High-titer influenza virus seed as well as preparation method and application thereof

Abstract

The invention relates to a high-titer influenza virus seed as well as a preparation method and application thereof, and the preparation method comprises the following steps: S1, obtaining a virus, inoculating the virus into a cell factory, and culturing to obtain a cell supernatant; and S2, carrying out microfiltration on the cell supernatant to remove cell debris in the cell supernatant, carrying out ultrafiltration concentration, and carrying out degerming filtration to obtain the high-titer influenza virus seed. According to the technical scheme, the cell factory is adopted for culturing the virus, the virus is inoculated to the ten-layer cell factory, after culturing, the cell supernatant in the cell factory is harvested, microfiltration is carried out, and the cell debris is removed, so that compared with a conventional method for removing the cell debris through centrifugation, microfiltration needs shorter time, the volume of the supernatant which can be treated at a time is larger, and the cell debris can be removed at a time. The sterile risk is lower; and concentrating the supernate, and sterilizing and filtering after concentration. The virus titer of the virus seed subjected to the preparation process can be increased by 5-10 times, and the impurity content is lower.

Description

Technical Field

[0001] The present invention relates to the field of virus technology, and in particular to a high-titer influenza virus strain, a preparation method and an application thereof. Background Art

[0002] The production process for inactivated influenza virus vaccines based on MDCK cells typically involves culturing MDCK cells in a bioreactor, inoculating them with a working seed batch of influenza virus. The virus is then cultured and harvested, and the harvested fluid is purified and inactivated before production. The virus seed used in influenza vaccines is prepared and distributed by WHO collaborating laboratories, with strains updated each epidemic season. After obtaining a recommended vaccine strain, vaccine companies should quickly prepare a working seed batch and produce influenza vaccine, striving to bring the influenza vaccine to market as soon as possible to meet market demand for the new season's influenza vaccine. Therefore, companies should minimize the time required from obtaining a recommended vaccine strain to producing a high-titer working seed batch. Currently, conventional seed preparation methods involve inoculating MDCK cells with the virus seed at varying MOIs, harvesting the cell supernatant, removing the cell pellet by centrifugation, and testing the viral titer. The seed with the highest titer is then aliquoted and packaged as the vaccine seed batch. Frequent strain changes and the poor proliferation of some strains on MDCK cells can limit virus yields and result in low titers in the prepared seed.

[0003] Influenza vaccine viruses are usually inoculated into the reactor at a certain multiplicity of infection (MOI). The MOI calculation formula is: MOI = virus titer (TCID 50 / mL) × virus volume (mL) ÷ number of cells. Currently, with the expansion of the production scale of cell-based influenza vaccines, the culture volume of bioreactors continues to increase, and therefore the amount of virus seeds used in vaccine production also increases. The lower the virus titer of the virus seed, the larger the volume of the virus seed required for vaccine production, sometimes even greater than 3L / batch. Since the virus seeds need to be stored in ultra-low temperature refrigerators at -60°C or below, a large amount of virus seeds will take up limited storage space. If the volume of the virus seeds is too large, more ultra-low temperature refrigerators will be needed to preserve the virus seeds. In addition, the output of a batch of virus seeds is limited. Considering the current use of virus seeds, it is easy for a batch of virus seeds to be unable to support the production of vaccines for an entire epidemic season. If a batch of virus seeds is used up, a new batch of virus seeds will need to be used.

[0004] After a batch of virus strains is prepared and passes comprehensive testing, they should be securely labeled with the strain number, name, generation, batch number, and preparation date, and managed uniformly by the company. Changing the strain batch constitutes a major change, requiring comprehensive analysis of the new strain to demonstrate that its quality attributes are identical to those of the previous batch. Therefore, changing strains should be avoided as much as possible during vaccine production, as this requires extensive validation work, increasing production costs and time pressures for the company. Summary of the Invention

[0005] Based on the above description, the present invention provides a high-titer influenza virus strain and its preparation method and application to solve the technical problem of how to increase the virus titer.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: The present invention provides a method for preparing a high-titer influenza virus seed, comprising the following steps: S1. Obtain the virus, inoculate the virus into the cell factory, culture it, and obtain the cell supernatant; S2. The cell supernatant is microfiltered to remove cell debris in the cell supernatant, concentrated by ultrafiltration, and sterilized and filtered to obtain a high-titer influenza virus seed.

