CN111281972A - Combined vaccine consisting of ABCYW135 group meningococcus vaccine and encephalitis B vaccine - Google Patents
Combined vaccine consisting of ABCYW135 group meningococcus vaccine and encephalitis B vaccine Download PDFInfo
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Abstract
The invention provides an ABCYW135 group meningococcus/encephalitis B combined vaccine, capsular polysaccharide is extracted from A group C group W135 group Y meningococcus respectively, and after purification, activated capsular polysaccharide is combined with diphtheria CRM197 protein; using PorA protein on outer membrane vesicles of group B meningococcus; vero cells are adopted to culture the Japanese encephalitis virus, and after inactivation and purification, the components are mixed according to a certain proportion for preventing infection caused by ABCYW135 group meningococcus and Japanese encephalitis virus.
Description
Technical Field
The invention relates to a medicine prepared from ABCYW135 meningitis vaccine and encephalitis B vaccine. Specifically, the invention relates to a novel combined vaccine prepared by respectively coupling stock solutions of A, C, W135 and Y group meningococcal polysaccharides and a protein carrier and a B group meningococcal PorA protein and Japanese encephalitis virus purification stock solution. More specifically, the encephalitis B virus is subjected to Vero cell culture, harvesting, inactivation, purification and other steps to obtain a virus stock solution. The A, C, W135 and Y meningococcal combined stock solution is purified A, C, W135 and Y meningococcal polysaccharide and diphtheria toxin protein CRM with non-toxic mutation197The conjugate stock solution is formed by chemical coupling. The B group meningococcus PorA protein stock solution is obtained by fermenting, harvesting, extracting, purifying and the like the B group meningococcus. The stock solutions are combined with a protective agent in proper dosage to prepare a combined vaccine for preventing meningococcus and encephalitis B.
Background
Epidemic meningitis is the only disease causing epidemic among bacterial cerebrospinal meningitis, and causes high mortality and disability rate. Over the past 100 years, epidemic encephalitis pathogens have been distributed worldwide, with perhaps 30-35 million cases appearing worldwide each year, which has been a worldwide public health problem.
Epidemic encephalitis is caused by Neisseria meningitidis (Nm). Based on the differences in chemical and antigenic properties of Nm Capsular Polysaccharides (CPS), 13 serogroups have been identified, a, B, C, D, 29E, H, I, K, L, W135, X, Y and Z13. Of these, group A, B, C, W135, Y Nm cause about 95% of the cases of infection, with group A, group C Neisseria meningitidis being the most contagious and the most common strain causing epidemic encephalomyelitis.
Meningococcal Polysaccharide Vaccine (MPV) is prepared from extracted Meningococcal capsular polysaccharide, the capsular polysaccharide determines the virulence of Meningococcal thallus, mutant Meningococcal without capsular polysaccharide is quickly eliminated by complement system in serum, and has no pathogenicity, and serum antibodies generated by immune system and aiming at capsular polysaccharide are main action substances for resisting Meningococcal. The existing polysaccharide vaccines are developed based on the principle. Capsular polysaccharide is a type of TI antigen, and immune response induced by the capsular polysaccharide does not need participation of T lymphocytes and cannot generate immune memory, especially for infants under two years old. Therefore, the WHO has suggested that the epidemic encephalitis polysaccharide vaccine is not used for routine vaccination of infants under 2 years of age.
Meningococcal polysaccharide conjugate vaccines (MCV) are vaccines in which the capsular polysaccharide of meningococcus is covalently bound to a protein carrier by means of a chemical linker, and are antigen T cell-dependent antigens that induce an immune response involving T lymphocytes that can produce immunological memory. The epidemic cerebrospinal meningitis polysaccharide conjugate vaccine has a wider vaccination range than that of the epidemic cerebrospinal meningitis polysaccharide vaccine, and can be used for routine immunization of infants under 2 years old.
The meningococcal group B capsular polysaccharide has weak immunogenicity due to the fact that the meningococcal group B capsular polysaccharide contains epitopes which are potentially cross-immunized with human antigens, and can induce autoimmune diseases, so that vaccine research using the meningococcal group B capsular polysaccharide as immunogen meets serious challenges. Currently, international research on group B meningococcal vaccines mainly adopts two strategies, namely Outer Membrane Vesicle (OMV) vaccines based on Outer Membrane proteins, i.e. clonal populations directed against certain specific circulating strains; the other is a recombinant protein vaccine based on reverse vaccinology technology. Successful marketing of group B epidemic encephalitis vaccines developed based on OMVs in cuba, new zealand, etc. has demonstrated the effectiveness of such vaccines, and it is worth our attention that the OMV vaccines developed so far have no versatility due to the distinct regional characteristics of the serological types and subtypes of group B epidemic encephalitis in different countries and regions. Therefore, for the research and development of group B epidemic encephalitis OMV vaccines, the selection of strains suitable for the pathogenesis characteristics of local diseases is particularly important. At the present stage, the B-group epidemic cerebrospinal meningitis cases separated in China mostly belong to CC-4821 clone groups, and are represented by P1.7-2,4, P1.22,14, P1.20,23 and other types in a sporadic package, which is different from clone groups and serosubtypes of main epidemic strains in other countries and regions in the world to a certain extent. Therefore, based on the current situation of epidemiology of the epidemic brain in China, the selection of the target strain is particularly important.
Encephalitis b is an acute viral infectious disease of the nervous system, and currently, about 30 million people live in epidemic areas of encephalitis worldwide, with at least 5 million clinical cases reported each year, and the vast majority originating from children. The Virus causing Encephalitis B is epidemic Encephalitis B Virus (Japanese Encephalitis Virus), which is called Encephalitis B Virus for short. Is a positive-sense single-stranded RNA virus, and the virus has an envelope. For the prevention and treatment of the encephalitis B virus, no specific medicine exists at present, and the control from the aspect of transmission through the vaccine is the most mainstream mode adopted at present.
The combined vaccine is prepared by mixing a plurality of living and inactivated organisms or related antigens according to a certain proportion and is used for preventing a plurality of diseases. The goal of combination vaccine development is to reduce the number of vaccine injections while preventing a wider variety of diseases. The significance of the method can not only improve the coverage rate and the inoculation rate of the vaccine, reduce the physiological and psychological pains brought to the inoculated personnel by multiple injections, reduce the difficulty in vaccine management and reduce the inoculation and management cost; can also reduce the dosage of preservatives, adjuvants and the like which are necessary to be contained in the production of the vaccine, reduce the adverse reaction of the vaccine and the like. It is believed that with the continuous development of vaccinology and the continuous enhancement of the cognition of people on the immune mechanism, the combined vaccine has no replaceable advantages by itself and will become the main preparation type for vaccine development and application in the future.
The combined vaccine researched by the invention comprises an ABCYW135 meningococcus vaccine and an encephalitis B vaccine, the main diseases prevented by the combined vaccine are epidemic meningitis A group, B group, C group, W135 group and Y group bacteria and encephalitis B virus, and the 6 diseases mainly have high morbidity in children groups and seriously affect the body health and development of infants. After the 6 vaccines are combined, the immunization times are effectively reduced, the immunization efficiency is improved, psychological and physiological pains brought to children and parents by immunization are reduced, meanwhile, after the B group epidemic meningitis vaccine containing 9 PorAs is combined, the overall protection range of epidemic meningitis bacteria is improved, and the situation that the epidemic trend of the B group epidemic meningitis bacteria is increased due to the immunization of the ACYW135 group vaccine without preventive measures is filled.
Disclosure of Invention
The invention provides a combined vaccine preparation containing A group, C group, W135 group Y meningococcal polysaccharide conjugate vaccine, B group meningococcal OMV vaccine and Japanese encephalitis inactivated vaccine, which has the characteristics of safety, effectiveness, controllability and prevention of multiple diseases by one injection and meets the relevant regulations in the three parts of 'Chinese pharmacopoeia' of 2015 edition.
