CN109908335A - A kind of preparation method of meningococcal capsular polysaccharide conjugate - Google Patents
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Abstract
The invention belongs to production of vaccine technical fields, specifically disclose a kind of preparation method of meningococcal capsular polysaccharide conjugate, the present invention uses meningococcal capsular polysaccharide derivates and diphtheria CRM197 albumen for raw material, it is coupled using carbodiimide EDAC as crosslinking agent, it is prepared by the technique of derivative solution preparation, mixing match, association reaction, ageing treatment, ultrafiltration, degerming, reduce process flow, the polysaccharide yield and protein yield for effectively increasing conjugate, achieve the purpose that reduce cost, improve benefit.
Description
Technical field
The invention belongs to production of vaccine technical field, in particular to a kind of preparation of meningococcal capsular polysaccharide conjugate
Method.
Background technique
Epidemic meningitis is the epidemic disease disease of the meninx inflammation as caused by Neisseria meningitidis, is not obtained yet so far effectively
Control.Neisseria meningitidis mainly via cough, sneeze or propagation of kissing, invades blood circulation by pharynx nasalis, finally limits to
In meninx and membrane of spinal cord, suppurative meninges pathology is formed.Common symptom include have a high fever, severe headache, posterior neck it is stiff；
Also have it is drowsiness, vomit, fear the appearance of situations such as light or fash.This disease can be led to that brain injury is even dead, and case fatality rate is higher, dimension
It holds between 5-10%, is height with child morbidity in distributing or large and small prevalence all over seeing countries in the world.
Neisseria meningitidis according to the specificity of its capsular polysaccharide meningococcus can be divided into A, B, C, D, 29E, H,
I, 13 sero-groups of K, L, W135, X, Y, Z, the bacterium of all sero-groups can cause a disease, but A, C, W135 and Y virulence are most strong,
Above-mentioned 4 sero-groups account for 95% of case load or more, are to cause the popular most common bacterial strain of meningitis.
Capsular polysaccharide can shield bacterial cell surface functional component, make it from being identified by host immune system, prevent
Complement system is swallowed by the protein activation of bacterium surface and immunocyte.If bacterium is swallowed by immunocyte, capsular polysaccharide
It can be avoided bacterium to be killed.Capsular polysaccharide is one of major antigen ingredient of meninx Neisseria, is used as epidemic meningitis polysaccharide vaccine
Main component, have certain protection to biggish children.However, epidemic meningitis polysaccharide vaccine is in 2 one full year of life infants below
Interior the induced antibody duration is short, is unable to inducing immunological memory, therefore acted on without lasting immunity infant, needs to take
Immunity inoculation is repeated several times, infant's peace is made to cross meningitis popular season.
Currently, some researches show that the capsular polysaccharide CPS of epidemic meningitis polysaccharide vaccine is thymus dependent antigen TI-Ag, using general
Polysaccharide and protein carrier are coupled, and thymus independent antigen TI-Ag can be made to be changed into thymus dependent antigen TD-Ag, to change
The antigenic property for becoming capsular polysaccharide CPS, can enhance immunogenicity, induce immunological memory.For this purpose, people put forth effort on epidemic meningitis combination
The development of vaccine.
Existing epidemic meningitis combined vaccine during the preparation process, first using polysaccharide derivates crosslinking agent mediation under with carrier egg
It is white to carry out coupling and form stable conjugate, then ultrafiltration and purifying are carried out to conjugate, successively to obtain the conjugate of high-purity.
However, the polysaccharide derivates, carrier protein, crosslinking agent and different, the prepared knot of preparation process that are used due to each producer
Polysaccharide yield and the protein yield for closing object are also different, and the polysaccharide yield and protein yield of the prior art are respectively 10% and 12% left side
There is polysaccharide yield and protein yield be low, at high cost, benefit is low and deficiency in the right side.
Summary of the invention
Aiming at the problems and shortcomings existing in the prior art, the object of the present invention is to provide a kind of meningococcal capsulars
The preparation method of polysaccharide conjugate uses meningococcal capsular polysaccharide derivates and diphtheria CRM197 albumen for raw material, adopts
Carbodiimide EDAC is used to be coupled as crosslinking agent, at derivative solution preparation, mixing match, association reaction, timeliness
Reason, ultrafiltration, degerming technique prepared, reduce process flow, effectively increase conjugate polysaccharide yield and albumen receive
Rate achievees the purpose that reduce cost, improve benefit.
