Method for purifying influenza virus harvest liquid
By using specific contents of EDTA solution and phosphate buffer in the influenza virus harvest solution for preparation and filtration, the problems of low recovery rate and high complex proteins of influenza virus harvest solution are solved, efficient purification is achieved and the safety and production efficiency of the vaccine are improved.
Patent Information
- Application Number
- CN202510080827.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-20
- Publication Date
- 2025-04-25
AI Technical Summary
The recovery rate of influenza virus harvesting solution in production is low, there are many mixed proteins and are not easy to remove, resulting in an increased risk of side reactions of the vaccine and affecting safety.
The influenza harvest solution was prepared with a specific content of EDTA solution and phosphate buffer. It was crudely filtered by 10μm filter element, clarified and filtered by 1.0±0.5μm filter element, and concentrated 10 times through a 1000KD membrane package to remove small molecule impurities and ovalbumin.
It significantly improves the protein content and hemoglobin titer of influenza virus, meets production requirements, reduces the risk of side reactions, and improves the safety and production efficiency of the vaccine.
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of virus purification, and in particular to a method for purifying influenza virus harvested liquid. Background Art
[0002] Influenza is a common respiratory infectious disease that can cause a high mortality rate, especially the emergence of new strains, which can cause a worldwide influenza pandemic. Influenza vaccines include whole virus vaccines, inactivated vaccines, live attenuated vaccines, split vaccines, subunit vaccines and genetically recombinant vaccines.
[0003] Influenza virus harvest liquid is a liquid containing a large number of influenza virus particles harvested from the allantoic cavity of chicken embryos. The impurities that may be present in the harvest liquid mainly include egg white, eggshell fragments, chicken blood cells, and possible exogenous microorganisms from the eggshell surface and the chicken embryo itself. These impurities may increase the complexity of subsequent inactivation and purification processes during the preparation of influenza viruses. In particular, if there is a lot of microbial contamination, a large amount of bacterial endotoxins may be produced, thereby increasing the risk of adverse reactions to the vaccine and affecting the safety of the vaccine. Therefore, in the vaccine preparation process, the removal and purification steps of these impurities are particularly important.
[0004] At present, different manufacturers use different process technologies in the process of processing influenza virus harvest liquid, including silk cloth filtration, tubular centrifuge separation and clarification (or disc centrifugation), or 20μm-1μm multi-stage filtration, 0.6+0.45μm filter element filtration (or 0.45μm membrane filter), sterilization grade 0.2μm filter element filtration, etc., or several combinations. The above methods 1) have high production costs, especially the disc centrifuge requires millions of production costs. The equipment maintenance cost is high. 2) The yield of antigen components in these methods for processing influenza virus harvest liquid is low, and the results are not ideal. Summary of the invention
[0005] In order to solve the recovery rate of influenza virus harvest liquid in production, the present invention provides a method for preparing and coarse filtering of influenza virus harvest liquid, which solves the problems of low recovery rate in the clarification process of influenza virus harvest liquid, many impurity proteins in the harvest liquid, and difficulty in removing the impurity proteins, and the added trace EDTA solution and pH 6.5 phosphate buffer solution are removed in the next ultrafiltration concentration process. The method is economical and practical, and has good implementation effect and stability.
[0006] To achieve the above object, the present invention provides a method for purifying influenza virus harvest fluid, comprising the following steps:
[0007] (1) Mix the influenza harvest fluid and EDTA solution in a volume ratio of 1 L:80-120 mL; mixing means mixing evenly;
[0008] (2) Adjust the pH to 7.2 ± 0.2 with a phosphate buffer of pH 6.0-6.5;
[0009] (3) Coarse filtration through a 10 μm filter element, and then clarify filtration through a 1.0 ± 0.5 μm filter element;
[0010] (4) The filtrate obtained in step (3) is mixed evenly with a phosphate buffer of pH 7.0-7.4 of 2-2.5 times the volume of the influenza harvest liquid, passed through a 1000KD membrane package, and concentrated 10 times to remove small molecule impurities, ovalbumin, etc. in the harvest liquid. After purification, samples are taken to test the protein content and hemagglutination titer according to the "Chinese Pharmacopoeia" (2020 edition). 10-fold concentration means that the volume is concentrated 10 times.