The technical scheme for realizing the invention is as follows: the combined vaccine provided by the invention consists of a group A epidemic meningitis coccus capsular polysaccharide-protein conjugate, a group C epidemic meningitis coccus capsular polysaccharide-protein conjugate, a group W135 epidemic meningitis coccus capsular polysaccharide-protein conjugate, a group Y epidemic meningitis coccus capsular polysaccharide-protein conjugate, a group B epidemic meningitis bacteria PorA protein and a group B encephalitis inactivated vaccine component.
The components are prepared according to the following scheme.
Preparing A, C, W135, Y group meningococcal polysaccharide conjugate vaccine stock solution:
group a meningococcal polysaccharide conjugate vaccine stock solution: the A group meningococcus working seed batch is cultured by liquid in a fermentation tank, and the culture is terminated at the later stage of logarithmic phase or the earlier stage of stationary phase. Sampling, measuring the concentration of a bacterial liquid, checking pure bacteria, sterilizing after the pure bacteria are qualified, and adding a formaldehyde solution into the harvested culture solution for sterilization or sterilizing by adopting a proper method such as heating and the like; centrifuging the sterilized culture solution, collecting supernatant, adding cetyl trimethyl ammonium bromide, and mixing to form precipitate; adding a proper amount of calcium chloride solution into the centrifuged precipitate, adding cooled ethanol to a final concentration of 25% (v/v) after the precipitate is fully dissociated, and centrifuging to collect supernatant; adding cooled ethanol into the supernatant to a final concentration of 75% (v/v), shaking thoroughly, centrifuging to collect precipitate, washing with anhydrous ethanol and acetone, and drying to obtain crude polysaccharide. Adsorbing protein and nucleic acid on a gel medium by adopting a column chromatography method for crude polysaccharide, collecting chromatography flow-through liquid, wrapping the chromatography flow-through liquid with a 100KD ultrafiltration membrane, performing ultrafiltration desalination by using injection water, collecting ultrafiltrate, adding a proper amount of calcium chloride solution, adding cooled ethanol to the final concentration of 75% (v/v), fully shaking up, centrifuging, collecting precipitate, washing with absolute ethyl alcohol and acetone, drying, and dissolving with the injection water to obtain purified polysaccharide stock solution; according to the calibration requirements in 'A group C group meningococcus polysaccharide vaccine' in 'Chinese pharmacopoeia' of 2015 edition, the polysaccharide stock solution is calibrated; activating qualified polysaccharide stock solution by CDAP, adding 1, 6-Adipic Dihydrazide (ADH), reacting for 2-4 hr, removing CDAP and underivatized ADH, and ultrafiltering or dialyzing; after the detection is qualified, mixing the polysaccharide derivative with carrier protein and the like, adding a proper amount of carbodiimide (EDAC) into the mixture to react for 2 to 5 hours in an ice-water bath, performing ultrafiltration or dialysis by using 0.2mol/L NaCl solution, performing Sepharose 4FF chromatography purification on the collected ultrafiltrate or dialysate, collecting a conjugate at an elution peak, performing aseptic filtration to obtain a group A meningococcal polysaccharide conjugate vaccine stock solution, and storing the group A meningococcal polysaccharide conjugate vaccine stock solution at 2 to 8 ℃;
group C meningococcal polysaccharide conjugate vaccine stock solution: the working seed batch of the C group meningococcus is cultured by liquid in a fermentation tank, and the culture is terminated at the later stage of logarithmic phase or the earlier stage of stationary phase. Sampling, measuring the concentration of a bacterial liquid, checking pure bacteria, sterilizing after the pure bacteria are qualified, and adding a formaldehyde solution into the harvested culture solution for sterilization or sterilizing by adopting a proper method such as heating and the like; centrifuging the sterilized culture solution, collecting supernatant, adding cetyl trimethyl ammonium bromide, and mixing to form precipitate; adding a proper amount of calcium chloride solution into the centrifuged precipitate, adding cooled ethanol to a final concentration of 25% (v/v) after the precipitate is fully dissociated, and centrifuging to collect supernatant; adding cooled ethanol into the supernatant to a final concentration of 75% (v/v), shaking thoroughly, centrifuging to collect precipitate, washing with anhydrous ethanol and acetone, and drying to obtain crude polysaccharide. Adsorbing protein and nucleic acid onto gel medium by column chromatography, collecting chromatography flow-through liquid, ultrafiltering with 100KD ultrafiltration membrane and water for injection for desalting, collecting ultrafiltrate, adding appropriate amount of calcium chloride solution, adding cooled ethanol to final concentration of 75% (v/v), shaking, and separatingCollecting the precipitate, washing with anhydrous ethanol and acetone, drying, and dissolving with injectable water to obtain purified polysaccharide stock solution; according to the requirements of the certification in the 'A group C group meningococcus polysaccharide vaccine' in the 'Chinese pharmacopoeia' of 2015 edition, the polysaccharide stock solution is certified; adding 1, 6-Adipic Dihydrazide (ADH) into qualified polysaccharide stock solution, reacting for 2-4 hr, removing underivatized ADH, and ultrafiltering or dialyzing; after the test is qualified, mixing the polysaccharide derivative with carrier protein in equal mass, adding appropriate amount of carbodiimide (EDAC) to react for 2-5 hours in an ice-water bath, performing ultrafiltration or dialysis by using 0.2mol/L NaCl solution, and subjecting the collected ultrafiltrate or dialysate to SepharoseTM4FF chromatography purification, collecting conjugate at elution peak, sterilizing, filtering to obtain C group meningococcus polysaccharide conjugate vaccine stock solution, and storing at 2-8 deg.C;