To realize above-mentioned first purpose, the present invention provides the following technical scheme that
A kind of preparation method of meningococcal capsular polysaccharide conjugate, comprising the following steps:
Step 1: derivative solution preparation
Meningococcal capsular polysaccharide derivates are put into beaker, temperature control are carried out using water-bath, temperature is controlled in 4~8 DEG C of models
In enclosing, stirring and adjusting pH value adjusts the pH value of liquid using the sodium chloride solution of 0.2mol/L and the hydrochloric acid solution of 0.1mol/L
To 6.00 ± 0.30 ranges, derivative solution is obtained；
Step 2: mixing match
Temperature controls under conditions of 4~8 DEG C, and diphtheria CRM197 albumen, stirring is added in the derivative solution into beaker
Mixing, until the meningococcal capsular polysaccharide derivates of object solution derived from beaker and the volume ratio of diphtheria CRM197 albumen reach
1:0.90~1:2.10, the reaction density of meningococcal capsular polysaccharide derivates are balanced between 0.5~2.5mg/mL
Solution；
Step 3: association reaction
Temperature controls under conditions of 4~8 DEG C, and EDAC is added in the balance solution into beaker, is stirred, until in beaker
The concentration of tolerant middle EDAC reaches 20~80mmol/L, starts association reaction, and control reaction temperature is maintained at 4~8 DEG C, and reaction is used
When 2~4 hours, obtain initial action object；
Step 4: ageing treatment
Beaker is placed in 2~8 DEG C of refrigerator together with the initial action object, stands 18 hours progress ageing treatments, obtains timeliness
Reactant；
Step 5: ultrafiltration
By the timeliness reactant, 6 times are carried out with the ultrafiltration membrane packet of ultrafilter and 100kD and changes liquid ultrafiltration, every time with the timeliness
The distilled water of 5 times of volumes of reactant is diluted, during removing association reaction after extra remaining reagent, in conjunction with
Object concentrate；
Step 6: degerming
Aseptic filtration is carried out to the conjugate refined solution with 0.22 μm of filter membrane, it is former to obtain meningococcal capsular polysaccharide conjugate
Liquid.
Further, in step 1, the meningococcal capsular polysaccharide derivates are A groups of capsular polysaccharide derivatives, C groups
One of capsular polysaccharide derivative, W135 groups of capsular polysaccharide derivatives, Y groups of capsular polysaccharide derivatives.
Further, in step 2, the ratio of A groups of capsular polysaccharide derivatives and diphtheria CRM197 albumen is 1:0.90~1:
1.60。
Further, in step 2, the ratio of C groups of capsular polysaccharide derivatives and diphtheria CRM197 albumen is 1:0.90~1:
1.40。
Further, in step 2, the ratio of W135 groups of capsular polysaccharide derivatives and diphtheria CRM197 albumen is 1:0.95
~1:1.30.
Further, in step 2, the ratio of Y groups of capsular polysaccharide derivatives and diphtheria CRM197 albumen is 1:0.95~1:
1.30。
Further, the meningococcal capsular polysaccharide conjugate is suitable for preparation monovalence epidemic meningitis combined vaccine and multivalence
Application during epidemic meningitis combined vaccine.
The invention has the following advantages:
CRM in the present invention197For the non-toxic variant of diphtheria toxin, there is no the risks that toxicity reply occurs in vaccine；EDAC conduct
The carboxyl group activator activation of protein of diphtheria CRM197 albumen, the carboxyl on diphtheria CRM197 albumen first reacted with EDAC after again with
The primary amine reaction of meningococcal capsular polysaccharide derivates linking arm end forms amido bond, thus by meningococcal capsular
Polysaccharide derivates and diphtheria CRM197 albumen connect into polysaccharide-protein conjugate, the spy with easy to operate, high efficiency, safety
Point.
During the preparation process, the technological parameter being related to is numerous for meningococcal capsular polysaccharide conjugate, as long as adjustment is wherein
One Xiang Zhibiao, there are biggish floatings for obtained yield.The present invention is that applicant is passing through creative work and largely testing
What confirmation obtained after testing, when meeting simultaneously in preparation step, the concentration of EDAC is 20-80mmol/L, the pH of derivative solution is
6.00 ± 0.30, meningococcal capsular polysaccharide derivates and diphtheria CRM197The volume ratio of albumen is 1:0.90~1:2.10, knot
When conjunction temperature is 4~8 DEG C, the polysaccharide yield of conjugate and protein yield can be promoted to significantly improve.
Wherein, the capsular polysaccharide derivative as corresponding to the meningococcus of different sero-groups is different, using the present invention
When preparing the conjugate of single sero-group, the polysaccharide yield average value of conjugate corresponding to A groups, C groups, W135 groups, Y groups is successively
Be 87.2%, 56.8%, 74.4% and 66.6%, protein yield average is followed successively by 83.8%, 66.9%, 67.9%,
69.6%, hence it is evident that polysaccharide yield higher than in the prior art 10% and 12% protein yield.
