CN105031643A - Preparation method and application of meningococcal A capsular polysaccharide conjugate vaccine - Google Patents
Preparation method and application of meningococcal A capsular polysaccharide conjugate vaccine Download PDFInfo
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- CN105031643A CN105031643A CN201510543858.7A CN201510543858A CN105031643A CN 105031643 A CN105031643 A CN 105031643A CN 201510543858 A CN201510543858 A CN 201510543858A CN 105031643 A CN105031643 A CN 105031643A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/095—Neisseria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/385—Haptens or antigens, bound to carriers
Abstract
The invention relates to a preparation method and application of a meningococcal A capsular polysaccharide conjugate vaccine. The method includes the following steps of diluting and activating meningococcal A capsular polysaccharide, coupling the meningococcal A capsular polysaccharide and sebacic dihydrazide to obtain meningococcal A polysaccharide-sebacic dihydrazide derivatives, and making the meningococcal A polysaccharide-sebacic dihydrazide derivatives and a tetanus toxoid solution have a catalytic reaction through carbodiimide to obtain conjugates of the meningococcal A capsular polysaccharide and carrier protein. The sebacic dihydrazide serves as the coupling agent in the meningococcal A conjugate vaccine preparation process, and compared with adipic dihydrazide, the sebacic dihydrazide has two more segments of methylene on a carbon chain, is longer in molecular chain, and can better overcome the steric hindrance between biomacromolecules and improve the conjugate yield.
Description
Technical field
The present invention relates to biomedicine field, particularly, relate to a kind of preparation method and application of A group meningitis cocci capsular polysaccharide conjugate vaccine.
Background technology
Neisseria meningitidis (Neisseriameningitidis, Nm) be the primary pathogenic bacterium of meningitis and explosive septicemia, the morbidity of epidemic cerebrospinal meningitis (hereinafter referred to as epidemic encephalitis) is often in sporadic, obvious relation between persistence is there is no between each case, usually break out in small-scale mode, endemic conditions that is catastrophic, that be difficult to expect can be changed in some areas sick.The annual epidemic encephalitis number of the infected in the whole world is about 500,000, dead 50,000.Nm is aerobe, and Gram's staining is negative, has pod membrane, general paired appearance (diplococcus).Nm can be divided into l3 sero-group according to the chemical composition of capsular polysaccharide, according to the antigenic specificity of outer membrane protein (Outermem-braneprotein, OMP) PorB and P0rA, can be divided into different serotype and blood serum subtype again.A, B, C, Y and W135 group meningitis cocci is main pathogenic sero-group, and sero-group is distributed with significant geographical difference.Caused by A group meningitis Neisseria in the epidemic encephalitis case of China more than 90%.
Research shows, the capsular polysaccharide vaccine of A group meningitis cocci is applicable to child and the adult of more than 2 years old.For the child being less than for 18 monthly ages, the effective percentage of the vaccine of this employing PRP is extremely low.
Succeeding in developing of A group's epidemic encephalitis combined vaccine solves vaccine problem invalid in infant.Combined vaccine has stronger immunogenicity and better immune effect than polysaccharide vaccine in the past, very effective in infant, even if use A group's epidemic encephalitis combined vaccine in little age baby, is also safely and effectively.
In process prepared by A group's epidemic encephalitis combined vaccine, the preparation method generally used at present is: utilize the method for liquid fermentation to prepare A group's epidemic encephalitis bacterium liquid, precipitate through CDAP again after sterilization, alcohol settling, cold phenol extracting, the step such as dehydrated alcohol and washing with acetone obtains refining A group's epidemic encephalitis capsular polysaccharide, adipic dihydrazide is connected after activating Hib capsular polysaccharide with CNBr again, then under the catalytic action of carbodiimide, A group's epidemic encephalitis capsular polysaccharide is combined with tetanus toxoid protein by the bridging effect of adipic dihydrazide, macromolecule polysaccharide protein conjugates is collected finally by gel filtration chromatography, be GL-PP combined vaccinogen liquid.
At present, in process prepared by A group's epidemic encephalitis combined vaccine, A group meningitis cocci capsular polysaccharide conjugate vaccine low in conjunction with polysaccharide yield.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of preparation method of A group meningitis cocci capsular polysaccharide conjugate vaccine, to overcome the problem low in conjunction with polysaccharide yield of existing A group meningitis cocci capsular polysaccharide conjugate vaccine.
In addition, the present invention also comprises a kind of application of preparation method of A group meningitis cocci capsular polysaccharide conjugate vaccine.