w135 meningococcal polysaccharide conjugate vaccine stock solution: the W135 group meningococcus working seed batch is cultured in a fermentation tank by liquid, and the culture is stopped at the later stage of logarithmic phase or the earlier stage of stationary phase. Sampling, measuring the concentration of a bacterial liquid, checking pure bacteria, sterilizing after the pure bacteria are qualified, and adding a formaldehyde solution into the harvested culture solution for sterilization or sterilizing by adopting a proper method such as heating and the like; centrifuging the sterilized culture solution, collecting supernatant, adding cetyl trimethyl ammonium bromide, and mixing to form precipitate; adding a proper amount of calcium chloride solution into the centrifuged precipitate, adding cooled ethanol to a final concentration of 25% (v/v) after the precipitate is fully dissociated, and centrifuging to collect supernatant; adding cooled ethanol into the supernatant to a final concentration of 75% (v/v), shaking thoroughly, centrifuging to collect precipitate, washing with anhydrous ethanol and acetone, and drying to obtain crude polysaccharide. Adsorbing protein and nucleic acid on a gel medium by adopting a column chromatography method for crude polysaccharide, collecting chromatography flow-through liquid, wrapping the chromatography flow-through liquid with a 100KD ultrafiltration membrane, performing ultrafiltration desalination by using injection water, collecting ultrafiltrate, adding a proper amount of calcium chloride solution, adding cooled ethanol to the final concentration of 75% (v/v), fully shaking up, centrifuging, collecting precipitate, washing with absolute ethyl alcohol and acetone, drying, and dissolving with the injection water to obtain purified polysaccharide stock solution; according to the requirements of the assay in "ACYW 135 group meningococcal polysaccharide vaccine" in "Chinese pharmacopoeia" of 2015 edition, the polysaccharide stock solution is assayed;activating qualified polysaccharide stock solution by CDAP, mixing the activated polysaccharide with carrier protein, reacting at room temperature for 1-2-hr, stopping with glycine solution, reacting for 1-2 hr, collecting the combined solution after reaction, ultrafiltering or dialyzing with 0.2mol/L NaCl solution, and subjecting the collected ultrafiltrate or dialysate to SepharoseTM4FF chromatography purification, collecting conjugate at elution peak, sterilizing, filtering to obtain W135 meningococcal polysaccharide conjugate vaccine stock solution, and storing at 2-8 deg.C;
meningococcal polysaccharide group Y conjugate vaccine stock solution: the W135 group meningococcus working seed batch is cultured in a fermentation tank by liquid, and the culture is stopped at the later stage of logarithmic phase or the earlier stage of stationary phase. Sampling, measuring the concentration of a bacterial liquid, checking pure bacteria, sterilizing after the pure bacteria are qualified, and adding a formaldehyde solution into the harvested culture solution for sterilization or sterilizing by adopting a proper method such as heating and the like; centrifuging the sterilized culture solution, collecting supernatant, adding cetyl trimethyl ammonium bromide, and mixing to form precipitate; adding a proper amount of calcium chloride solution into the centrifuged precipitate, adding cooled ethanol to a final concentration of 25% (v/v) after the precipitate is fully dissociated, and centrifuging to collect supernatant; adding cooled ethanol into the supernatant to a final concentration of 75% (v/v), shaking thoroughly, centrifuging to collect precipitate, washing with anhydrous ethanol and acetone, and drying to obtain crude polysaccharide. Adsorbing protein and nucleic acid on a gel medium by adopting a column chromatography method for crude polysaccharide, collecting chromatography flow-through liquid, wrapping the chromatography flow-through liquid with a 100KD ultrafiltration membrane, performing ultrafiltration desalination by using injection water, collecting ultrafiltrate, adding a proper amount of calcium chloride solution, adding cooled ethanol to the final concentration of 75% (v/v), fully shaking up, centrifuging, collecting precipitate, washing with absolute ethyl alcohol and acetone, drying, and dissolving with the injection water to obtain purified polysaccharide stock solution; according to the requirements of the assay in "ACYW 135 group meningococcal polysaccharide vaccine" in "Chinese pharmacopoeia" of 2015 edition, the polysaccharide stock solution is assayed; activating qualified polysaccharide stock solution by CDAP, mixing the activated polysaccharide with carrier protein, reacting at room temperature for 1-2-hr, stopping with glycine solution, reacting for 1-2 hr, collecting the combined solution after reaction, ultrafiltering or dialyzing with 0.2mol/L NaCl solution, and subjecting the collected ultrafiltrate or dialysate to SepharoseTM4FF chromatography purityAnd (3) dissolving, collecting the conjugate at the elution peak, sterilizing and filtering to obtain the Y-group meningococcus polysaccharide conjugate vaccine stock solution, and storing at 2-8 ℃.
The B group meningococcus PorA protein vaccine is prepared by the following method:
the group B meningococcus should be selected to contain the following 9 PorA types: p1.7,16, P1.5-1,2-2, P1.19,15-1, P1.5-2,10, P1.12-1,13, P1.7-2,4, P1.21-2,28, P1.22,14, P1.20,23, and prepared by the following method: the B group meningococcus working seed batch is cultured by adopting fermentation tank liquid, and the culture is stopped at the later stage of logarithmic phase or the earlier stage of stationary phase. Sampling, measuring the concentration of the bacteria liquid, and checking the pure bacteria. After cooling the culture, the volume was reduced by microfiltration through a hollow fiber device, the concentrated product was diafiltered to pH 8.4. + -. 0.4 using Tris-HCl, concentrated EDTA solution was added and incubated for 30 minutes at 20 ℃ with stirring, and the supernatant was centrifuged. After further volume reduction of the 100kD ultrafiltration supernatant, EDTA was removed by diafiltration with Tris-HCl, pH 8.6. Using nucleases in Mg2+In the presence of the cofactor (incubation at 21 ℃ for 18 hours), the DNA fragment was digested. Any precipitate formed during nuclease treatment was removed with a clarification filter, followed by removal of DNA and small molecules by Sepharose 6FF, and the buffer was changed to storage buffer (Tris-HCl, 3% (w/v) sucrose), which was a group B meningococcal PorA protein vaccine stock, and stored at 2-8 ℃.
The Japanese encephalitis inactivated vaccine is prepared by the following method:
vero cells in a Japanese encephalitis working cell bank are taken, revived, amplified for 2-3 generations, mixed with a certain amount of microcarrier Cytodex-1 to prepare cell inoculation liquid, inoculated into a biological reaction tank for continuous culture for 5-7 days, then inoculated with a Japanese encephalitis virus seed working seed batch according to the proportion of 0.002 MOI, virus liquid is continuously harvested after virus infection, a filter element of 0.65 mu m is used for clarifying the virus harvest liquid, ultrafiltration concentration is carried out by a 300KD pore size membrane, β -propiolactone is added into the virus concentrated liquid according to the proportion of 1:4000 to inactivate for 24 hours, after inactivation, the virus inactivated liquid is hydrolyzed at 37 ℃ for 2 hours, the virus inactivated liquid is purified by Sepharose 6FF column chromatography or other suitable methods to obtain Japanese encephalitis inactivated vaccine stock solution, the Japanese encephalitis inactivated vaccine stock solution is stored at 2-8 ℃, and the Japanese encephalitis vaccine stock solution is diluted after the Japanese encephalitis vaccine is verified according to the verification requirement of the Japanese encephalitis inactivated vaccine (Vero cells) in the three parts of the version of China pharmacopoeia 2015.
The vaccine provided by the invention is prepared into a liquid dosage form and/or a freeze-dried dosage form according to requirements, and the components are mixed before use.