In addition, also eliminating purifying this operation step in the present invention, although the present invention does not purify, it is made
Conjugate remain unchanged the requirement of every quality of conjugate stoste when meeting vaccine preparation, the preparation step of conjugate is reduced with this
Suddenly, the production cycle for shortening conjugate has the characteristics that operation simplifies, production cost reduces, benefit improves.
Detailed description of the invention
Fig. 1 is the process flow chart for preparing meningococcal capsular polysaccharide conjugate.
Specific embodiment
Below in conjunction with attached drawing, invention is further described in detail.
1, material and its meningococcal capsular polysaccharide derivates are prepared:
A group's capsular polysaccharide derivative, C groups of capsular polysaccharide derivatives, W135 groups of capsular polysaccharide derivatives and Y groups of pods in the present invention
Film polysaccharide derivates are that the applicant voluntarily prepares, specific preparation method referring to application publication number be CN106317245A,
Documented by CN106366208A and CN106699914A.
Diphtheria CRM197 albumen, carbodiimide EDAC, sodium chloride, hydrochloric acid are commercial product, wherein sodium chloride and hydrochloric acid
It is that analysis is pure；Distilled water is prepared in situ by water purification machine, and it is pure to be similarly analysis.
The sodium chloride solution of 0.2mol/L: it is deployed by sodium chloride and distilled water.
The hydrochloric acid solution of 0.1mol/L: it is deployed by hydrochloric acid and distilled water.
2, a kind of preparation method of meningococcal capsular polysaccharide conjugate of embodiment is suitable for preparation monovalence epidemic meningitis and combines
Application during vaccine and multivalence epidemic meningitis combined vaccine, comprising the following steps:
Step 1: meningococcal capsular polysaccharide derivates are put into beaker by derivative solution preparation, temperature is carried out using water-bath
Control, temperature control within the scope of 4~8 DEG C, and stirring and adjusting pH value uses the sodium chloride solution of 0.2mol/L and the salt of 0.1mol/L
Acid solution is adjusted in the pH value to 6.00 ± 0.30 ranges of liquid, obtains derivative solution.
Step 2: diphtheria is added into beaker under conditions of 4~8 DEG C for the control of mixing match temperature in derivative solution
CRM197 albumen, is stirred, until the meningococcal capsular polysaccharide derivates of object solution derived from beaker and diphtheria CRM197
The volume ratio of albumen reaches 1:0.90~1:2.10, the reaction densities of meningococcal capsular polysaccharide derivates 0.5~
Between 2.5mg/mL, balance solution is obtained.
Step 3: the control of association reaction temperature is added EDAC into beaker inner equilibrium solution, stirs under conditions of 4~8 DEG C
Mixing is mixed, until the concentration of EDAC reaches 20~80mmol/L in beaker contents, starts association reaction, control reaction temperature is kept
It at 4~8 DEG C, reacts the used time 2~4 hours, obtains initial action object.
Step 4: beaker is placed in 2~8 DEG C of refrigerator by ageing treatment together with initial action object, when standing progress in 18 hours
Effect processing, obtains timeliness reactant.
Step 5: timeliness reactant is carried out 6 times with the ultrafiltration membrane packet of ultrafilter and 100kD and changes liquid ultrafiltration, every time by ultrafiltration
It is diluted with the distilled water of 5 times of volumes of timeliness reactant, during removing association reaction after extra remaining reagent, is obtained
Obtain conjugate concentrate.
Step 6: degerming carries out aseptic filtration to conjugate refined solution with 0.22 μm of filter membrane, meningococcal capsular is obtained
Polysaccharide conjugate stoste.
Embodiment 1 to embodiment 3 is prepared as described above method, the corresponding meningitis ball for preparing A groups, C groups, W135 groups and Y groups
Bacterium capsular polysaccharide conjugate measures its corresponding polysaccharide yield, protein yield by the examination criteria in " Chinese Pharmacopoeia 2015 editions "
With every qualitative items of conjugate stoste.The technological parameter specifically prepared see the table below one, and testing result see the table below two and table three.
One embodiment 1 of table to embodiment 3 meningococcal capsular polysaccharide conjugate technological parameter
Two embodiment 1 of table to embodiment 3 meningococcal capsular polysaccharide conjugate polysaccharide yield and protein yield
Three embodiment 1 of table to embodiment 3 meningococcal capsular polysaccharide conjugate quality measurements
Referring to table two, through detecting, the meningococcal capsular using A crowds prepared by the present invention, C groups, W135 groups and Y groups is more
The polysaccharide yield average value of glycoconjugate is followed successively by 87.2%, 56.8%, 74.4% and 66.6%, and protein yield average is successively
It is 83.8%, 66.9%, 67.9%, 69.6%, further calculating can obtain, meningitis capsular polysaccharide conjugate prepared by the present invention
Polysaccharide yield average value be 73.86%, protein yield average value be 69.96%, hence it is evident that it is more higher than in the prior art 10%
Sugared yield and 12% protein yield, effectively increase the polysaccharide yield and protein yield of conjugate.
