CN103623401A - Multivalent pneumococcus capsular polysaccharide-protein conjugated composition and preparation method thereof - Google Patents
Multivalent pneumococcus capsular polysaccharide-protein conjugated composition and preparation method thereof Download PDFInfo
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Abstract
The invention provides a multivalent pneumococcus capsular polysaccharide-protein conjugated composition and a preparation method thereof. The conjugated composition is formed through covalent linkages between pneumococcus capsular polysaccharides of 14 different serotypes and a carrier protein, wherein the 14 different serotypes are: 1, 3, 4, 5, 6A, 6B, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F. The conjugated composition has a good absorption effect and stability, and has multiple immunogenicity and protective property aiming at the invasion of pneumococcus of the 14 serotypes; the effect of the conjugated composition is better than that of the low price and low quality pneumonia-treating compositions on the market, and moreover the immune response of the conjugated composition is higher than that of the composition that has not been combined. By using the multivalent pneumococcus capsular polysaccharide conjugate vaccine containing the conjugated composition, the inoculation times can be reduced, the immune procedure is simplified, diseases of humans and animals caused by the pneumococcus of the 14 serotypes can be effectively prevented, moreover the disease coverage is wider, and the immune effect is better.
Description
Technical field
The invention belongs to microbiology and immunology field, specifically, relate to a kind of multivalent pneumococcal capsular polysaccharide-protein conjugation composition and method of making the same.
Background technology
Streptococcus pneumoniae (Streptococcus pneumoniae) can cause the Noninvasive diseases such as the invasive diseases such as meningitis, pneumonia and otitis media.According to the United Nations statistical data, show have every year and surpass two years old following child of 1,000,000 coccigenic disease that dies of pneumonia.At present, for the main mean of defense of streptococcus pneumoniae, be polysaccharide vaccine and combined vaccine, verified the former the protection effect in adult, but for the main Susceptible population of streptococcus pneumoniae, the protection in baby and child is but very weak.Combined vaccine can cause effective protection and immunological memory because of it in infant, and since 2000 come out first, in the whole world, many countries are included into Immunization programme or approval listing.
Only there are 7 valent pneumococcal conjugate vaccines in China at present
go through to go on the market, only can protect 7 valencys, protection domain is limited, and resembles other 10 valent pneumococcal conjugate vaccines
13 valent pneumococcal conjugate vaccine Prevenar
its serotype combination designs the distinctive serotype of ，Dui China and lacks protection mainly for European and American developed countries' popularity.
Summary of the invention
The object of this invention is to provide a kind of multivalent pneumococcal capsular polysaccharide-protein conjugation compositions, its preparation method and application.
Another object of the present invention is to provide a kind of polyvalent pneumococcal capsular polysaccharide conjugate vaccine that contains above-mentioned conjugation compositions.
In order to realize the object of the invention, a kind of multivalent pneumococcal capsular polysaccharide-protein conjugation compositions of the present invention, it is by 14 kinds of different serotypes streptococcus pneumoniae (Streptococcus pneumoniae) capsular polysaccharide and the covalently bound conjugation compositions forming of carrier protein; Wherein, described 14 kinds of different serotypes be 1,3,4,5,6A, 6B, 9V, 14,18C, 19A, 19F, 22F, 23F and 33F.
Aforesaid multivalent pneumococcal capsular polysaccharide-protein conjugation compositions, the mass ratio of capsular polysaccharide and carrier protein is 1:2-2:1, and the quality of each serotype capsular polysaccharide is serotype 1: serotype 3: serotype 4: serotype 5: serotype 6A: serotype 9V: serotype 14: serotype 18C: serotype 19A: serotype 19F: serotype 22F: serotype 23F: serotype 33F: serotype 6B=1:1:1:1:1:1:1:1:1:1:1:1:1:2.
Aforesaid multivalent pneumococcal capsular polysaccharide-protein conjugation compositions, described carrier protein should be avirulence, and can be combined with pneumonia capsular polysaccharide and can cause the protein that polysaccharide immunogenic significantly raises, and is easy to a large amount of acquisitions simultaneously.Carrier protein of the present invention is preferably diphtheria toxoid or tetanus toxin, more preferably diphtheria toxoid CRM197.The acquisition of above-mentioned protein carrier, needs, through steps such as thalline fermentation, ultrafiltration, purification, detoxifications, to be this area routine techniques.