Detailed Description
The present invention includes, but is not limited to, the following examples
Preparation of a stock solution of a group a, group C, group W135, group Y meningococcal polysaccharide conjugate vaccine:
group a meningococcal polysaccharide conjugate vaccine stock solution: the A group meningococcus working seed batch is cultured by liquid in a fermentation tank, and the culture is terminated at the later stage of logarithmic phase or the earlier stage of stationary phase. Sampling, measuring the concentration of a bacterial liquid, checking pure bacteria, sterilizing after the pure bacteria are qualified, and adding a formaldehyde solution into the harvested culture solution for sterilization or sterilizing by adopting a proper method such as heating and the like; centrifuging the sterilized culture solution, collecting supernatant, adding cetyl trimethyl ammonium bromide, and mixing to form precipitate; adding a proper amount of calcium chloride solution into the centrifuged precipitate to enable the final concentration to be 1mol/L, stirring for more than 3 hours at the temperature of 2-8 ℃, adding cooled ethanol to the final concentration of 25% (v/v) after the precipitate is fully dissociated, stirring for more than 3 hours at the temperature of 2-8 ℃, and centrifuging to collect supernatant; adding cooled ethanol into the supernatant to a final concentration of 75% (v/v), shaking thoroughly, centrifuging to collect precipitate, washing with anhydrous ethanol and acetone, and drying to obtain crude polysaccharide. Adsorbing protein and nucleic acid on a gel medium by adopting a column chromatography method for crude polysaccharide, collecting chromatography flow-through liquid, carrying out ultrafiltration desalination by using 100KD ultrafiltration membrane coated with injection water, collecting ultrafiltrate, adding a proper amount of calcium chloride solution to enable the final concentration to be 0.1mol/L, adding cooled ethanol to enable the final concentration to be 75% (v/v), fully shaking up, centrifuging, collecting precipitate, washing with absolute ethyl alcohol and acetone, drying, and dissolving with injection water to obtain a purified polysaccharide stock solution; according to the calibration requirements in 'A group C group meningococcus polysaccharide vaccine' in 'Chinese pharmacopoeia' of 2015 edition, the polysaccharide stock solution is calibrated; activating qualified polysaccharide stock solution by CDAP, adding 1, 6-Adipic Dihydrazide (ADH), reacting for 1-4 hr, removing CDAP and underivatized ADH, and ultrafiltering or dialyzing; after the detection is qualified, mixing the polysaccharide derivative with carrier protein and the like, adding a proper amount of carbodiimide (EDAC) into the mixture to react for 2 to 5 hours in an ice-water bath, performing ultrafiltration or dialysis by using 0.2mol/L NaCl solution, performing Sepharose 4FF chromatography purification on the collected ultrafiltrate or dialysate, collecting a conjugate at an elution peak, performing aseptic filtration to obtain a group A meningococcal polysaccharide conjugate vaccine stock solution, and storing the group A meningococcal polysaccharide conjugate vaccine stock solution at 2 to 8 ℃;
group C meningococcal polysaccharide conjugate vaccine stock solution: the working seed batch of the C group meningococcus is cultured by liquid in a fermentation tank, and the culture is terminated at the later stage of logarithmic phase or the earlier stage of stationary phase. Sampling, measuring the concentration of a bacterial liquid, checking pure bacteria, sterilizing after the pure bacteria are qualified, and adding a formaldehyde solution into the harvested culture solution for sterilization or sterilizing by adopting a proper method such as heating and the like; centrifuging the sterilized culture solution, collecting supernatant, adding cetyl trimethyl ammonium bromide, and mixing to form precipitate; adding a proper amount of calcium chloride solution into the centrifuged precipitate to enable the final concentration to be 1mol/L, stirring for more than 3 hours at the temperature of 2-8 ℃, adding cooled ethanol to the final concentration of 25% (v/v) after the precipitate is fully dissociated, stirring for more than 3 hours at the temperature of 2-8 ℃, and centrifuging to collect supernatant; adding cooled ethanol into the supernatant to a final concentration of 75% (v/v), shaking thoroughly, centrifuging to collect precipitate, washing with anhydrous ethanol and acetone, and drying to obtain crude polysaccharide. Adsorbing protein and nucleic acid on a gel medium by adopting a column chromatography method for crude polysaccharide, collecting chromatography flow-through liquid, carrying out ultrafiltration desalination by using 100KD ultrafiltration membrane coated with injection water, collecting ultrafiltrate, adding a proper amount of calcium chloride solution to enable the final concentration to be 0.1mol/L, adding cooled ethanol to enable the final concentration to be 75% (v/v), fully shaking up, centrifuging, collecting precipitate, washing with absolute ethyl alcohol and acetone, drying, and dissolving with injection water to obtain a purified polysaccharide stock solution; according to the requirements of the certification in the 'A group C group meningococcus polysaccharide vaccine' in the 'Chinese pharmacopoeia' of 2015 edition, the polysaccharide stock solution is certified; adding 1, 6-adipic acid hydrazide (ADH) into the qualified polysaccharide stock solution, reacting for 2-4 hr, and removing residuesDerivatized ADH, ultrafiltration or dialysis; after the test is qualified, mixing the polysaccharide derivative with carrier protein in equal mass, adding appropriate amount of carbodiimide (EDAC) to react for 2-5 hours in an ice-water bath, performing ultrafiltration or dialysis by using 0.2mol/L NaCl solution, and subjecting the collected ultrafiltrate or dialysate to SepharoseTM4FF chromatography purification, collecting conjugate at elution peak, sterilizing, filtering to obtain C group meningococcus polysaccharide conjugate vaccine stock solution, and storing at 2-8 deg.C;
w135 meningococcal polysaccharide conjugate vaccine stock solution: the W135 group meningococcus working seed batch is cultured in a fermentation tank by liquid, and the culture is stopped at the later stage of logarithmic phase or the earlier stage of stationary phase. Sampling, measuring the concentration of a bacterial liquid, checking pure bacteria, sterilizing after the pure bacteria are qualified, and adding a formaldehyde solution into the harvested culture solution for sterilization or sterilizing by adopting a proper method such as heating and the like; centrifuging the sterilized culture solution, collecting supernatant, adding cetyl trimethyl ammonium bromide, and mixing to form precipitate; adding a proper amount of calcium chloride solution into the centrifuged precipitate to enable the final concentration to be 1mol/L, stirring for more than 3 hours at the temperature of 2-8 ℃, adding cooled ethanol to the final concentration of 25% (v/v) after the precipitate is fully dissociated, stirring for more than 3 hours at the temperature of 2-8 ℃, and centrifuging to collect supernatant; adding cooled ethanol into the supernatant to a final concentration of 75% (v/v), shaking thoroughly, centrifuging to collect precipitate, washing with anhydrous ethanol and acetone, and drying to obtain crude polysaccharide. Adsorbing protein and nucleic acid on a gel medium by adopting a column chromatography method for crude polysaccharide, collecting chromatography flow-through liquid, carrying out ultrafiltration desalination by using 100KD ultrafiltration membrane coated with injection water, collecting ultrafiltrate, adding a proper amount of calcium chloride solution to enable the final concentration to be 0.1mol/L, adding cooled ethanol to enable the final concentration to be 75% (v/v), fully shaking up, centrifuging, collecting precipitate, washing with absolute ethyl alcohol and acetone, drying, and dissolving with injection water to obtain a purified polysaccharide stock solution; according to the requirements of the assay in "ACYW 135 group meningococcal polysaccharide vaccine" in "Chinese pharmacopoeia" of 2015 edition, the polysaccharide stock solution is assayed; activating qualified polysaccharide stock solution by CDAP, mixing the activated polysaccharide with carrier protein, reacting at room temperature for 1-2-hr, terminating with glycine solution for 1-2 hr, collecting the binding solution with concentration of 0.2mol/LUltrafiltering or dialyzing NaCl solution, and passing the collected ultrafiltrate or dialysate through SepharoseTM4FF chromatography purification, collecting conjugate at elution peak, sterilizing, filtering to obtain W135 meningococcal polysaccharide conjugate vaccine stock solution, and storing at 2-8 deg.C;
meningococcal polysaccharide group Y conjugate vaccine stock solution: the Y group meningococcus working seed batch is cultured by liquid in a fermentation tank, and the culture is terminated at the later stage of logarithmic phase or the earlier stage of stationary phase. Sampling, measuring the concentration of a bacterial liquid, checking pure bacteria, sterilizing after the pure bacteria are qualified, and adding a formaldehyde solution into the harvested culture solution for sterilization or sterilizing by adopting a proper method such as heating and the like; centrifuging the sterilized culture solution, collecting supernatant, adding cetyl trimethyl ammonium bromide, and mixing to form precipitate; adding a proper amount of calcium chloride solution into the centrifuged precipitate to enable the final concentration to be 1mol/L, stirring for more than 3 hours at the temperature of 2-8 ℃, adding cooled ethanol to the final concentration of 25% (v/v) after the precipitate is fully dissociated, stirring for more than 3 hours at the temperature of 2-8 ℃, and centrifuging to collect supernatant; adding cooled ethanol into the supernatant to a final concentration of 75% (v/v), shaking thoroughly, centrifuging to collect precipitate, washing with anhydrous ethanol and acetone, and drying to obtain crude polysaccharide. Adsorbing protein and nucleic acid on a gel medium by adopting a column chromatography method for crude polysaccharide, collecting chromatography flow-through liquid, carrying out ultrafiltration desalination by using 100KD ultrafiltration membrane coated with injection water, collecting ultrafiltrate, adding a proper amount of calcium chloride solution to enable the final concentration to be 0.1mol/L, adding cooled ethanol to enable the final concentration to be 75% (v/v), fully shaking up, centrifuging, collecting precipitate, washing with absolute ethyl alcohol and acetone, drying, and dissolving with injection water to obtain a purified polysaccharide stock solution; according to the requirements of the assay in "ACYW 135 group meningococcal polysaccharide vaccine" in "Chinese pharmacopoeia" of 2015 edition, the polysaccharide stock solution is assayed; activating qualified polysaccharide stock solution by CDAP, mixing the activated polysaccharide with carrier protein, reacting at room temperature for 1-2-hr, stopping with glycine solution, reacting for 1-2 hr, collecting the combined solution after reaction, ultrafiltering or dialyzing with 0.2mol/L NaCl solution, and subjecting the collected ultrafiltrate or dialysate to SepharoseTM4FF chromatography purification, collecting the conjugate at the elution peak, degerming and filtering to obtain the Y-group meningococcus polysaccharide conjugate vaccine stock solutionAnd storing at 2-8 ℃.
