Preparation method of group B meningococcus outer membrane vesicle protein
Through OMV dissolution technology without EDTA and multi-step purification method, group B meningococci outer membrane vesicle protein was prepared, solving the problems of immunogenic protein loss and high process cost in traditional methods, and achieving efficient and safe vaccine preparation.
Patent Information
- Application Number
- CN202411738148.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-29
- Publication Date
- 2025-03-11
AI Technical Summary
The traditional method of preparing the outer membrane vesicle protein of group B meningococci uses detergent EDTA, which leads to the loss of immunogenic proteins and high process costs.
Using a unique OMV dissolution technology without EDTA, the supernatant was collected and washed and ultrafiltration was performed using a unique OMV dissolution technology without EDTA, which retained the integrity of the outer membrane vesicle structure.
Effectively dissolution of immunogenic antigen proteins, reduce the dissolution of impurities such as nucleic acids, reduce the pressure of downstream purification process, and improve the immunogenicity and safety of the vaccine.
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of cell biology, and in particular to a method for preparing group B meningococcal outer membrane vesicle protein. Background Art
[0002] Neisseria meningitidis (Nm), commonly known as meningococcus, is a pathogenic Gram-negative diplococcus. The bacteria are kidney-shaped or bean-shaped, with a diameter of 0.6μm to 0.8μm, without spores and flagella.
[0003] The prevalent bacterial groups are mainly A, B, C and W135 groups. The proportion of group A meningococcal cases is generally decreasing, while group B and W135 are on the rise. The meningococcal vaccines available globally mainly include group A, group A+C, and quadrivalent vaccines of groups A, C, W135 and Y. The effective immunogenic components of these vaccines are all capsular polysaccharides of meningococci. However, the capsular polysaccharides of group B meningococci are similar to the polymer structure of human N-acetylneuraminic acid, which may cause severe autoimmune reactions. In addition, the capsular polysaccharides of group B meningococci have low immunogenicity and are difficult to induce effective immune protection in children. Therefore, they are not suitable as candidate vaccine antigens.
[0004] There are two main types of group B meningococcal vaccines on the market. One is the outer membrane vesicle (OMV) vaccine with outer membrane protein (OMP) as the main component; the other is a recombinant protein vaccine based on reverse vaccinology technology.
[0005] OMV is a product secreted from the surface of Gram-negative bacteria. It is a vesicle-like structure formed on the surface of bacteria by budding from the bacterial outer membrane under certain mechanisms. Most outer membrane vesicles are spherical, with a diameter of about 20 to 250 nm. Their components include lipopolysaccharide, outer membrane proteins, phospholipids, DNA, and periplasmic components that are wrapped by the outer membrane during the formation process.
[0006] Traditionally, the detergent EDTA extraction method is used to prepare group B meningococcal outer membrane vesicles, that is, sodium deoxycholate is used for extraction in the presence of ethylenediaminetetraacetic acid (EDTA) to obtain OMV (dOMV). The lipopolysaccharide (LPS) of meningococci is highly toxic, and detergent treatment can reduce the LPS content and thus reduce endotoxin activity. However, there are some defects in the OMV vaccine after detergent treatment. While reducing endotoxin activity, it also removes some potentially effective antigenic proteins with immunogenicity, such as factor H binding protein (fHbp) and genome-derived Neisseria protein (GNA2132). Therefore, people gradually try to use genetic methods to modify strain-related genes to prepare attenuated strains or extract natural OMV (nOMV) only after EDTA treatment (including similar process steps as dOMV but without detergent steps) to prepare vaccines. Compared with dOMV, nOMV has better immunogenicity. For commercial applications, a scalable production and preparation process is needed. In order to improve the yield, an EDTA extraction step is required. However, the cell fusion effect of EDTA leads to the release of nucleic acid substances, which is a problem. Ultracentrifugation can be used to remove nucleic acids, but this method has limited processing capacity and high process costs. Summary of the invention
[0007] The purpose of the present invention is to provide a method for preparing group B meningococcal outer membrane vesicle protein, thereby avoiding the technical problems of immunogenic protein loss and high process cost caused by the use of detergent EDTA in traditional processes.