The present invention's adopted technical scheme that solves the problem is:
A preparation method for A group meningitis cocci capsular polysaccharide conjugate vaccine, comprises the following steps:
B1: solution A group's epidemic encephalitis capsular polysaccharide water for injection being diluted to 8mg/ml, constant temperature to 20 DEG C, regulates pH to 10.50;
B2: add and carry out activation processing with the Bromine cyanide. of the quality such as polysaccharide described in B1, maintains pH10.5 and activates 20 minutes, regulate pH to 8.5;
B3: add and carry out coupling reaction with polysaccharide solution equal-volume, isocyatic sebacic dihydrazide solution described in B1, maintains pH8.5 and reacts 15 minutes;
B4: the reactant of B3 gained is repeatedly diluted-ultrafiltration and concentration, removes remaining Bromine cyanide., obtains A group's epidemic encephalitis polysaccharide-sebacic dihydrazide derivant;
B5: add in the A group's epidemic encephalitis polysaccharide-sebacic dihydrazide derivant of B4 gained and the tetanus toxoid protein solution with the quality such as polysaccharide described in B1, then with water for injection dilution, make polysaccharide final concentration be 3mg/ml;
B6: B5 gained solution is mixed in rearmounted ice bath, adding carbodiimide powder to final concentration is 30mmol/L, maintains pH5.5, in 2 ~ 8 DEG C of reactions 60 minutes, adjusts PH to 7.0 to put 2 ~ 8 DEG C and leaves standstill 10 hours;
B7: by the reactant of B6 gained through the separation and purification of Sepharose4FF gel column, collects V
0neighbouring eluent, by the eluent aseptic filtration of collecting, obtains the conjugate of A group's epidemic encephalitis capsular polysaccharide and carrier protein.
In capsular polysaccharide and tetanus toxoid protein cohesive process, bridging agent is very important, the sterically hindered of capsular polysaccharide and tetanus toxoid protein can be overcome by bridging effect, make polysaccharide more easily and tetanus toxoid protein linked together by covalent bond.The bridging agent that existing combined vaccine uses is adipic dihydrazide, adipic dihydrazide molecule two ends respectively have a hydrazide group, polysaccharide (or tetanus toxoid) easily and after activation generates amido link, together covalently bound, at present, in process prepared by A group's epidemic encephalitis combined vaccine, the efficiency that adipic dihydrazide is connected on capsular polysaccharide is very low, need excessive interpolation, and the efficiency that obtained capsular polysaccharide-adipic dihydrazide derivant is combined with tetanus toxoid protein is not high yet, cause certain wasting of resources, cause the low in conjunction with polysaccharide yield of A group meningitis cocci capsular polysaccharide conjugate vaccine, the coupling agent adipic dihydrazide that tradition uses by the present invention replaces to sebacic dihydrazide, sebacic dihydrazide is a kind of structure micromolecular compound similar to adipic dihydrazide, difference is many 2 methylene in carbochain, strand is longer, as combined vaccine bridging agent, more can overcome mutual sterically hindered of biomacromolecule, improve in conjunction with yield.
An application for the preparation method of A group meningitis cocci capsular polysaccharide conjugate vaccine, is applied to the preparation of the conjugate of A group's epidemic encephalitis capsular polysaccharide and carrier protein by a kind of preparation method of A group meningitis cocci capsular polysaccharide conjugate vaccine.
To sum up, the invention has the beneficial effects as follows:
The present invention using sebacic dihydrazide as the coupling agent in process prepared by A group's epidemic encephalitis combined vaccine, sebacic dihydrazide compares adipic dihydrazide many 2 methylene in carbochain, strand is longer, more can overcome mutual sterically hindered of biomacromolecule, improves in conjunction with yield.
Detailed description of the invention
Below in conjunction with embodiment, to the detailed description further of invention do, but embodiments of the present invention are not limited thereto.