Preparation of group B meningococcal PorA protein vaccine:
the group B meningococcus should be selected to contain the following 9 PorA types: p1.7,16, P1.5-1,2-2, P1.19,15-1, P1.5-2,10, P1.12-1,13, P1.7-2,4, P1.21-2,28, P1.22,14, P1.20,23, and prepared by the following method: the B group meningococcus working seed batch is cultured by adopting fermentation tank liquid, and the culture is stopped at the later stage of logarithmic phase or the earlier stage of stationary phase. Sampling, measuring the concentration of the bacteria liquid, and checking the pure bacteria. After cooling the culture, the volume was reduced by microfiltration through a hollow fiber device, the concentrated product was diafiltered to pH 8.4. + -. 0.4 using Tris-HCl, concentrated EDTA solution was added and incubated for 30 minutes at 20 ℃ with stirring, and the supernatant was centrifuged. After further volume reduction of the 100kD ultrafiltration supernatant, EDTA was removed by diafiltration with Tris-HCl, pH 8.6. Using nucleases in Mg2+In the presence of the cofactor (incubation at 21 ℃ for 18 hours), the DNA fragment was digested. Any precipitate formed during nuclease treatment was removed with a clarification filter, followed by removal of DNA and small molecules by Sepharose 6FF, and the buffer was changed to storage buffer (Tris-HCl, 3% (w/v) sucrose), which was a group B meningococcal PorA protein vaccine stock, and stored at 2-8 ℃.
Preparation of inactivated Japanese encephalitis vaccine:
vero cells in a Japanese encephalitis working cell bank are taken, revived, amplified for 2-3 generations, mixed with a certain amount of microcarrier Cytodex-1 to prepare cell inoculation liquid, inoculated into a biological reaction tank for continuous culture for 5-7 days, then inoculated with a Japanese encephalitis virus seed working seed batch according to the proportion of 0.002 MOI, virus liquid is continuously harvested after virus infection, a filter element of 0.65 mu m is used for clarifying the virus harvest liquid, ultrafiltration concentration is carried out by a 300KD pore size membrane, β -propiolactone is added into the virus concentrated liquid according to the proportion of 1:4000 to inactivate for 24 hours, after inactivation, the virus inactivated liquid is hydrolyzed at 37 ℃ for 2 hours, the virus inactivated liquid is purified by Sepharose 6FF column chromatography or other suitable methods to obtain Japanese encephalitis inactivated vaccine stock solution, the Japanese encephalitis inactivated vaccine stock solution is stored at 2-8 ℃, and the Japanese encephalitis vaccine stock solution is diluted after the Japanese encephalitis vaccine is verified according to the verification requirement of the Japanese encephalitis inactivated vaccine (Vero cells) in the three parts of the version of China pharmacopoeia 2015.
Subpackaging: the A group C group W135 group Y meningococcal polysaccharide conjugate vaccine, the B group meningococcal PorA protein vaccine and the Japanese encephalitis inactivated vaccine prepared in the above way are respectively filled in 1 or 2 independent penicillin bottles according to dosage forms (liquid dosage forms and/or freeze-dried dosage forms).
And (4) finished product verification: in addition to the determination of the water content, 0.5ml of the liquid formulation is sucked and added into the freeze-dried formulation for redissolving and then the rest of the tests are carried out.
1. Physical examination
1.1 appearance: the lyophilized preparation should be a white loose body; the group B meningococcus PorA protein vaccine is a microemulsion white semitransparent liquid without foreign matters, and the group B encephalitis inactivated vaccine is a transparent liquid without foreign matters; after the freeze-dried preparation is added with the group B PorA protein vaccine and the Japanese encephalitis vaccine, the freeze-dried preparation is quickly dissolved without shaking and scattering foreign matters;
1.2 filling difference: checking according to law (2015 edition of general rules 0102 of Chinese pharmacopoeia), wherein the limit of the loading difference is less than or equal to +/-15%;
1.3 identification test: the product should form obvious precipitation lines with ACYW135 group Neisseria meningitidis diagnostic serum and diphtheria antitoxin respectively by adopting an immune double diffusion method (2015 edition, general rule of Chinese pharmacopoeia 3403). The ELISA method is adopted for examination, and the epidemic encephalitis B virus antigen and the group B epidemic encephalitis PorA protein antigen are proved to be contained.
2. Chemical examination
2.1 moisture: should not be higher than 3.0% (2015 version 0832, the general rule of Chinese pharmacopoeia);
2.2 pH value: measured according to law (2015 version of general rule 0631 of Chinese pharmacopoeia), should be 6.0-8.0;
2.3 osmolality: the measurement according to the law (2015 edition of general rule 0632 of Chinese pharmacopoeia) should be 280-550 mOsmol/kg;
2.4 polysaccharide content: measuring the content of phosphorus by a method (2015 edition of Tonghe 3103 in Chinese pharmacopoeia), and calculating the content of polysaccharide in group A; measuring the sialic acid content by the method (2015 edition of Tonghe 3102 in Chinese pharmacopoeia), and calculating the total sialic acid content in the sample by taking N-acetylneuraminic acid as a reference substance; determining the content of glucose by the method, and calculating the content of polysaccharide in the Y group, wherein the molar mass of the glucose in the Y group is the same as the molar mass of the sialic acid, so that the content of the sialic acid in the Y group can be calculated; determining galactose content by the method, calculating polysaccharide content of W group, wherein the molar mass of glucose in W group is the same as that of sialic acid, calculating sialic acid content in W group, subtracting sialic acid content in W group and Y group from total sialic acid content to obtain sialic acid content in C group, and calculating sialic acid content in C group with N-acetylneuraminic acid as reference. According to the dosage for each time, the polysaccharide containing group A is 10-15 mu g; 10-15 μ g of group C polysaccharide; 10-15 μ g of W135 polysaccharide; 10-15 μ g of group Y polysaccharide;
2.5 content of free polysaccharide:
group A: precipitating polysaccharide combined with protein in conjugate stock solution with cold phenol, respectively measuring phosphorus content in stock solution before precipitation and supernatant after precipitation (2015 edition of Tonghe 3103 in Chinese pharmacopoeia), and calculating free polysaccharide content in group A to be not higher than 20%;
group C, group W135, group Y: precipitating polysaccharides combined with proteins in the conjugate stock solution by using cold phenol, respectively measuring sialic acid content in the stock solution before precipitation and supernatant after precipitation (2015 edition of Chinese pharmacopoeia general regulation 3102), simultaneously measuring glucose and galactose content in the supernatant before precipitation and after precipitation, and respectively calculating free polysaccharide content of group C, group W and group Y according to a measuring method of polysaccharide content in 5.2.4, wherein the content should not be higher than 15%;
the phosphorus content, the sialic acid content, the glucose content and the galactose content before and after the precipitation of polysaccharide stock solutions of the A group, the C group, the W group and the Y group are detected by the same method, and the polysaccharide recovery rate is respectively calculated and is 80-100 percent.