[0007] Furthermore, in step S1: In the step of obtaining the virus, the target influenza virus is diluted with a virus maintenance solution; The virus maintenance solution includes VP culture medium, DMEM culture medium and recombinant trypsin, the volume ratio of the VP culture medium to the DMEM culture medium is 1: (1-2), and the concentration of the recombinant trypsin is 2-5 μg / mL.

[0008] Furthermore, in step S1: The cell factory has ten layers and includes MDCK cells. In each cell factory, the inoculation amount of the MDCK cells is 5×10 8 ~1×10 9 indivual; The MOI value of virus inoculation was 0.001~0.1.

[0009] Furthermore, in step S1: the culture temperature is 33-35°C, and the culture time is 60-108 h.

[0010] Furthermore, in step S2: In the microfiltration step, a 0.45 μm microfilter was used for microfiltration; The pressure of microfiltration is not higher than 0.1 MPa.

[0011] Furthermore, in step S2: In the ultrafiltration and concentration step, a 300KD membrane package ultrafiltration system is used for ultrafiltration and concentration; During ultrafiltration concentration, the flow rate of the seed solution is 20-50 mL / min, and the ultrafiltration pressure is not higher than 0.3 MPa; Before ultrafiltration and concentration, the volume of the toxin seed solution of each cell factory is 1500 mL. After ultrafiltration and concentration, the volume of the toxin seed solution of each cell factory is not higher than 500 mL.

[0012] Furthermore, in step S2: In the sterilization filtration step, filtration was performed using a 0.45 μm + 0.2 μm filter; The pressure of sterile filtration shall not exceed 0.1 MPa.

[0013] The present invention also provides a high-titer influenza virus seed, which is prepared according to the above-mentioned method for preparing the high-titer influenza vaccine virus seed.

[0014] The present invention also provides a use of the aforementioned high-titer influenza virus strain in the preparation of influenza vaccines.

[0015] Compared with the prior art, the technical solution of this application has the following beneficial technical effects: 1. In this application, a highly efficient cell factory system is used for virus culture. This design greatly increases the culture capacity per unit area, enabling higher virus yields within the same laboratory footprint. Subsequently, microfiltration is used to separate cell debris. Compared with traditional centrifugation, the use of microfiltration technology to remove cells can process large amounts of supernatant in a shorter time, has good adaptability to samples of different volumes, and has lower sterility risks. 2. In this application, the supernatant from the microfiltration process is concentrated using a membrane ultrafiltration system with a specific molecular weight cutoff to increase the concentration of viral particles. This concentration process helps to increase the viral titer in the final product while reducing the proportion of non-target components, thereby improving the purity and potency of the product; 3. In this application, the concentrated virus solution is finally sterilized and filtered to ensure the sterility of the final product, which can effectively remove bacteria, fungi and other microbial contaminants, ensuring the safety and quality stability of the biological product; 4. After the entire preparation process is completed, the titer of the virus seed can increase 5 to 10 times compared to the initial state, thereby significantly improving the production efficiency of the virus; and because the impurities are separated to varying degrees in each step, the final virus product has a lower impurity content and is purer; this method provides reliable technical support for the efficient, large-scale production of high-quality viral vaccines or other virus-based therapeutic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A schematic flow chart of a method for preparing high-titer influenza virus strains provided in an embodiment of the present invention; Figure 2 This is a schematic diagram of the process for preparing high-titer influenza virus strains provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0017] To facilitate understanding of the present application, the present application will be described more fully below with reference to the accompanying drawings. The accompanying drawings provide embodiments of the present application. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to make the disclosure of the present application more thorough and comprehensive.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0019] As cell-based influenza vaccine production scales up, bioreactor culture volumes continue to increase, leading to a corresponding increase in the amount of virus seed used in vaccine production. The lower the seed's viral titer, the larger the volume required for vaccine production, sometimes exceeding 3L per batch. Because virus seed must be stored in ultra-low-temperature freezers at -60°C or below, large quantities of virus seed occupy limited storage space. Excessively large seed volumes require additional ultra-low-temperature freezers to preserve the seed. Furthermore, the production volume of a single batch of virus seed is limited. Given the current volume of virus seed used, it's easy for a batch to be insufficient to support vaccine production for an entire epidemic season. Once a batch of virus seed is used up, a new batch will be needed.