Embodiment:
A preparation method for A group meningitis cocci capsular polysaccharide conjugate vaccine, comprises the following steps:
B1: solution A group's epidemic encephalitis capsular polysaccharide water for injection being diluted to 8mg/ml, constant temperature to 20 DEG C, regulates pH to 10.50;
B2: add and carry out activation processing with the Bromine cyanide. of the quality such as polysaccharide described in B1, maintains pH10.5 and activates 20 minutes, regulate pH to 8.5;
B3: add and carry out coupling reaction with polysaccharide solution equal-volume, isocyatic sebacic dihydrazide solution described in B1, maintains pH8.5 and reacts 15 minutes;
B4: the reactant of B3 gained is repeatedly diluted-ultrafiltration and concentration, removes remaining Bromine cyanide., obtains A group's epidemic encephalitis polysaccharide-sebacic dihydrazide derivant;
B5: add in the A group's epidemic encephalitis polysaccharide-sebacic dihydrazide derivant of B4 gained and the tetanus toxoid protein solution with the quality such as polysaccharide described in B1, then with water for injection dilution, make polysaccharide final concentration be 3mg/ml;
B6: B5 gained solution is mixed in rearmounted ice bath, adding carbodiimide powder to final concentration is 30mmol/L, maintains pH5.5, in 8 DEG C of reactions 60 minutes, adjusts PH to 7.0 to put 8 DEG C and leaves standstill 10 hours;
B7: by the reactant of B6 gained through the separation and purification of Sepharose4FF gel column, collects V
0neighbouring eluent, by the eluent aseptic filtration of collecting, obtains the conjugate of A group's epidemic encephalitis capsular polysaccharide and carrier protein.
An application for the preparation method of A group meningitis cocci capsular polysaccharide conjugate vaccine, is applied to the preparation of the conjugate of A group's epidemic encephalitis capsular polysaccharide and carrier protein by a kind of preparation method of A group meningitis cocci capsular polysaccharide conjugate vaccine.
Sample the conjugate of the A group's epidemic encephalitis capsular polysaccharide obtained and carrier protein and detect polyoses content, protein content, free polysaccharide, relative molecular mass by official method, in 8 DEG C of preservations, prepare 3 batches altogether, result is as shown in table 1:
Table 1
The another adipic dihydrazide that uses in the same way is as coupling agent, and carry out association reaction, prepare the conjugate of combined vaccinogen liquid and A group's epidemic encephalitis capsular polysaccharide and carrier protein, indices is as shown in table 2:
Table 2
The present invention prepares the polysaccharide yield of A group's epidemic encephalitis capsular polysaccharide conjugate more than 30%, contrast with the yield of traditional associated methods 15 ~ 20%, polysaccharide yield improves a lot, and saves raw materials for production, and the manpower of single dose finished product vaccine, raw material, energy cost are reduced significantly.
As mentioned above, the present invention can be realized preferably.
Claims (2)
1. a preparation method for A group meningitis cocci capsular polysaccharide conjugate vaccine, is characterized in that, comprises the following steps:
B1: solution A group's epidemic encephalitis capsular polysaccharide water for injection being diluted to 8mg/ml, constant temperature to 20 DEG C, regulates pH to 10.50;
B2: add and carry out activation processing with the Bromine cyanide. of the quality such as polysaccharide described in B1, maintains pH10.5 and activates 20 minutes, regulate pH to 8.5;
B3: add and carry out coupling reaction with polysaccharide solution equal-volume, isocyatic sebacic dihydrazide solution described in B1, maintains pH8.5 and reacts 15 minutes;
B4: the reactant of B3 gained is repeatedly diluted-ultrafiltration and concentration, removes remaining Bromine cyanide., obtains A group's epidemic encephalitis polysaccharide-sebacic dihydrazide derivant;
B5: add in the A group's epidemic encephalitis polysaccharide-sebacic dihydrazide derivant of B4 gained and the tetanus toxoid protein solution with the quality such as polysaccharide described in B1, then with water for injection dilution, make polysaccharide final concentration be 3mg/ml;
B6: B5 gained solution is mixed in rearmounted ice bath, adding carbodiimide powder to final concentration is 30mmol/L, maintains pH5.5, in 2 ~ 8 DEG C of reactions 60 minutes, adjusts PH to 7.0 to put 2 ~ 8 DEG C and leaves standstill 10 hours;
B7: by the reactant of B6 gained through the separation and purification of Sepharose4FF gel column, collects V
0neighbouring eluent, by the eluent aseptic filtration of collecting, obtains the conjugate of A group's epidemic encephalitis capsular polysaccharide and carrier protein.
2. the application of the preparation method of an A group meningitis cocci capsular polysaccharide conjugate vaccine, it is characterized in that, the preparation method of a kind of A group meningitis cocci capsular polysaccharide conjugate vaccine according to claim 1 is applied to the preparation of the conjugate of A group's epidemic encephalitis capsular polysaccharide and carrier protein.