[0008] To achieve the above object, the present invention provides the following technical solutions:
[0009] The present invention provides a method for preparing a group B meningococcal outer membrane vesicle protein, comprising the following steps:
[0010] (1) Resuscitation of serogroup B meningococcal bacteria;
[0011] (2) transferring the revived bacterial strains to a culture device containing semi-integrated fluid for bacterial strain expansion and culture;
[0012] (3) transferring the amplified bacterial strain to a fermentation tank, starting aeration and stirring for fermentation, and terminating the culture at the end of the bacterial logarithmic growth phase or the beginning of the stationary phase;
[0013] (4) Collection of outer membrane vesicle proteins of group B meningococci
[0014] The bacteria and the extraction buffer were stirred and mixed at a ratio of 1:10-20 g / ml, and oscillated at a constant temperature, and then centrifuged to collect the supernatant. An equal volume of sodium chloride solution was added to the supernatant, and the supernatant was washed and filtered twice;
[0015] (5) Purification of outer membrane vesicle proteins of group B meningococci
[0016] Add polyoxyethylene mono-tert-octylphenyl ether to the supernatant after washing and filtration to a final concentration of 1%, mix well, centrifuge and let stand, absorb the upper liquid, and use a 30KD ultrafiltration membrane bag and 20mM PB as the ultrafiltration solution for three times to replace the liquid;
[0017] The ultrafiltration solution was loaded onto a Sepharose Q FF chromatography column that had been pre-equilibrated with solution A. After loading, solution B was used for elution, and the elution peak was collected;
[0018] The collected solution was loaded onto a hydroxyapatite chromatography column that had been pre-equilibrated with solution B. After loading, solution C was used for elution, and the flow-through peak was collected and stored after sterilization and filtration.
[0019] Furthermore, the resuscitation method in (1) comprises: taking a working seed of group B meningococcus 440910, inoculating it into a cell culture bottle filled with meningococcal semi-comprehensive liquid culture medium, placing it in a carbon dioxide cell culture incubator, and culturing it at 35-38° C. for 18-26 hours.
[0020] Furthermore, the amplification culture conditions in (2) are: a constant temperature shaking incubator, culturing at 35-38° C. for 3-5 hours at a rotation speed of 150-250 rpm.
[0021] Furthermore, the fermentation conditions in (3) are as follows: an aeration rate of 10 to 100 L / min, a stirring speed of 100 to 500 rpm, a temperature of 35 to 38° C., and a culture time of 6 to 8 hours.
[0022] Furthermore, the extraction buffer in (4) is 0.2 mol / L lithium chloride + 0.1 mol / L sodium acetate solution; the constant temperature oscillation conditions are: temperature 45°C to 55°C, speed 150 rpm, oscillation 1 to 2 hours; the centrifugation conditions are speed 11000 rpm, temperature 2 to 8°C, centrifugation for 1 hour; when collecting the supernatant, a 0.45 μm filter is used for clarification filtration.
[0023] Furthermore, the solution A in (5) is a 20 mM PB, pH 7.0 solution; the solution B is a 20 mM PB, 0.45 M NaCl, pH 7.0 solution; and the solution C is a 500 mM PB, 0.45 M NaCl, pH 7.0 solution.
[0024] Furthermore, polyoxyethylene mono-tert-octylphenyl ether is added to the (5) to a final concentration of 1%, and then placed in a 2-8°C cold storage for oscillation and mixing, and then placed for 60 minutes, and then centrifuged at 15000g centrifugal force at room temperature for 15 minutes and allowed to stand.
[0025] Based on the above technical solution, the embodiments of the present invention can at least produce the following technical effects:
[0026] (1) The method for preparing group B meningococcal outer membrane vesicle protein provided by the present invention adopts a unique OMV dissolution technology and does not use EDTA. It can dissolve the effective antigen protein components well, which not only reduces the dissolution of impurity components such as bacterial intracellular nucleic acids and reduces the pressure of downstream purification processes, but also can retain the integrity of the outer membrane vesicle structure and effectively preserve its immunogenicity.