[0020] After a batch of virus strains is prepared and passes comprehensive testing, they should be securely labeled with the strain number, name, generation, batch number, and preparation date, and managed uniformly by the company. Changing the strain batch constitutes a major change, requiring comprehensive analysis of the new strain to demonstrate that its quality attributes are identical to those of the previous batch. Therefore, changing strains should be avoided as much as possible during vaccine production, as this requires extensive validation work, increasing production costs and time pressures for the company.

[0021] In view of this, the present invention provides a method for preparing high-titer influenza virus strains, comprising the following steps: S1. Obtain the virus, inoculate the virus into the cell factory, culture it, and obtain the cell supernatant; S2. The cell supernatant is microfiltered to remove cell debris in the cell supernatant, concentrated by ultrafiltration, and sterilized and filtered to obtain a high-titer influenza virus seed.

[0022] In the technical scheme of the present application, the virus is cultured by using a cell factory, the virus is inoculated into a ten-layer cell factory, after culture, the cell supernatant in the cell factory is harvested, microfiltration is carried out to remove cell debris, compared with the conventional centrifugal removal of cell debris, the microfiltration requires a shorter time, and a larger volume of supernatant can be treated at one time; the supernatant is then concentrated, and after concentration, sterilization filtration is carried out. Through the above preparation process, the titer of the virus seed can be increased by 5-10 times, and the impurity content is lower.

[0023] Further, in step S1: In the step of obtaining the virus, the target influenza virus is diluted with a virus maintenance solution; The virus maintenance solution comprises a VP culture medium, a DMEM culture medium and recombinant trypsin, the volume ratio of the VP culture medium to the DMEM culture medium is 1: (1-2), and the concentration of the recombinant trypsin is 2-5 μg / mL.

[0024] In the technical scheme of the present application, the VP culture medium and the DMEM culture medium are combined, which can provide sufficient nutrient sources, suitable osmotic pressure and buffer environment for the growth and development of the virus in the cells; by adding recombinant trypsin, an appropriate concentration of recombinant trypsin can effectively promote the lysis and activation of influenza virus hemagglutinin (HA), thereby enhancing the infection ability of the virus, which is beneficial to the efficiency and consistency of subsequent virus infection, amplification or vaccine preparation and other operations.

[0025] Further, in step S1: The number of layers of the cell factory is ten, and the cell factory comprises MDCK cells, in each cell factory, the inoculation amount of the MDCK cells is 5×10 8 ~1×10 9 cells; The MOI value of virus inoculation is 0.001-0.1.

[0026] In the technical scheme of the present application, by controlling the inoculation amount of the MDCK cells in the cell factory, it is ensured that the cells form a uniform and dense monolayer on the culture surface, which provides sufficient target cell resources for subsequent efficient virus infection; at the same time, it avoids the nutrition competition and growth inhibition caused by too high inoculation amount, thereby maintaining the good physiological state of the cells; by controlling the MOI value of virus inoculation to be 0.01-0.1, the cytotoxicity and early lysis caused by high MOI can be avoided under the premise of ensuring the virus infection efficiency, which is beneficial to the stable amplification and high-titer harvesting of the virus.