[0011] Preferably, the pH of the phosphate buffer in step (2) is 6.5. The pH 6.5 phosphate buffer is prepared by adding 32.6 g Na2HPO4 and 37.2 g NaH2PO4·H2O to 800 mL water for injection and stirring to mix well, then diluting to 1000 mL with water for injection and shaking well.
[0012] Preferably, the pH of the phosphate buffer in step (4) is 7.2. The phosphate buffer of pH 7.2 is prepared by adding 27.7 g Na2HPO4 and 7.6 g NaH2PO4·H2O into 800 mL water for injection and stirring to mix well, then diluting to 1000 mL with water for injection and shaking well.
[0013] The EDTA solution is prepared by adding 50-60 g EDTA-3Na and 50-60 g EDTA-4Na into 800 mL water for injection, stirring and mixing evenly, and then making the volume to 1000 mL with water for injection and shaking well.
[0014] Preferably, the EDTA solution is prepared by adding 56.7 g EDTA-3Na and 50.9 g EDTA-4Na into 800 mL water for injection, stirring and mixing evenly, and then making the volume to 1000 mL with water for injection and shaking well.
[0015] The inventors have found that the yield of influenza harvest fluid purification can be effectively improved by preparing influenza harvest fluid with EDTA-3Na and EDTA-4Na solutions with specific contents.
[0016] Further preferably, the EDTA solution is prepared by adding 56.7 g EDTA-3Na, 50.9 g EDTA-4Na and 7.51 g glycine into 800 mL water for injection, stirring and mixing evenly, and then making the volume to 1000 mL with water for injection and shaking well.
[0017] Further preferably, the EDTA solution is prepared by adding 56.7 g EDTA-3Na, 50.9 g EDTA-4Na and 11.71 g betaine into 800 mL water for injection, stirring and mixing the mixture evenly, and then making the volume to 1000 mL with water for injection and shaking the mixture evenly.
[0018] Most preferably, the EDTA solution is prepared by adding 56.7 g EDTA-3Na, 50.9 g EDTA-4Na, 3.75 g glycine, and 5.86 g betaine to 800 mL water for injection, stirring and mixing the mixture evenly, and then making the volume to 1000 mL with water for injection and shaking the mixture evenly.
[0019] Beneficial effects of the present invention:
[0020] The present invention provides technical support for influenza virus harvest fluid. The results show that the harvest fluid prepared by the present invention, the influenza virus protein content and hemagglutination titer recovered after clarification and filtration are significantly increased, meeting the production requirements. In addition, the method is easy to scale up and has the characteristics of significantly improving the productivity and economic benefits of influenza virus industrial production. DETAILED DESCRIPTION
[0021] As an example, a method for purifying influenza virus harvest fluid comprises the following steps:
[0022] (1) Mix the influenza harvest fluid and EDTA solution in a volume ratio of 1 L:80-120 mL; mixing means mixing evenly;
[0023] (2) Adjust the pH to 7.2 ± 0.2 with a phosphate buffer of pH 6.0-6.5;
[0024] (3) Coarse filtration through a 10 μm filter element, and then clarify filtration through a 1.0 ± 0.5 μm filter element;
[0025] (4) The filtrate obtained in step (3) is mixed evenly with a phosphate buffer of pH 7.0-7.4 of 2-2.5 times the volume of the influenza harvest liquid, passed through a 1000KD membrane package, and concentrated 10 times to remove small molecule impurities, ovalbumin, etc. in the harvest liquid. After purification, samples are taken to test the protein content and hemagglutination titer according to the "Chinese Pharmacopoeia" (2020 edition). 10-fold concentration means that the volume is concentrated 10 times.
[0026] The EDTA solution is prepared by adding 50-60 g EDTA-3Na and 50-60 g EDTA-4Na into 800 mL water for injection, stirring and mixing evenly, and then making the volume to 1000 mL with water for injection and shaking well.
[0027] Ethylenediaminetetraacetic acid, or EDTA for short, is a metal chelator with poor solubility in water. It is usually used in the form of sodium salt in buffers, which can help remove these metal ions in the buffer and prevent them from interfering with other reagents in the experiment. The inventors found that the yield of influenza harvest fluid purification can be effectively improved by preparing influenza harvest fluid with a specific content of EDTA-3Na and EDTA-4Na solutions.
