage: serum-free suspension MDCK was cultured in shake flasks and infected with Influenza virus infectious B / Phuket / 3073 / 2013, By type virus at an MOI of 0.0001. The infection parameters were a cell density of 4.0*10 6 cells / ml, cell survival rate 98.9%, TPCK trypsin concentration 2.0μg / ml, pH 7.4, shaking flask rotation number 90 rpm, culture temperature 34°C, and harvest time 48 hours.
[0049] The vaccine virus harvest liquid contaminated with mycoplasma is centrifuged at 10,000-15,000 rpm in a high-speed centrifuge to precipitate some of the mycoplasma and harvest the virus supernatant.
[0050] The supernatant of the vaccine virus seed centrifugation is inoculated into MDCK monolayer cells, adsorbed at 37°C for 1-2 hours, rinsed 3-5 times with 0.01M PBS or CD MDCK Production Medium (serum-free) cell culture medium, replaced with cholesterol-free CD MDCK Production Medium (serum-free) maintenance medium, TPCK trypsin concentration of 2μg / ml, maintenance medium pH of 6.9-7.0, and cultured at 34°C for 48-72 hours to harvest the vaccine virus seed liquid.
[0051] Centrifuge again The vaccine virus harvest fluid was centrifuged at 10,000-15,000 rpm in a high-speed centrifuge to precipitate some of the mycoplasmas and harvest the virus supernatant.
[0052] Second cell culture The supernatant of the vaccine virus seed centrifugation was inoculated into MDCK monolayer cells, adsorbed at 37°C for 1-2 hours, rinsed 3-5 times with 0.01M PBS or cholesterol-free CD MDCK Production Medium (serum-free) cell culture medium, replaced with cholesterol-free CD MDCK Production Medium (serum-free) maintenance medium, the TPCK trypsin concentration was 2 μg / ml, the pH of the maintenance medium was 6.9-7.0, and the vaccine virus seed liquid was harvested after culturing at 34°C for 48-72 hours. Detection indicators at each step step Mycoplasma removal rate% Hemagglutination titer Centrifugation 85.9 1024 Cell culture 96.7 2048 Centrifuge again 99.9 1024 Second cell culture 100 2048
[0053] Embodiment 5
[0054] This embodiment provides a complex multi-step method for removing mycoplasma from influenza vaccine virus fluid, which includes the following steps: Normal virus passage: MDCK cells were cultured in shake flasks and infected with InfluenzaVirus Infectious NIBRG-14 (H5N1) virus at an MOI of 0.001 and an MDCK cell density of 3.0*10 6 cells / ml, TPCK trypsin concentration was 2.0μg / ml, pH was 7.4, culture temperature was 34℃, and harvest time was 72 hours.
[0055] The vaccine virus harvest liquid contaminated with mycoplasma is centrifuged at 10,000-15,000 rpm in a high-speed centrifuge to precipitate some of the mycoplasma and harvest the virus supernatant.
[0056] The supernatant of the vaccine virus seed centrifugation was inoculated into MDCK monolayer cells, adsorbed at 37°C for 1-2 hours, rinsed 3-5 times with 0.01M PBS or CD MDCK Production Medium (serum-free) cell culture medium, replaced with cholesterol-free CD MDCK Production Medium (serum-free) maintenance medium, TPCK trypsin concentration of 2μg / ml, maintenance medium pH of 6.9-7.0, and cultured at 34°C for 72 hours to harvest the vaccine virus seed liquid.
[0057] Centrifuge again The vaccine virus harvest fluid was centrifuged at 10,000-15,000 rpm in a high-speed centrifuge to precipitate some of the mycoplasmas and harvest the virus supernatant.
[0058] Second cell culture The supernatant of the vaccine virus seed centrifugation was inoculated into Vero monolayer cells, adsorbed at 37°C for 1-2 hours, rinsed 3-5 times with 0.01M PBS or cholesterol-free CD MDCK Production Medium (serum-free) cell culture medium, replaced with cholesterol-free CD MDCK Production Medium (serum-free) maintenance medium, the TPCK trypsin concentration was 5 μg / ml, the pH of the maintenance medium was 6.9-7.0, and the vaccine virus seed liquid was harvested after culturing at 34°C for 72 hours. Detection indicators at each step step Mycoplasma removal rate% Hemagglutination titer Centrifugation 85.1 256 Cell culture 96.8 512 Centrifuge again 99.9 256 Second cell culture 100 512
Claims
1. A serum-free suspension MDCK cell culture influenza vaccine virus contamination mycoplasma removal process The method comprises the following steps: Centrifugation: Influenza vaccine strains contaminated with mycoplasma are centrifuged at 10,000-15,000 rpm in a high-speed centrifuge to precipitate some of the mycoplasma and harvest the vaccine virus culture supernatant. The supernatant of influenza vaccine virus centrifugation is inoculated into MDCK monolayer cells, adsorbed at 37°C for 1-2 hours, rinsed 3-5 times with buffer or cholesterol-free and serum-free cell culture medium, replaced with cholesterol-free and serum-free maintenance medium with a pH of 6.9-7.0, and cultured at 34°C for 48-72 hours to harvest the vaccine virus seed liquid. Centrifuge again, centrifuge the influenza vaccine virus harvest liquid at 10,000-15,000 rpm in a high-speed centrifuge to precipitate some mycoplasmas and harvest the virus supernatant. After cell culture again, the supernatant of the vaccine virus seed centrifuged was inoculated into MDCK monolayer cells, adsorbed at 37°C for 1-2 hours, rinsed 3-5 times with buffer or cholesterol-free and serum-free cell culture medium, replaced with cholesterol-free serum-free maintenance medium with a pH of 6.9-7.0, and cultured at 34°C for 48-72 hours to harvest the vaccine virus seed liquid.
2. A method for removing mycoplasma contamination from influenza vaccine strains according to claim 1, characterized in that: The viral antigen is any one of the following: H1N1 influenza virus, H3N2 influenza virus, By influenza B virus, Bv influenza B virus, H5N1 influenza virus, etc.
3. A method for removing mycoplasma contamination from influenza vaccine strains according to claim 1, wherein the mycoplasma is removed by centrifugation, and the centrifugal force is preferably 10,000-15,000 rpm.
4. A method for removing mycoplasma contaminated by influenza vaccine virus strains according to claim 1, wherein the mycoplasma is removed by electrostatic and specific adsorption of the virus to the cell receptor at 37°C for 1-2 hours, and then washed with a buffer or a cholesterol-free and serum-free cell culture medium.
5. A method for removing mycoplasma contamination from influenza vaccine strains according to claim 1, wherein: The purpose of inactivating mycoplasma is achieved by adjusting the pH of the cell virus culture maintenance medium, preferably pH 6.9-7.0, to a slightly acidic environment.
6. A method for removing mycoplasma contamination from influenza vaccine strains according to claim 1, wherein: Choose a cholesterol-free maintenance medium, preferably a cholesterol-free serum-free maintenance medium. Mycoplasma reproduction depends on cholesterol, so the purpose of reducing mycoplasma replication is achieved.
7. A method for removing mycoplasma contamination from influenza vaccine strains according to claim 1, wherein: Repeat the above method once continuously.