[0027] Further, in step S1, the culture temperature is 33-35℃, and the culture time is 60-108 h. Preferably, the culture temperature is 34℃, and the culture time is 72 h.

[0028] Further, in step S2: In the microfiltration step, a 0.45 μm microfilter was used for microfiltration; The pressure of microfiltration is not higher than 0.1 MPa.

[0029] In the technical solution of the present invention, the use of a microfiltration membrane with a pore size of 0.45 μm can achieve a good clarification effect without affecting the permeability of the virus, significantly improving the efficiency and product quality of subsequent purification or concentration steps; at the same time, by controlling the operating pressure below 0.1 MPa, damage to the virus structure or inactivation caused by high pressure can be avoided, further ensuring the stability and biological potency of the virus.

[0030] Furthermore, in step S2: In the ultrafiltration and concentration step, a 300KD membrane package ultrafiltration system is used for ultrafiltration and concentration; During ultrafiltration concentration, the flow rate of the seed solution is 20-50 mL / min, and the ultrafiltration pressure is not higher than 0.3 MPa; Before ultrafiltration and concentration, the volume of the toxin seed solution of each cell factory is 1500 mL. After ultrafiltration and concentration, the volume of the toxin seed solution of each cell factory is not higher than 500 mL.

[0031] In the technical solution of the present invention, by using a 300KD membrane package ultrafiltration system for ultrafiltration concentration, viral particles can be effectively intercepted, while small molecular impurities, culture medium residues and some soluble interfering substances are removed, thereby achieving efficient concentration and preliminary purification of the viral liquid; by adjusting the speed and pressure of ultrafiltration concentration, the filtration flux and concentration efficiency are improved while avoiding the destruction of the virus structure or loss of activity due to high pressure, thereby effectively maintaining the virus's infectivity and stability.

[0032] Furthermore, in step S2: In the sterilization filtration step, filtration was performed using a 0.45 μm + 0.2 μm filter; The pressure of sterile filtration shall not exceed 0.1 MPa.

[0033] In the technical scheme of the present application, a series multi-stage filtering mode is adopted, and the virus liquid is subjected to sterilization treatment by sequentially passing through 0.45 μm and 0.2 μm pore size filters, the filtering combination can effectively remove bacteria, fungi and other microbial contaminants in the process liquid, and ensure that the sterility of the final virus product meets the quality and safety requirements of biological products; wherein the 0.45 μm filter is mainly used for preliminary removal of larger particles and part of microorganisms, and plays a pre-filtering role; then the 0.2 μm filter is used for fine filtering, and reliable sterilization filtering is realized, and the clarity and microbial safety of the virus liquid are significantly improved. At the same time, the operating pressure of the whole sterilization filtering process is controlled in the range of not higher than 0.1 MPa, avoiding the shear force damage to the virus structure caused by high pressure, so as to effectively maintain the integrity and infectivity of the virus.

[0034] The present application also provides a high-titer influenza virus seed prepared by the method for preparing high-titer influenza vaccine virus seed.

[0035] The present application also provides the use of the high-titer influenza virus seed in the preparation of an influenza vaccine.

[0036] The technical scheme of the present application will be further described in detail in combination with specific examples, and it should be understood that the following examples are only used to explain the present application, and are not used to limit the present application.