[0028] As an embodiment, the EDTA solution is prepared by adding 56.7 g EDTA-3Na, 50.9 g EDTA-4Na, 3.75 g glycine, and 5.86 g betaine into 800 mL water for injection, stirring and mixing evenly, and then making the volume to 1000 mL with water for injection and shaking well.
[0029] Glycine is a natural amino acid with good compatibility with cells and biomolecules. It helps stabilize the influenza virus harvest solution. Betaine, an alkaloid, is widely found in animals and plants. Compared with glycine, betaine has a better inhibitory effect on the nonspecific adsorption of influenza virus to solid surfaces and impurities (egg white, eggshell fragments, chicken blood cells, etc.) during the purification process, which helps to reduce the loss of influenza virus and improve the purification yield.
[0030] Example 1, a method for purifying influenza virus harvest fluid, the specific process steps are as follows:
[0031] S1 divides the same batch of harvested fluid into two samples, A and B;
[0032] Sample A was not prepared, but sample B was prepared: 90 mL of EDTA solution was added to 1 L of sample B for preparation; then pH 6.5 phosphate buffer was added to adjust the pH to 7.2; EDTA solution: 56.7 g of EDTA-3Na and 50.9 g of EDTA-4Na were added to 800 mL of water for injection, stirred and mixed evenly, and then the volume was made up to 1000 mL with water for injection and shaken well;
[0033] S2 performs coarse filtration, clarifying filtration, and ultrafiltration concentration on sample A and the prepared sample B respectively;
[0034] Coarse filtration: The prepared harvest liquid is coarsely filtered through a 10μm filter element to remove some macromolecular impurities in the harvest liquid, so as to increase the throughput of the 1.0μm filter element;
[0035] Clarification and filtration: The crude filtrate is clarified and filtered through a 1.0 μm filter element to remove macromolecular impurities in the harvested liquid;
[0036] Ultrafiltration concentration: Add 2L of pH 7.2 phosphate buffer to the clarified filtrate and shake well, dialyze with a 300KD membrane package, and concentrate 10 times to remove small molecular impurities, ovalbumin, etc. in the harvest liquid.
[0037] The concentrated solutions of purified sample A and purified sample B were sampled respectively to detect the protein content and hemagglutination titer.
[0038] The above purification process was repeated 3 times to take the average value. The test results are as follows:
[0039] Table 1 Test results
[0040] serial number Concentrate volume Protein content μg / mL Hemagglutination titer A 1000mL 873 1:8960 B 1000mL 2416 1:25600
[0041] The experimental results show that the protein content and hemagglutination titer of the harvested liquid obtained by the purification process of the present invention are significantly better than those of the harvested liquid that has not been prepared. During the purification process of the influenza virus harvested liquid, the influenza virus is easily adhered to impurities (egg white, eggshell fragments, chicken blood cells, etc.). The presence of ETDA-3Na and EDTA-4Na can avoid the nonspecific adsorption of the virus to the solid surface, which helps to improve the yield of the influenza virus after purification. In addition, the presence of ETDA-3Na and EDTA-4Na in a specific ratio can stabilize the pH value of the solution, which is conducive to providing suitable environmental conditions, thereby maintaining the stability of the virus.
[0042] Therefore, after the harvest liquid is prepared according to the process of the present invention, the purification yield of influenza virus can be effectively improved, and most of the impurities in the harvest liquid can be removed.
[0043] Example 2, a method for purifying influenza virus harvested fluid, the specific process steps are as follows:
[0044] S1: dividing the same batch of harvested liquid into two samples, C and D; the harvested liquid is the same batch of harvested liquid as the harvested liquid in Example 1;
[0045] Samples C and D are prepared:
[0046] Add 90 mL of EDTA solution to 1 L of sample C for preparation; then add pH 6.5 phosphate buffer to adjust the pH to 7.2; EDTA solution: add 56.7 g of EDTA-3Na to 800 mL of water for injection and stir to mix evenly, then dilute to 1000 mL with water for injection and shake well;
[0047] Add 90 mL of EDTA solution to 1 L of sample D for preparation; then add pH 6.5 phosphate buffer to adjust the pH to 7.2; EDTA solution: add 50.9 g of EDTA-4Na to 800 mL of water for injection and stir to mix evenly, then dilute to 1000 mL with water for injection and shake well;
[0048] S2 respectively performs coarse filtration, clarifying filtration, and ultrafiltration concentration on the prepared sample C and the prepared sample D;
[0049] Coarse filtration: The prepared harvest liquid is coarsely filtered through a 10μm filter element to remove some macromolecular impurities in the harvest liquid, so as to increase the throughput of the 1.0μm filter element;
[0050] Clarification and filtration: The crude filtrate is clarified and filtered through a 1.0 μm filter element to remove macromolecular impurities in the harvested liquid;
[0051] Ultrafiltration concentration: Add 2L of pH 7.2 phosphate buffer to the clarified filtrate and shake well, dialyze with a 300KD membrane package, and concentrate 10 times to remove small molecular impurities, ovalbumin, etc. in the harvest liquid.