[0027] (2) The method for preparing group B meningococcal outer membrane vesicle protein provided by the present invention does not use the detergent EDTA for extraction, can effectively remove impurities such as nucleic acid and LPS, can reduce fever reaction after vaccination, and improve the safety of the vaccine. DETAILED DESCRIPTION
[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0029] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.
[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.
[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.
[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0033] PB: Sodium phosphate buffer (containing sodium dihydrogen phosphate and disodium hydrogen phosphate)
[0034] Extraction buffer: 0.2mol / L lithium chloride + 0.1mol / L sodium acetate solution
[0035] Triton X-114 Alias: Polyoxyethylene mono-tert-octylphenyl ether, CAS#: 9036-19-5
[0036] The above-mentioned drugs are all commercially available.
[0037] Example 1: Fermentation culture of group B meningococci
[0038] 1. Open a working seed of group B meningococcus 440910 in a biosafety cabinet, inoculate it into a cell culture bottle containing semi-comprehensive liquid medium for meningococcal disease, place it in a carbon dioxide cell culture incubator, and culture it at 35-38°C for 18-26 hours.
[0039] 2. After the recovery and cultivation of the strains, use a sterile pipette to transfer the first-generation bacterial solution to a conical flask containing semi-comprehensive liquid culture medium for meningococcal disease, place it in a constant temperature shaking incubator, and culture it at 35-38°C for 3-5 hours at a speed of 150-250 rpm for strain amplification and cultivation.
[0040] 3. Transfer the amplified strain to the fermentation tank, start ventilation and stirring, the ventilation volume is 10-100L / min, and the stirring speed is 100-500rpm. Control the temperature at 35-38℃, culture for 6-8 hours, monitor the bacterial growth during the process, and terminate the culture at the end of the bacterial logarithmic growth period or the early stage of the stationary period.
[0041] Example 2: Purification of Group B Meningococcal OMV
[0042] 1. Collect the cells by centrifugation, add 10-20 ml of extraction buffer per gram of wet cells, stir at room temperature for 30 minutes to mix. Then place in a constant temperature oscillator, set the temperature to 45℃-55℃, the speed to 150 rpm, and oscillate for 1-2 hours.
[0043] 2. Centrifuge the bacterial suspension at 11000 rpm and 2-8°C for 1 hour, collect the supernatant, and filter it with a 0.45 μm filter. Then add an equal volume of sodium chloride solution to the supernatant and wash it twice.
[0044] 3. Add Triton X-114 to a final concentration of 1%, place in a 2-8℃ cold storage, shake and mix, and place for 60 minutes, then centrifuge at 15000g for 15 minutes at room temperature, let stand overnight, and carefully aspirate the upper liquid. Then use a 30KD ultrafiltration membrane package and ultrafiltration three times with 20mM PB as the ultrafiltration solution to change the liquid.
[0045] 4. Load the ultrafiltration solution onto a Sepharose QFF chromatography column that had been pre-equilibrated with solution A (20 mM PB, pH 7.0 solution). After loading, elute with solution B (20 mM PB, 0.45 M NaCl, pH 7.0 solution) and collect the elution peak.
[0046] 5. Load the mixed solution onto a hydroxyapatite (CHT) chromatography column pre-equilibrated with solution B. After loading, use solution C (500 mM PB, 0.45 M NaCl, pH 7.0 solution) for elution, collect the flow-through peak, sterilize and filter, and store at 2-8°C to obtain the stock solution.
[0047] Example 3: Quality Inspection
[0048] The original solution was quality tested and its nucleic acid and pyrogen were tested in accordance with General Rules 0401 and General Rules 1142 of the "Chinese Pharmacopoeia 2020 Edition". The results showed that the quality met the expected requirements.