[0037] Example 1 The present application provides a H5N1 type influenza virus seed, which is prepared by the method as follows: Figure 2 1. Take 5 ten-layer cell factories, inoculate 5 10^8 MDCK cells in each cell factory, and place them in a 37℃ carbon dioxide incubator for 24h; 2. When the cell confluence reaches 90%, discard the cell culture liquid, rinse with PBS for 2 times, and add 1.5L virus maintenance liquid (VP:DMEM 1:2, containing 2 μg / ml recombinant trypsin); 3. Dilute the H5N1 pandemic influenza master seed batch virus (H5N1 (A / Vietnam / 1194 / 2004 - NIBRG-14), the original strain is purchased from the National Institute for Biological Standards and Control (NIBSC), and then the master seed batch is established on the MDCK cell substrate) with the virus maintenance liquid (VP:DMEM 1:2, containing 2 μg / ml recombinant trypsin), inoculate the diluted virus liquid into the cell factory, and the inoculation MOI is 0.1; 4. Mix the cell factories, and place them in a 33℃ carbon dioxide incubator for 72h; 5. Collect the virus liquid, and centrifuge at 3000rpm for 10min to remove the cell debris; 6. The virus liquid is subjected to sterilization treatment by sequentially passing through 0.45 μm and 0.2 μm pore size filters, and the filtering combination can effectively remove bacteria, fungi and other microbial contaminants in the process liquid, and ensure that the sterility of the final virus product meets the quality and safety requirements of biological products; wherein the 0.45 μm filter is mainly used for preliminary removal of larger particles and part of microorganisms, and plays a pre-filtering role; then the 0.2 μm filter is used for fine filtering, and reliable sterilization filtering is realized, and the clarity and microbial safety of the virus liquid are significantly improved. At the same time, the operating pressure of the whole sterilization filtering process is controlled in the range of not higher than 0.1 MPa, avoiding the shear force damage to the virus structure caused by high pressure, so as to effectively maintain the integrity and infectivity of the virus.5. After the culture is completed, the cell supernatant is collected in a pre-microfiltration glass bottle connected to a 0.45 μm microfiltration filter. The pre-microfiltration glass bottle is connected to a positive pressure air connector, and the liquid outlet is connected to the liquid inlet of the post-microfiltration glass bottle. The air pressure is adjusted to 0.01 MPa and microfiltration is started. 6. After microfiltration is completed, rinse the 300KD membrane ultrafiltration system with PBS solution until the pH value of the permeate is neutral as detected by pH test paper; 7. After the ultrafiltration system is flushed, ultrafiltration concentration is started. The flow rate of the seed solution is controlled at 20 mL / min. The pressure valves at the inlet and reflux ends are adjusted to make the transmembrane pressure 0.1 MPa. 8. When the volume of the concentrated virus seed reaches 1000 ml, stop the peristaltic pump and transfer the concentrated virus seed solution into the collection bottle; 9. Rinse the membrane with 0.5L virus maintenance solution for 4 minutes and transfer the washing solution into the collection bottle; 10. Rinse the membrane package with 0.5L virus maintenance solution for 4 minutes and transfer the membrane washing solution into the collection bottle. 11. Connect the collection bottle to the 0.45μm+0.2μm sterilizing filter, connect the liquid inlet end of the collection bottle to the positive pressure air connector, open the hemostatic forceps, adjust the air pressure to 0.01Mpa, and start sterilization filtration.

[0038] 12. After sterilization and filtration, a total of 1.9 L of H5N1 influenza virus seed for production was obtained, and the virus titer was 1 g 8.1 TCID 50 / ml, hemagglutination titer was 1:1024, and total protein content was 198μg / mL.

[0039] The supernatant of the H5N1 cell factory was tested and the virus titer was Lg7.4TCID 50 / ml, and the hemagglutination titer was 1:256. After microfiltration and ultrafiltration of the virus seed, the virus titer increased by 5 times, the hemagglutination titer increased by 4 times, and the virus volume was reduced from 7.5L to 1.9L, which can greatly increase the virus seed titer and reduce the space required for storage.