[0052] The concentrated solutions of purified sample C and purified sample D were sampled respectively to detect the protein content and hemagglutination titer.
[0053] The above purification process was repeated 3 times to take the average value. The test results are as follows:
[0054] Table 2 Test results
[0055] serial number Concentrate volume Protein content μg / mL Hemagglutination titer C 1000mL 1760 1:20640 D 1000mL 1835 1:18820
[0056] The experimental results show that the protein content and hemagglutination titer of the harvest fluid obtained by preparing only one of EDTA-3Na and EDTA-4Na are significantly lower than those of the harvest fluid prepared by mixing the two.
[0057] Example 3, a method for purifying influenza virus harvested fluid, the specific process steps are as follows:
[0058] S1: dividing the same batch of harvested liquid into three samples, E, F, and G; the harvested liquid is the same batch of harvested liquid as the harvested liquid in Example 1;
[0059] Samples E, F, and G are prepared:
[0060] Add 90 mL of EDTA solution to 1 L of sample E for preparation; then add pH 6.5 phosphate buffer to adjust the pH to 7.2; EDTA solution: add 56.7 g of EDTA-3Na, 50.9 g of EDTA-4Na, and 7.51 g (0.1 mol) of glycine to 800 mL of water for injection, stir and mix evenly, then dilute to 1000 mL with water for injection and shake well;
[0061] Add 90 mL of EDTA solution to 1 L of sample F for preparation; then add pH 6.5 phosphate buffer to adjust the pH to 7.2; EDTA solution: add 56.7 g of EDTA-3Na, 50.9 g of EDTA-4Na, and 11.71 g (0.1 mol) of betaine to 800 mL of water for injection, stir and mix evenly, then dilute to 1000 mL with water for injection and shake well;
[0062] Add 90 mL of EDTA solution to 1 L of sample G for preparation; then add pH 6.5 phosphate buffer to adjust the pH to 7.2; EDTA solution: add 56.7 g of EDTA-3Na, 50.9 g of EDTA-4Na, 3.75 g (0.05 mol) of glycine, and 5.86 g (0.05 mol) of betaine to 800 mL of water for injection, stir and mix evenly, then dilute to 1000 mL with water for injection and shake well;
[0063] S2 respectively performs coarse filtration, clarifying filtration, and ultrafiltration concentration on the prepared samples E, F, and G;
[0064] Coarse filtration: The prepared harvest liquid is coarsely filtered through a 10μm filter element to remove some macromolecular impurities in the harvest liquid, so as to increase the throughput of the 1.0μm filter element;
[0065] Clarification and filtration: The crude filtrate is clarified and filtered through a 1.0 μm filter element to remove macromolecular impurities in the harvested liquid;
[0066] Ultrafiltration concentration: Add 2L of pH 7.2 phosphate buffer to the clarified filtrate and shake well, dialyze with a 300KD membrane package, and concentrate 10 times to remove small molecular impurities, ovalbumin, etc. in the harvest liquid.
[0067] The concentrated solutions of purified samples E, F, and G were sampled respectively to detect the protein content and hemagglutination titer.