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| CN201510543858.7A CN105031643A (en) | 2015-08-31 | 2015-08-31 | Preparation method and application of meningococcal A capsular polysaccharide conjugate vaccine |
| PCT/CN2016/078088 WO2017036141A1 (en) | 2015-08-31 | 2016-03-31 | Preparation method and use of group a meningococcal capsular polysaccharide conjugate vaccine |
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| CN201510543858.7A CN105031643A (en) | 2015-08-31 | 2015-08-31 | Preparation method and application of meningococcal A capsular polysaccharide conjugate vaccine |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106344915A (en) * | 2016-08-29 | 2017-01-25 | 成都欧林生物科技股份有限公司 | Preparation method of group A meningococcal capsular polysaccharide conjugate vaccine |
| WO2017036141A1 (en) * | 2015-08-31 | 2017-03-09 | 成都欧林生物科技股份有限公司 | Preparation method and use of group a meningococcal capsular polysaccharide conjugate vaccine |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102633898A (en) * | 2012-04-23 | 2012-08-15 | 成都欧林生物科技股份有限公司 | Optimization method of Hib polysaccharide and protein binding process |
| CN102861330A (en) * | 2012-06-29 | 2013-01-09 | 成都欧林生物科技股份有限公司 | Haemophilus influenzae type b (Hib) polysaccharide and refined tetanus toxoid coupling process |
| CN103083652A (en) * | 2013-02-06 | 2013-05-08 | 中国科学院过程工程研究所 | Meningococcal polysaccharide conjugate vaccine treating heterobifunctional reagent as conjugation bridge, and its preparation method |
| CN103251943A (en) * | 2013-05-20 | 2013-08-21 | 成都欧林生物科技股份有限公司 | Method for preparing type b Haemophilus influenzae and polysaccharide conjugate vaccine |
| CN104548090A (en) * | 2015-01-27 | 2015-04-29 | 中国科学院过程工程研究所 | Beta-glucan modified meningitis polysaccharide conjugate vaccine and preparation method thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105056228A (en) * | 2015-08-31 | 2015-11-18 | 成都欧林生物科技股份有限公司 | Method for preparing group A meningococcal capsular polysaccharide conjugate vaccine |
| CN105031643A (en) * | 2015-08-31 | 2015-11-11 | 成都欧林生物科技股份有限公司 | Preparation method and application of meningococcal A capsular polysaccharide conjugate vaccine |
| CN105031642A (en) * | 2015-08-31 | 2015-11-11 | 成都欧林生物科技股份有限公司 | Meningococcal A capsular polysaccharide conjugate vaccine and preparation method thereof |
-
2015
- 2015-08-31 CN CN201510543858.7A patent/CN105031643A/en active Pending
-
2016
- 2016-03-31 WO PCT/CN2016/078088 patent/WO2017036141A1/en active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102633898A (en) * | 2012-04-23 | 2012-08-15 | 成都欧林生物科技股份有限公司 | Optimization method of Hib polysaccharide and protein binding process |
| CN102861330A (en) * | 2012-06-29 | 2013-01-09 | 成都欧林生物科技股份有限公司 | Haemophilus influenzae type b (Hib) polysaccharide and refined tetanus toxoid coupling process |
| CN103083652A (en) * | 2013-02-06 | 2013-05-08 | 中国科学院过程工程研究所 | Meningococcal polysaccharide conjugate vaccine treating heterobifunctional reagent as conjugation bridge, and its preparation method |
| CN103251943A (en) * | 2013-05-20 | 2013-08-21 | 成都欧林生物科技股份有限公司 | Method for preparing type b Haemophilus influenzae and polysaccharide conjugate vaccine |
| CN104548090A (en) * | 2015-01-27 | 2015-04-29 | 中国科学院过程工程研究所 | Beta-glucan modified meningitis polysaccharide conjugate vaccine and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| 刘方蕾 等: "A群奈瑟氏脑膜炎球菌荚膜多糖-破伤风类毒素结合疫苗的制备及其免疫原性研究", 《微生物学免疫学进展》 * |
| 杨丽华 等: "A群脑膜炎球菌多糖结合疫苗的研制", 《中国生物制品学杂志》 * |
| 潘超 等: "病原细菌多糖疫苗的多糖结合疫苗研究进展", 《生物技术通讯》 * |
| 熊慧玲等: "A群脑膜炎球菌多糖结合疫苗的制备及其稳定性考察", 《预防医学情报杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017036141A1 (en) * | 2015-08-31 | 2017-03-09 | 成都欧林生物科技股份有限公司 | Preparation method and use of group a meningococcal capsular polysaccharide conjugate vaccine |
| CN106344915A (en) * | 2016-08-29 | 2017-01-25 | 成都欧林生物科技股份有限公司 | Preparation method of group A meningococcal capsular polysaccharide conjugate vaccine |
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