[0040] Example 2 This embodiment provides an H1N1 influenza virus seed, and the preparation method thereof is as follows: 1. Take 10 ten-layer cell factories and inoculate 8 MDCK cells in each cell factory. 10^8 MDCK cells were then cultured in a 37°C CO2 incubator for 36 h; 2. When the cell confluence reaches 90%, discard the cell culture medium, rinse twice with PBS, and add 1.5L of virus maintenance medium (VP:DMEM 1:2, containing 4μg / ml recombinant trypsin); 3. Dilute the H1N1 influenza master seed batch virus (H1N1 (A / Victoria / 4897 / 2022 IVR-238), the original strain was purchased from the China Influenza Center and then established as a master seed batch on MDCK cell matrix) in virus maintenance medium (VP:DMEM 1:2, containing 4 μg / ml recombinant trypsin) and inoculate the diluted virus solution into the cell factory at an MOI of 0.01. 4. After mixing the cell factory, place it in a 34°C CO2 incubator and culture for 80 hours; 5. After the culture is completed, the cell supernatant is collected in a pre-microfiltration glass bottle connected to a 0.45 μm microfiltration filter. The pre-microfiltration glass bottle is connected to a positive pressure air connector, and the liquid outlet is connected to the liquid inlet of the post-microfiltration glass bottle. The air pressure is adjusted to 0.02 MPa and microfiltration is started. 6. After microfiltration is completed, rinse the 300KD membrane ultrafiltration system with PBS solution until the pH value of the permeate is neutral when detected by pH test paper; 7. After the ultrafiltration system is flushed, ultrafiltration concentration is started. The flow rate of the seed solution is controlled at 35 mL / min. The pressure valves at the inlet and reflux ends are adjusted to make the transmembrane pressure 0.2 MPa. 8. When the volume of the concentrated virus seed reaches 800 ml, stop the peristaltic pump and transfer the concentrated virus seed solution into the collection bottle; 9. Rinse the membrane package with 1L virus maintenance solution for 5 minutes and transfer the washing solution into the collection bottle; 10. Rinse the membrane package with 1L virus maintenance solution for 5 minutes and transfer the washing solution into the collection bottle. 11. Connect the collection bottle to the 0.45μm+0.2μm sterilizing filter, connect the liquid inlet end of the collection bottle to the positive pressure air connector, open the hemostatic forceps, adjust the air pressure to 0.02Mpa, and start sterilization filtration.

[0041] 12. After sterilization and filtration, a total of 2.7 L of H1N1 influenza virus seed for production was obtained. The virus titer was 1 g8.3 TCID50 / ml, the hemagglutination titer was 1:1024, and the total protein content was 227 μg / mL.

[0042] The supernatant of the H1N1 cell factory was tested, and the virus titer was Lg7.5TCID50 / ml, and the hemagglutination titer was 1:256. After microfiltration and ultrafiltration of the virus seed, the virus titer increased by 6.3 times, the hemagglutination titer increased by 4 times, and the virus volume was reduced from 15L to 2.7L, which can greatly increase the virus seed titer and reduce the space required for storage.

[0043] Example 3 This embodiment provides a BV influenza virus seed, the preparation method of which is as follows: 1. Take 15 ten-layer cell factories and inoculate MDCK cells in each cell factory. 10^9 MDCK cells were then cultured in a 37°C CO2 incubator for 48 h; 2. When the cell confluence reaches 90%, discard the cell culture medium, rinse twice with PBS, and add 1.5L of virus maintenance medium (VP:DMEM 1:2, containing 5μg / ml recombinant trypsin); 3. Dilute the influenza BV master seed batch virus (the original BV (B / Austria / 1359417 / 2021 BVR-26) strain was purchased from the Victorian Infectious Diseases Reference Laboratory (VIDRL) ​​and then established as a master seed batch on MDCK cell substrate) with virus maintenance medium (VP:DMEM 1:2, containing 5 μg / ml recombinant trypsin) and inoculate the diluted virus solution into the cell factory at an MOI of 0.001; 4. After mixing the cell factory, place it in a 34°C CO2 incubator and culture for 96 hours; 5. After the culture is completed, the cell supernatant is collected in a pre-microfiltration glass bottle connected to a 0.45 μm microfiltration filter. The pre-microfiltration glass bottle is connected to a positive pressure air connector, and the liquid outlet is connected to the liquid inlet of the post-microfiltration glass bottle. The air pressure is adjusted to 0.03 MPa and microfiltration is started. 6. After microfiltration is completed, rinse the 300KD membrane ultrafiltration system with PBS solution until the pH value of the permeate is neutral when detected by pH test paper; 7. After the ultrafiltration system is flushed, ultrafiltration concentration is started. The flow rate of the seed solution is controlled at 50 mL / min. The pressure valves at the inlet and reflux ends are adjusted to make the transmembrane pressure 0.25 MPa. 8. When the volume of the concentrated virus seed reaches 500 ml, stop the peristaltic pump and transfer the virus seed concentrate into the collection bottle; 9. Rinse the membrane package with 2L virus maintenance solution for 6 minutes and transfer the washing solution into the collection bottle; 10. Rinse the membrane package with 2L virus maintenance solution for 6 minutes and transfer the membrane washing solution into the collection bottle. 11. Connect the collection bottle to the 0.45μm+0.2μm sterilizing filter, connect the liquid inlet end of the collection bottle to the positive pressure air connector, open the hemostatic forceps, adjust the air pressure to 0.03Mpa, and start sterilization filtration.