[0068] The above purification process was repeated 3 times to take the average value. The test results are as follows:
[0069] Table 3 Test results
[0070] serial number Concentrate volume Protein content μg / mL Hemagglutination titer E 1000mL 2820 1:28670 F 1000mL 3180 1:32400 G 1000mL 3650 1:36280
[0071] The experimental results show that the purification yield of influenza virus harvest liquid can be further improved by mixing EDTA-3Na and EDTA-4Na in a specific ratio and then preparing it with glycine and betaine. Glycine and betaine can protect influenza viruses, help maintain the structural integrity of influenza viruses, enhance the stability of influenza virus particles, and help prevent the dissociation or degradation of influenza viruses during the purification process, thereby increasing the protein yield and the number of effective influenza viruses. The introduction of glycine and betaine also helps to reduce the nonspecific adsorption of influenza viruses to solid surfaces such as pipes and utensils during the purification process, maintain the stability of the virus in the liquid phase, and help improve the yield.
[0072] Configuration of the phosphate buffer solution in Example 1-3:
[0073] pH 6.5 phosphate buffer: Add 32.6 g Na2HPO4 and 37.2 g NaH2PO4·H2O into 800 mL water for injection and stir to mix evenly. Then dilute to 1000 mL with water for injection and shake well.
[0074] pH 7.2 phosphate buffer: Add 27.7 g Na2HPO4 and 7.6 g NaH2PO4·H2O into 800 mL water for injection and stir to mix evenly. Then dilute to 1000 mL with water for injection and shake well.
Claims
1. A method for purifying influenza virus harvested fluid, characterized in that: The following steps are involved: (1) The influenza harvest fluid and the EDTA solution are prepared in a volume ratio of 1L:80-120mL; the preparation means mixing evenly; the EDTA solution is 50-60g EDTA-3Na and 50-60g EDTA-4Na added to 800mL water for injection, stirred and mixed evenly, and then the volume is made up to 1000mL with water for injection and shaken to obtain; (2) Adjust the pH to 7.2 ± 0.2 with a phosphate buffer of pH 6.0-6.5; (3) Coarse filtration through a 10 μm filter element, and then clarify filtration through a 1.0 ± 0.5 μm filter element; (4) The filtrate obtained in step (3) was mixed evenly with 2-2.5 times the volume of influenza harvest fluid and pH 7.0-7.4 phosphate buffer, passed through a 1000KD membrane package, and concentrated 10 times.
2. The influenza virus harvest liquid purification method according to claim 1, wherein: The EDTA solution is prepared by adding 56.7 g EDTA-3Na and 50.9 g EDTA-4Na into 800 mL water for injection, stirring and mixing evenly, and then making the volume to 1000 mL with water for injection and shaking well.
3. The influenza virus harvest liquid purification method according to claim 1, wherein: Step (1) can also be prepared by mixing the influenza harvest fluid and the EDTA solution in a volume ratio of 1L:80-120mL; the preparation means mixing evenly; the EDTA solution is 56.7g EDTA-3Na, 50.9g EDTA-4Na, and 7.51g glycine added to 800mL water for injection, stirred and mixed evenly, and then the volume is made up to 1000mL with water for injection and shaken well.
4. The influenza virus harvest liquid purification method according to claim 1, wherein: Step (1) can also be prepared by mixing the influenza harvest fluid and the EDTA solution in a volume ratio of 1L:80-120mL; the preparation means mixing evenly; the EDTA solution is prepared by adding 56.7g EDTA-3Na, 50.9g EDTA-4Na, and 11.71g betaine to 800mL water for injection, stirring and mixing evenly, and then diluting the volume to 1000mL with water for injection and shaking well.
5. The influenza virus harvest liquid purification method according to claim 1, wherein: The pH of the phosphate buffer in step (2) is 6.
5.
6. The method for purifying influenza virus harvested liquid as claimed in claim 5, characterized in that: The pH 6.5 phosphate buffer solution is prepared by adding 32.6 g Na2HPO4 and 37.2 g NaH2PO4·H2O into 800 mL water for injection and stirring to mix evenly. The solution is then diluted to 1000 mL with water for injection and shaken well.
7. The method for purifying influenza virus harvested liquid as claimed in claim 1, characterized in that: The pH of the phosphate buffer in step (4) is 7.
2.
8. The method for purifying influenza virus harvested liquid as claimed in claim 7, wherein: The pH 7.2 phosphate buffer solution is prepared by adding 27.7 g Na2HPO4 and 7.6 g NaH2PO4·H2O into 800 mL water for injection and stirring to mix evenly. The solution is then diluted to 1000 mL with water for injection and shaken well.