3. Efficacy testing: injecting 12-14g NIH (or Balb/C) mice subcutaneously into each batch of vaccine, each group comprises 10 mice, taking another 10 mice in the same batch as a control, injecting a physiological sodium chloride solution, injecting subcutaneously for 1 time on the 0 th day and the 14 th day respectively, wherein each injection dose respectively comprises polysaccharide of group A, group C, group W and group Y by 2.5 mu g, collecting blood on the 21 st to 28 th days after the first injection, measuring IgG antibody titer of the polysaccharide of the group A, the group C, the group W and the group Y in serum by an ELISA method, and calculating Cutoff value by using the absorbance value of the serum of the mice in the control group of the physiological sodium chloride solution. The positive conversion rate of the antibody in the vaccine group is not less than 80%.
4. And (3) testing the encephalitis B titer: the neutralizing antibody was determined in a plaque reduction neutralization assay using an immunized mouse neutralizing antibody assay. Reference vaccines (RA and RB) and neutralization test positive sera were provided by the chinese food and drug assay institute. Diluting the vaccine (T) to be tested into 1: 32 reference vaccine (R) according to required dilution, immunizing 10 mice with abdominal cavity with weight of 12-14g, each 0.5ml, 2 times, and 7 days at intervals. Blood was collected on day 7 after the second immunization, and serum was separated, mixed with the same amount of serum from the same group of mice, and inactivated at 56 ℃ for 30 minutes. Diluting the positive serum, the serum of the tested vaccine and the serum of the reference vaccine, respectively mixing the diluted positive serum, the serum of the tested vaccine and the serum of the reference vaccine with the same amount of diluted virus (about 200 PFU/0.4ml) to be used as virus control, placing the virus control in a water bath at 37 ℃ for 90 minutes, inoculating 0.4ml of 6-hole cell culture plate BHK21 cells in each hole, placing the virus control in a water bath at 37 ℃ for 90 minutes, adding a culture medium covering containing methylcellulose, culturing the virus control in a 5% carbon dioxide incubator at 37 ℃ for 5 days, dyeing, counting plaques, and calculating the plaque reduction rate of the tested vaccine and the reference vaccine on the virus control. The average number of plaques in the virus control group should be between 50 and 150.
5. Thermal stability test: the vaccine is subjected to stability test before leaving the factory, placed at 37 ℃ for 7 days, and subjected to titer determination according to the titer determination method, and still qualified.
6. Bovine serum albumin residual quantity: should not exceed 50 ng/dose (2015 edition of general rule 3411 of Chinese pharmacopoeia).
7. Antibiotic residual quantity: the dosage of the enzyme linked immunosorbent assay is not higher than 50 ng/dose.
Residual amount of Vero cell DNA: should be no more than 100 pg/dose (2015 edition "Chinese pharmacopoeia" general rule 3407 first method).
The residual amount of Vero cell protein: the enzyme linked immunosorbent assay is adopted, and the concentration should be not higher than 2 mu g/ml.
10. And (4) sterile inspection: should meet the regulations (2015 edition of the general rule 1101 of Chinese pharmacopoeia).
11 abnormal toxicity test: should meet the regulations (2015 edition of general rule 1141 of Chinese pharmacopoeia).
12. And (3) checking bacterial endotoxin: the inactivated Japanese encephalitis vaccine should be no higher than 50 EU/ml; A. c, W135, group Y meningococcal polysaccharide should be administered in a dose of not more than 500EU (2015 edition "Chinese pharmacopoeia" general rule 1143) for each 1 human administration.
13. Heat source inspection: examination was performed by law (2015 edition "Chinese pharmacopoeia" general rule 1142). The injection dose is 1ml per 1kg rabbit body weight, and contains polysaccharide 1 μ g (containing group A polysaccharide 0.05 μ g, group C polysaccharide 0.05 μ g, group Y polysaccharide 0.05 μ g, group W135 polysaccharide 0.05 μ g, and group B OMV protein 0.05 μ g).
14. And (3) total protein determination: group B PorA protein vaccine was taken and tested as per method (general rule 0731 second method).
Detection of the content of PorA protein: taking group B PorA protein vaccine, and performing polyacrylamide gel electrophoresis (SDS-PAGE), wherein the content of PorA protein should not be less than 55% of the total protein content.
Group B PorA protein vaccine serum bactericidal antibody detection: taking a test sample, immunizing NIH mice of 8 weeks of age, each group comprising 10 mice, subcutaneously immunizing at 0 day and 14 days respectively, collecting serum 28 days after immunization, and performing bactericidal efficacy test, wherein the bactericidal antibody titer is not less than 1: 4. Bactericidal antibody test methods and requirements reference: WS295-2008 "diagnostic criteria for epidemic cerebrospinal meningitis".
Claims (13)
1. The combined vaccine is characterized by comprising a Neisseria meningitidis group A capsular polysaccharide-protein conjugate, a Neisseria meningitidis group C capsular polysaccharide-protein conjugate, a Neisseria meningitidis group Y capsular polysaccharide-protein conjugate, a Neisseria meningitidis group W135 capsular polysaccharide-protein conjugate, a Neisseria meningitidis group B OMV vaccine and an inactivated Japanese encephalitis vaccine.
2. Combination vaccine according to claim 1, characterized in that: the A group, C group, W135 group and Y group meningococcal polysaccharide conjugates are prepared by the following method:
a) preparing a stock solution of meningococcal polysaccharide conjugate vaccines of group A, group C, group W135 and group Y:
i.A group meningococcal polysaccharide conjugate vaccine stock solution: group A meningococcusThe working coccus seeds are cultured in a fermentation tank in liquid, the culture is stopped at the late logarithmic phase or the early stationary phase, the samples are taken for bacterial liquid concentration determination and pure bacteria inspection, and the bacteria are sterilized after the samples are qualified, and formaldehyde solution can be added into the harvested culture solution for sterilization or the bacteria can be sterilized by adopting a proper method such as heating and the like; centrifuging the sterilized culture solution, collecting supernatant, adding cetyl trimethyl ammonium bromide, and mixing to form precipitate; adding a proper amount of calcium chloride solution into the centrifuged precipitate, adding cooled ethanol to a final concentration of 25% (v/v) after the precipitate is fully dissociated, and centrifuging to collect supernatant; adding cooled ethanol into the supernatant to a final concentration of 75% (v/v), fully shaking up, centrifuging to collect precipitate, washing with absolute ethanol and acetone, obtaining dried precipitate as crude polysaccharide, adsorbing protein and nucleic acid on a gel medium by adopting a column chromatography method, collecting chromatography flow-through liquid, carrying out ultrafiltration desalination by using 100KD ultrafiltration membrane coated injection water, collecting ultrafiltrate, adding cooled ethanol to a final concentration of 75% (v/v), fully shaking up, centrifuging to collect precipitate, washing with absolute ethanol and acetone, dissolving with injection water after drying to obtain purified polysaccharide stock solution, and verifying the polysaccharide stock solution according to the verification requirement in 'ACYW 135 group meningococcus polysaccharide vaccine' in the third part of 'Chinese pharmacopoeia' of 2015 edition; activating qualified polysaccharide stock solution by CDAP, adding 1, 6-Adipic Dihydrazide (ADH), reacting for 2-4 hr, removing CDAP and underivatized ADH, and ultrafiltering or dialyzing; after the test is qualified, mixing the polysaccharide derivative with carrier protein in equal mass, adding appropriate amount of carbodiimide (EDAC) to react for 2-5 hours in an ice-water bath, performing ultrafiltration or dialysis by using 0.2mol/L NaCl solution, and subjecting the collected ultrafiltrate or dialysate to SepharoseTM4FF chromatography purification, collecting conjugate at elution peak, sterilizing, filtering to obtain A group meningococcus polysaccharide conjugate vaccine stock solution, and storing at 2-8 deg.C;