[0044] 12. After sterilization and filtration, a total of 4.5 L of BV influenza virus seed for production was obtained. The virus titer was 1 g9.3 TCID50 / ml, the hemagglutination titer was 1:2048, and the total protein content was 205 μg / mL.

[0045] The supernatant of the BV cell factory was tested, and the virus titer was Lg8.6TCID50 / ml, and the hemagglutination titer was 1:512. After microfiltration and ultrafiltration of the virus seed, the virus titer increased by 5 times, the hemagglutination titer increased by 4 times, and the virus volume was reduced from 22.5L to 4.5L, which can greatly increase the virus seed titer and reduce the space required for storage.

[0046] Comparative Example 1 This embodiment provides an H5N1 influenza virus seed, and the preparation method thereof is as follows: 1. Take 40 T225 cell culture flasks and inoculate 1 MDCK cell per flask. 10^7 MDCK cells were then cultured in a 37°C CO2 incubator for 24 h; 2. When the cell confluence reaches 90%, discard the cell culture medium, rinse twice with PBS, and add 50 mL of virus maintenance medium (VP:DMEM 1:2, containing 2 μg / mL recombinant trypsin); 3. Dilute the H5N1 pandemic influenza master seed batch virus (H5N1 (A / Vietnam / 1194 / 2004 - NIBRG-14), the original strain was purchased from the National Institute for Biological Products Control (NIBSC), UK, and the master seed batch was established on MDCK cell matrix) in virus maintenance medium (VP:DMEM 1:2, containing 2 μg / ml recombinant trypsin) and inoculate the diluted virus solution into the cell factory at an MOI of 0.1. 4. After mixing the cell culture flask, place it in a 33°C CO2 incubator and culture for 72 hours; 5. After the culture is completed, transfer the cell supernatant in the culture flask to a centrifuge cup, place it in a centrifuge, and centrifuge at 2000 rpm / min for 10 minutes; 6. After centrifugation, carefully pour the supernatant from the centrifuge cup into the collection bottle. Finally, a total of 1.8L of H5N1 influenza virus seed for production was obtained, and the virus titer was 1g7.3TCID 50 / ml, hemagglutination titer was 1:256, and total protein content was 382μg / mL.