[0049] batch number Nucleic acid (≤3.5%) Pyrogen 20230603-3 2.84％ Comply with regulations 20230925-4 2.69％ Comply with regulations 20240403-1 2.45％ Comply with regulations
[0050] Finally, it should be noted that:
[0051] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for preparing group B meningococcal outer membrane vesicle protein, characterized in that: The following steps are involved: (1) Resuscitation of serogroup B meningococcal bacteria; (2) Transfer the revived bacterial strains to a culture device containing semi-integrated fluid for bacterial amplification culture; (3) The amplified bacterial strain is transferred to a fermentation tank, ventilation and stirring are started for fermentation, and the culture is terminated at the end of the bacterial logarithmic growth phase or the beginning of the stationary phase; (4) Collection of outer membrane vesicle proteins of group B meningococci The bacteria and the extraction buffer were stirred and mixed at a ratio of 1:10-20 g / ml, and oscillated at a constant temperature, and then centrifuged to collect the supernatant. An equal volume of sodium chloride solution was added to the supernatant, and the supernatant was washed and filtered twice; (5) Purification of outer membrane vesicle proteins of group B meningococci Add polyoxyethylene mono-tert-octylphenyl ether to the supernatant after washing and filtration to a final concentration of 1%, mix well, centrifuge and let stand, absorb the upper liquid, and use a 30KD ultrafiltration membrane bag and 20mM PB as the ultrafiltration solution for ultrafiltration three times to change the liquid; The ultrafiltration solution was loaded onto a Sepharose Q FF column that had been pre-equilibrated with solution A. After loading, solution B was used for elution, and the elution peak was collected. The collected solution was loaded onto a hydroxyapatite chromatography column that had been pre-equilibrated with solution B. After loading, solution C was used for elution, and the flow-through peak was collected and stored after sterilization and filtration.
2. The method for preparing group B meningococcal outer membrane vesicle protein according to claim 1, characterized in that: The resuscitation method in (1) comprises: taking a working seed of group B meningococcus 440910, inoculating it into a cell culture bottle filled with meningococcal semi-comprehensive liquid culture medium, placing it in a carbon dioxide cell culture incubator, and culturing it at 35-38° C. for 18-26 hours.
3. The method for preparing group B meningococcal outer membrane vesicle protein according to claim 1, characterized in that: The amplification culture conditions in (2) are: a constant temperature shaking incubator, culturing at 35-38° C. for 3-5 hours at a rotation speed of 150-250 rpm.
4. The method for preparing group B meningococcal outer membrane vesicle protein according to claim 1, characterized in that: The fermentation conditions in (3) are as follows: an aeration rate of 10 to 100 L / min, a stirring speed of 100 to 500 rpm, a temperature of 35 to 38° C., and a cultivation time of 6 to 8 hours.
5. The method for preparing group B meningococcal outer membrane vesicle protein according to claim 1, characterized in that: The extraction buffer in (4) is 0.2 mol / L lithium chloride + 0.1 mol / L sodium acetate solution; the constant temperature oscillation conditions are: temperature 45°C to 55°C, speed 150 rpm, oscillation 1 to 2 hours; the centrifugation conditions are speed 11000 rpm, temperature 2 to 8°C, centrifugation for 1 hour; when collecting the supernatant, a 0.45 µm filter is used for clarification filtration.
6. The method for preparing group B meningococcal outer membrane vesicle protein according to claim 1, characterized in that: The solution A in (5) is a 20 mM PB, pH 7.0 solution; the solution B is a 20 mM PB, 0.45 M NaCl, pH 7.0 solution; The C solution is 500 mM Pb, 0.45 M NaCl, pH 7.0 solution.
7. The method for preparing group B meningococcal outer membrane vesicle protein according to claim 1, characterized in that: Polyoxyethylene mono-tert-octylphenyl ether was added to the (5) to a final concentration of 1%, and then placed in a 2-8°C cold storage for oscillation and mixing, and then placed for 60 minutes, and then centrifuged at 15000g centrifugal force at room temperature for 15 minutes and allowed to stand.