group c meningococcal polysaccharide conjugate vaccine stock solution: culturing group C meningococcus working seed in fermentation tank, terminating culture in late logarithmic phase or early stationary phase, sampling, measuring bacterial liquid concentration, inspecting pure bacteria, sterilizing, and sterilizing by adding formaldehyde solution or heatingSterilizing by appropriate method; centrifuging the sterilized culture solution, collecting supernatant, adding cetyl trimethyl ammonium bromide, and mixing to form precipitate; adding a proper amount of calcium chloride solution into the centrifuged precipitate, adding cooled ethanol to a final concentration of 25% (v/v) after the precipitate is fully dissociated, and centrifuging to collect supernatant; adding cooled ethanol into the supernatant to a final concentration of 75% (v/v), fully shaking up, centrifugally collecting precipitate, washing with absolute ethanol and acetone, obtaining dried precipitate as crude polysaccharide, adsorbing protein and nucleic acid on a gel medium by adopting a column chromatography method, collecting chromatography flow-through liquid, carrying out ultrafiltration desalination by using 100KD ultrafiltration membrane coated injection water, collecting ultrafiltrate, adding a proper amount of sodium chloride solution, adding cooled ethanol to a final concentration of 75% (v/v), fully shaking up, centrifugally collecting precipitate, washing with absolute ethanol and acetone, dissolving with injection water after drying to obtain purified polysaccharide stock solution, and verifying the polysaccharide stock solution according to a verification requirement in ' A group C meningococcus polysaccharide vaccine ' in ' three parts of ' Chinese pharmacopoeia ' 2015 edition; adding 1, 6-Adipic Dihydrazide (ADH) into qualified polysaccharide stock solution, reacting for 2-4 hr, removing underivatized ADH, and ultrafiltering or dialyzing; after the test is qualified, mixing the polysaccharide derivative with carrier protein in equal mass, adding appropriate amount of carbodiimide (EDAC) to react for 2-5 hours in an ice-water bath, performing ultrafiltration or dialysis by using 0.2mol/L NaCl solution, and subjecting the collected ultrafiltrate or dialysate to SepharoseTM4FF chromatography purification, collecting conjugate at elution peak, sterilizing, filtering to obtain C group meningococcus polysaccharide conjugate vaccine stock solution, and storing at 2-8 deg.C;
group y meningococcal polysaccharide conjugate vaccine stock solution: the Y group meningococcus working seed batch is cultured by adopting fermentation tank liquid, the culture is terminated at the later stage of logarithmic phase or the earlier stage of stationary phase, the sample is taken for bacterial liquid concentration determination and pure bacteria inspection, and the sterilization is carried out after the sample is qualified, and formaldehyde solution can be added into the harvested culture solution for sterilization or the sterilization by adopting a proper method such as heating and the like; centrifuging the sterilized culture solution, collecting supernatant, adding cetyl trimethyl ammonium bromide, and mixing to form precipitate; adding appropriate amount of calcium chloride solution into the centrifuged precipitate, adding cooledEthanol to a final concentration of 25% (v/v), centrifuging and collecting the supernatant; adding cooled ethanol into the supernatant to a final concentration of 75% (v/v), fully shaking up, centrifuging to collect precipitate, washing with absolute ethanol and acetone, drying to obtain crude polysaccharide, adsorbing protein and nucleic acid on a gel medium by adopting a column chromatography method, collecting chromatography flow-through liquid, carrying out ultrafiltration desalination by using 100KD ultrafiltration membrane coated injection water, collecting ultrafiltrate, adding a proper amount of calcium chloride solution, adding cooled ethanol to a final concentration of 75% (v/v), fully shaking up, centrifuging to collect precipitate, washing with absolute ethanol and acetone, drying, and dissolving with injection water to obtain a purified polysaccharide stock solution: according to the requirements of the assay in "ACYW 135 group meningococcal polysaccharide vaccine" in "Chinese pharmacopoeia" of 2015 edition, the polysaccharide stock solution is assayed; activating qualified polysaccharide stock solution by CDAP, mixing the activated polysaccharide with carrier protein, reacting at room temperature for 1-2-hr, stopping with glycine solution, reacting for 1-2 hr, collecting the combined solution after reaction, ultrafiltering or dialyzing with 0.2mol/L NaCl solution, and subjecting the collected ultrafiltrate or dialysate to SepharoseTM4FF chromatography purification, collecting the conjugate at the elution peak, sterilizing and filtering to obtain Y-group meningococcus polysaccharide conjugate vaccine stock solution, and storing at 2-8 ℃;
a meningococcal polysaccharide of group w135 conjugate vaccine stock solution: the W135 group meningococcus working seed batch is cultured by adopting fermentation tank liquid, the culture is terminated at the later stage of logarithmic phase or the earlier stage of stationary phase, the sample is taken for bacterial liquid concentration determination and pure bacteria inspection, and the sterilization is carried out after the sample is qualified, and formaldehyde solution can be added into the harvested culture solution for sterilization or the sterilization by adopting a proper method such as heating and the like; centrifuging the sterilized culture solution, collecting supernatant, adding cetyl trimethyl ammonium bromide, and mixing to form precipitate; adding a proper amount of calcium chloride solution into the centrifuged precipitate, adding cooled ethanol to a final concentration of 25% (v/v) after the precipitate is fully dissociated, and centrifuging to collect supernatant; adding cooled ethanol into the supernatant to final concentration of 75% (v/v), shaking, centrifuging to collect precipitate, washing with anhydrous ethanol and acetone, drying to obtain crude polysaccharide, dissolving the crude polysaccharide in a solution containing protein and nucleic acid by column chromatography, and adsorbing protein and nucleic acidSeparating flow-through liquid on a gel medium, wrapping the gel medium with a 100KD ultrafiltration membrane, carrying out ultrafiltration desalination by using injection water, collecting ultrafiltrate, adding a proper amount of calcium chloride solution, adding cooled ethanol to a final concentration of 75% (v/v), fully shaking, centrifuging, collecting precipitate, washing by using absolute ethanol and acetone, drying, dissolving by using injection water to obtain purified polysaccharide stock solution, and verifying the polysaccharide stock solution according to the verification requirement in 'ACYW 135 meningococcal polysaccharide vaccine' in the third part of 'Chinese pharmacopoeia' of 2015 edition; activating qualified polysaccharide stock solution by CDAP, mixing the activated polysaccharide with carrier protein, reacting at room temperature for 1-2-hr, stopping with glycine solution, reacting for 1-2 hr, collecting the combined solution after reaction, ultrafiltering or dialyzing with 0.2mol/L NaCl solution, and subjecting the collected ultrafiltrate or dialysate to SepharoseTM4FF chromatography purification, collecting the conjugate at the elution peak, sterilizing and filtering to obtain the W135 meningococcal polysaccharide conjugate vaccine stock solution, and storing at 2-8 ℃.