[0047] In Comparative Example 1, T225 cell culture flasks were used to culture cells and viruses. The operation was cumbersome, requiring more manpower and time, and the virus seed yield was low, with a high risk of sterility. Impurities were subsequently removed only by centrifugation, and the impurity removal effect was poor. Compared with the virus seed after microfiltration and ultrafiltration concentration treatment after culturing in a 10-layer cell factory in Example 1, the virus seed titer obtained in Comparative Example 1 was reduced by 6.3 times, the hemagglutination titer was reduced by 4 times, and the total protein content was increased by 1.9 times. The above comparative results show that the influenza virus seed prepared by this scheme has a higher virus titer, a lower impurity content, and a higher purity.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0049] In summary, the present invention achieves the following technical effects: 1. In this invention, a highly efficient cell factory system is used for virus culture. This design greatly increases the culture capacity per unit area, enabling higher virus yields within the same laboratory footprint. Subsequently, microfiltration is used to separate cell debris. Compared with traditional centrifugation, the use of microfiltration technology to remove cells can process large amounts of supernatant in a shorter time and has good adaptability to samples of different volumes. 2. In this invention, the supernatant from the microfiltration process is concentrated using a membrane ultrafiltration system with a specific molecular weight cutoff to increase the concentration of viral particles. This concentration process helps to increase the viral titer in the final product while reducing the proportion of non-target components, thereby improving the purity and potency of the product. 3. In the present invention, the concentrated virus solution is finally sterilized and filtered to ensure the sterility of the final product, which can effectively remove bacteria, fungi and other microbial contaminants, thereby ensuring the safety and quality stability of the biological product; 4. After the entire preparation process is completed, the titer of the virus seed can increase 5 to 10 times compared to the initial state, thereby significantly improving the production efficiency of the virus; and because the impurities are separated to varying degrees in each step, the final virus product has a lower impurity content and is purer; this method provides reliable technical support for the efficient, large-scale production of high-quality viral vaccines or other virus-based therapeutic products.

Claims

1. A method for preparing high-titer influenza virus strains, characterized in that: The following steps are involved: S1. Obtain the virus, inoculate the virus into the cell factory, culture it, and obtain the cell supernatant; S2. The cell supernatant is microfiltered to remove cell debris in the cell supernatant, concentrated by ultrafiltration, and sterilized and filtered to obtain a high-titer influenza virus seed.

2. The method for preparing high-titer influenza vaccine virus according to claim 1, characterized in that: In step S1: In the step of obtaining the virus, the target influenza virus is diluted with a virus maintenance solution; The virus maintenance solution includes VP culture medium, DMEM culture medium and recombinant trypsin, the volume ratio of the VP culture medium to the DMEM culture medium is 1: (1-2), and the concentration of the recombinant trypsin is 2-5 μg / mL.

3. The method for preparing high-titer influenza vaccine virus according to claim 1, characterized in that: In step S1: The cell factory has ten layers and includes MDCK cells. In each cell factory, the inoculation amount of the MDCK cells is 5×10 8 ~1×10 9 indivual; The MOI value of virus inoculation was 0.001~0.

1.

4. The method for preparing high-titer influenza vaccine virus according to claim 1, characterized in that: In step S1, the culture temperature is 33-35°C, and the culture time is 60-108 h.

5. The method for preparing high-titer influenza vaccine virus according to claim 1, characterized in that: In step S2: In the microfiltration step, a 0.45 μm microfilter was used for microfiltration; The pressure of microfiltration is not higher than 0.1 MPa.

6. The method for preparing high-titer influenza vaccine virus according to claim 1, characterized in that: In step S2: In the ultrafiltration and concentration step, a 300KD membrane package ultrafiltration system is used for ultrafiltration and concentration; During ultrafiltration concentration, the flow rate of the seed solution is 20-50 mL / min, and the ultrafiltration pressure is not higher than 0.3 MPa; Before ultrafiltration and concentration, the volume of the toxin seed solution of each cell factory is 1500 mL. After ultrafiltration and concentration, the volume of the toxin seed solution of each cell factory is not higher than 500 mL.

7. The method for preparing high-titer influenza vaccine virus according to claim 1, characterized in that: In step S2: In the sterilization filtration step, filtration was performed using a 0.45 μm + 0.2 μm filter; The pressure of sterile filtration shall not exceed 0.1 MPa.

8. A high-titer influenza virus strain, characterized in that The high-titer influenza vaccine virus seed is prepared according to the method for preparing the high-titer influenza vaccine virus seed according to any one of claims 1 to 7.

9. Use of the high-titer influenza virus seed according to claim 8 in the preparation of influenza vaccine.

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