Referring to table three, A crowds produced by the present invention, C groups, W135 groups and Y groups meningococcal capsular polysaccharide conjugate
Endotoxin content, free polysaccharide, floating preteins, the eluent rate of recovery of molecular size KD value < 0.2, EDAC residual, cyanide
Residual and sterility test meet the standard of Chinese Pharmacopoeia, therefore, preparation method of the invention after saving purification step still
Be able to satisfy the standard of vaccine preparation, simplify the production technology of meningococcal capsular polysaccharide conjugate, reached reduce cost,
The purpose improved benefit.
This specific embodiment is only explanation of the invention, is not limitation of the present invention, those skilled in the art
Member can according to need the modification that not creative contribution is made to the present embodiment after reading this specification, but as long as at this
All by the protection of Patent Law in the scope of the claims of invention.
Claims (7)
1. a kind of preparation method of meningococcal capsular polysaccharide conjugate, which comprises the following steps:
Step 1: derivative solution preparation
Meningococcal capsular polysaccharide derivates are put into beaker, temperature control are carried out using water-bath, temperature is controlled in 4~8 DEG C of models
In enclosing, stirring and adjusting pH value adjusts the pH value of liquid using the sodium chloride solution of 0.2mol/L and the hydrochloric acid solution of 0.1mol/L
To 6.00 ± 0.30 ranges, derivative solution is obtained；
Step 2: mixing match
Temperature controls under conditions of 4~8 DEG C, and diphtheria CRM197 albumen, stirring is added in the derivative solution into beaker
Mixing, until the meningococcal capsular polysaccharide derivates of object solution derived from beaker and the volume ratio of diphtheria CRM197 albumen reach
1:0.90~1:2.10, the reaction density of meningococcal capsular polysaccharide derivates are balanced between 0.5~2.5mg/mL
Solution；
Step 3: association reaction
Temperature controls under conditions of 4~8 DEG C, and EDAC is added in the balance solution into beaker, is stirred, until in beaker
The concentration of tolerant middle EDAC reaches 20~80mmol/L, starts association reaction, and control reaction temperature is maintained at 4~8 DEG C, and reaction is used
When 2~4 hours, obtain initial action object；
Step 4: ageing treatment
Beaker is placed in 2~8 DEG C of refrigerator together with the initial action object, stands 18 hours progress ageing treatments, obtains timeliness
Reactant；
Step 5: ultrafiltration
By the timeliness reactant, 6 times are carried out with the ultrafiltration membrane packet of ultrafilter and 100kD and changes liquid ultrafiltration, every time with the timeliness
The distilled water of 5 times of volumes of reactant is diluted, during removing association reaction after extra remaining reagent, in conjunction with
Object concentrate；
Step 6: degerming
Aseptic filtration is carried out to the conjugate refined solution with 0.22 μm of filter membrane, it is former to obtain meningococcal capsular polysaccharide conjugate
Liquid.
2. a kind of preparation method of meningococcal capsular polysaccharide conjugate according to claim 1, which is characterized in that step
In rapid one, the meningococcal capsular polysaccharide derivates are A groups of capsular polysaccharide derivatives, C crowds of capsular polysaccharide derivatives, W135
One of group's capsular polysaccharide derivative, Y groups of capsular polysaccharide derivatives.
3. a kind of preparation method of meningococcal capsular polysaccharide conjugate according to claim 2, which is characterized in that step
In rapid two, the ratio of A groups of capsular polysaccharide derivatives and diphtheria CRM197 albumen is 1:0.90~1:1.60.
4. a kind of preparation method of meningococcal capsular polysaccharide conjugate according to claim 2, which is characterized in that step
In rapid two, the ratio of C groups of capsular polysaccharide derivatives and diphtheria CRM197 albumen is 1:0.90~1:1.40.
5. a kind of preparation method of meningococcal capsular polysaccharide conjugate according to claim 2, which is characterized in that step
In rapid two, the ratio of W135 groups of capsular polysaccharide derivatives and diphtheria CRM197 albumen is 1:0.95~1:1.30.
6. a kind of preparation method of meningococcal capsular polysaccharide conjugate according to claim 2, which is characterized in that packet
Include following steps: in step 2, the ratio of Y groups of capsular polysaccharide derivatives and diphtheria CRM197 albumen is 1:0.95~1:1.30.
7. a kind of preparation method of meningococcal capsular polysaccharide conjugate as claimed in any of claims 1 to 6,
It is characterized in that, being suitable for preparing the application during monovalence epidemic meningitis combined vaccine and multivalence epidemic meningitis combined vaccine.
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