3. Combination vaccine according to claim 1, characterized in that: the Japanese encephalitis inactivated vaccine is prepared by the following method:
taking Vero cells in a Japanese encephalitis working cell bank, recovering, amplifying for 2-3 generations, mixing with a certain amount of microcarrier Cytodex-1 to prepare a cell inoculation liquid, inoculating the cell inoculation liquid into a biological reaction tank, continuously culturing for 5-7 days, then inoculating a Japanese encephalitis virus seed working seed batch according to the proportion of 0.002 MOI, continuously harvesting virus liquid after virus infection, clarifying the virus harvest liquid by using a 0.65 mu m filter core, performing ultrafiltration concentration by using a 300KD pore size membrane pack, adding β -propiolactone into the virus concentrated liquid according to the proportion of 1:4000 of final concentration, inactivating the virus inactivated liquid for 24 hours, hydrolyzing the virus inactivated liquid at 37 ℃ for 2 hours after inactivation is finished, and passing the virus inactivated liquid through SepharoseTM6FF is purified by column chromatography or other suitable methods to obtain Japanese encephalitis inactivated vaccine stock solution, and the stock solution is stored at 2-8 ℃; according to the detection requirements in 'freeze-dried Japanese encephalitis inactivated vaccine (Vero cells)' in the 'Chinese pharmacopoeia' of 2015 edition, detecting the Japanese encephalitis inactivated vaccine stock solution; and (3) diluting the qualified vaccine stock solution in proportion to obtain the inactivated Japanese encephalitis vaccine.
4. The combination vaccine of claim 1, wherein the group B meningococcal PorA protein vaccine comprises the following 9 PorA types, preferably the 9 types are P1.7,16, P1.5-1,2-2, P1.19,15-1, P1.5-2,10, P1.12-1,13, P1.7-2,4, P1.21-2,28, P1.22,14, P1.20,23, and the group B meningococcal PorA protein vaccine is prepared by the following method:
culturing meningococcus group B in liquid in a fermentation tank, terminating the culture at late logarithmic phase or early stationary phase, sampling, measuring bacterial liquid concentration, checking pure bacteria, cooling the culture, performing microfiltration by a hollow fiber device to reduce the volume, percolating the concentrated product to pH8.4 +/-0.4 by using Tris-HCl, adding EDTA concentrated solution, stirring and incubating at 20 ℃ for 30 minutes, centrifuging to separate supernatant, reducing the volume of the ultrafiltration supernatant by 100KD, percolating and removing EDTA by using Tris-HCl with pH8.6, digesting DNA fragments by using nuclease in the presence of Mg2+ cofactor (incubating at 21 ℃ for 18 hours), removing any precipitate formed during nuclease treatment by using a clarifying filter, and then performing Sepharose treatment by using the nucleaseTM6FF removes DNA and small molecules, and changes the buffer into storage buffer (Tris-HCl, 3% (w/v) sucrose), which is the stock solution of PorA protein vaccine of meningococcus group B, and stores at 2-8 ℃.
5. The combination vaccine of claim 1, wherein the component ACYW135 meningococcal polysaccharide conjugate vaccine is calculated by polysaccharide, and the amount of meningococcal capsular polysaccharide group A, C, Y and W135 in each unit dose of preparation is 10-15 μ g.
6. The combination vaccine of claim 1, wherein the lyophilisation adjuvant of the component meningococcal polysaccharide conjugate vaccine is sucrose.
7. The combination vaccine according to claim 1, wherein the component inactivated Japanese encephalitis vaccine contains Japanese encephalitis virus protein in an amount of 6-9 μ g per unit dose of the preparation.
8. The combination vaccine of claim 1, wherein the component group B meningococcal protein vaccine is a 9-valent PorA vaccine comprising 9 PorAs as antigens, wherein each PorA protein is present at 15 μ g.
9. The combination vaccine of claim 1, wherein the component group B meningococcal PorA protein vaccine is in a liquid formulation.
10. Combination vaccine according to claim 1, wherein the component meningococcal capsular polysaccharides are each conjugated to a carrier protein, such as CRM197Proteins, tetanus toxoid, and the like.
11. The combination vaccine of claim 1, wherein the meningococcal capsular polysaccharides from group A, group C, group Y and group W135 are covalently bound to a carrier protein, wherein the ratio of polysaccharide to protein is 1:1, and adipic dihydrazide may be added during the coupling process.
12. The combination vaccine of claim 1, which is an injectable formulation for subcutaneous or intramuscular injection.
13. Combination vaccine according to claim 1, for use in a vaccine for the prevention of infection by group a, group B, group C, group Y, group W135 neisseria meningitidis, encephalitis B virus.
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1626242A (en) * | 2003-12-10 | 2005-06-15 | 薛平 | Combined vaccine composed of viral of Japanese b encephalitis and vaccine of brain fever cocci |
| CN101007167A (en) * | 2007-02-07 | 2007-08-01 | 薛平 | Mucosal meningococcal multivalence combined vaccines |
| CN101590226A (en) * | 2009-06-19 | 2009-12-02 | 长春长生生物科技股份有限公司 | ACY W 135Group meningitis cocci polysaccharide type-b hemophilus influenza combined vaccine |
| US20100297166A1 (en) * | 2006-07-19 | 2010-11-25 | Reddy Jeeri R | Meningococcal Oligosaccharide Linked Polysaccharides and Diptheria Protein Conjucate Vaccine for All Ages |
| CN103550773A (en) * | 2013-09-22 | 2014-02-05 | 成都康华生物制品有限公司 | Multivalent combined vaccine |
| CN104127869A (en) * | 2014-07-18 | 2014-11-05 | 北京祥瑞生物制品有限公司 | Multivalent group B meningococcal protein vaccine and preparation method thereof |
| CN104998255A (en) * | 2015-06-30 | 2015-10-28 | 北京祥瑞生物制品有限公司 | Novel ACYW135 meningococcal conjugate vaccine and preparation method thereof |
| CN108295253A (en) * | 2018-04-17 | 2018-07-20 | 北京成大天和生物科技有限公司 | A kind of A, C group meningitis cocci-b types haemophilus influenzae/encephalitis B combined vaccine |
| CN108939061A (en) * | 2018-08-03 | 2018-12-07 | 北京智飞绿竹生物制药有限公司 | A kind of multicomponent B group meningitis cocci vaccine and preparation method thereof |
-
2020
- 2020-03-24 CN CN202010212354.8A patent/CN111281972A/en not_active Withdrawn
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1626242A (en) * | 2003-12-10 | 2005-06-15 | 薛平 | Combined vaccine composed of viral of Japanese b encephalitis and vaccine of brain fever cocci |
| US20100297166A1 (en) * | 2006-07-19 | 2010-11-25 | Reddy Jeeri R | Meningococcal Oligosaccharide Linked Polysaccharides and Diptheria Protein Conjucate Vaccine for All Ages |
| CN101007167A (en) * | 2007-02-07 | 2007-08-01 | 薛平 | Mucosal meningococcal multivalence combined vaccines |
| CN101590226A (en) * | 2009-06-19 | 2009-12-02 | 长春长生生物科技股份有限公司 | ACY W 135Group meningitis cocci polysaccharide type-b hemophilus influenza combined vaccine |
| CN103550773A (en) * | 2013-09-22 | 2014-02-05 | 成都康华生物制品有限公司 | Multivalent combined vaccine |
| CN104127869A (en) * | 2014-07-18 | 2014-11-05 | 北京祥瑞生物制品有限公司 | Multivalent group B meningococcal protein vaccine and preparation method thereof |
| CN104998255A (en) * | 2015-06-30 | 2015-10-28 | 北京祥瑞生物制品有限公司 | Novel ACYW135 meningococcal conjugate vaccine and preparation method thereof |
| CN108295253A (en) * | 2018-04-17 | 2018-07-20 | 北京成大天和生物科技有限公司 | A kind of A, C group meningitis cocci-b types haemophilus influenzae/encephalitis B combined vaccine |
| CN108939061A (en) * | 2018-08-03 | 2018-12-07 | 北京智飞绿竹生物制药有限公司 | A kind of multicomponent B group meningitis cocci vaccine and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| WATERBEEMD ET AL.: "Improved OMV vaccine against Neisseria meningitidis using genetically engineered strains and a detergent-free purification process", 《VACCINE》 * |
| 吴根鹏等: "B群脑膜炎球菌疫苗开发与临床前评价", 《国际生物制品学杂志》 * |
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