The present invention also provides the method for the described multivalent pneumococcal capsular polysaccharide-protein conjugation compositions of preparation, respectively to 14 kinds of different serotypes (1, 3, 4, 5, 6A, 6B, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F) capsular polysaccharide of streptococcus pneumoniae carries out separation and purification, then by each serotype capsular polysaccharide respectively with carrier protein carry out covalently bound (for example, first by the capsular polysaccharide chemical activation of purification, by reductive amination method, activation capsular polysaccharide is attached on carrier protein separately respectively), form unit price conjugate, finally various unit price conjugate is mixed, obtain multivalent pneumococcal capsular polysaccharide-protein conjugation compositions.
Wherein, the step of streptococcus pneumoniae capsular polysaccharide separation and purification is:
1) set up streptococcus pneumoniae strain library, adopt work seed to carry out streptococcus pneumoniae fermentation;
2) in streptococcus pneumoniae fermentation liquid, add appropriate sodium deoxycholate to sterilize and peel off pneumonia capsular polysaccharide;
3) fermentation liquid after sterilization is carried out centrifugal, remove thalline, collect centrifugal supernatant, then carry out ultrafiltration and concentration;
4) in concentrated solution, add dehydrated alcohol, making ethanol final concentration is 25% ~ 45%, centrifugal removal nucleic acid after stirring;
5) in supernatant, add ethanol to make its final concentration reach 55% ~ 80%, after stirring, centrifugal, remove supernatant collecting precipitation;
6) precipitation that is dissolved in water, adds the phenol of 1-3 times of liquor capacity to carry out repeatedly extracting, removes albumen;
7) in supernatant, add ethanol, make its final concentration reach 55% ~ 80%, centrifugal rear collecting precipitation, obtains the pneumococcal capsular polysaccharide of purification.
Also can adopt other purification technique well known in the art, as the methods such as column chromatography are carried out purification to streptococcus pneumoniae capsular polysaccharide.
The present invention also provides the application of described multivalent pneumococcal capsular polysaccharide-protein conjugation compositions in the medicine for the preparation of prevention or treatment streptococcus pneumoniae associated diseases.
The present invention further provides a kind of polyvalent pneumococcal capsular polysaccharide conjugate vaccine, it contains described multivalent pneumococcal capsular polysaccharide-protein conjugation compositions, and optional excipient and/or the adjuvant based on aluminum.The described adjuvant based on aluminum, as aluminium oxide, aluminium hydroxide, aluminum phosphate, aluminum sulfate etc.The described adjuvant based on aluminum is commercially available, and also can adopt this area conventional method to be prepared, the preferred sodium chloride of described excipient and/or succinic acid etc.
In aforesaid polyvalent pneumococcal capsular polysaccharide conjugate vaccine, its final concentration of the adjuvant based on aluminum is 0.125-2.0mg/ml.
Aforesaid polyvalent pneumococcal capsular polysaccharide conjugate vaccine, it can be spray, injection, lyophilized preparation, capsule, tablet or pill etc.Can carry out in the following manner immunity: skin, injection (comprising subcutaneous injection, intramuscular injection, intravenous injection), oral, collunarium or mucosa spraying etc.
The preparation method of described polyvalent pneumococcal capsular polysaccharide conjugate vaccine comprises: after various unit price conjugate is adopted respectively to aluminium adjuvant absorption, mix; Or adopt again aluminium adjuvant absorption after various unit price conjugate mixing, obtain 14 valency pneumococcal capsular polysaccharide combined vaccines (14vPnC).
Polyvalent pneumococcal capsular polysaccharide conjugate vaccine of the present invention can for the protection of or the treatment patient that causes pneumonia due to pneumococcal infection, vaccine can pass through muscle, subcutaneous, intradermal routes injection or pass through mucosa delivery.This multivalence combined vaccine is suitable for inoculating 2,4,6 and the infant at 12-15 monthly age, is also more suitable in child, youth and adult.
The present invention be directed to the pneumococcal popularity of the peculiar serotype of China; creationaryly selected 1,3,4,5, the serotype combination of 6A, 6B, 9V, 14,18C, 19A, 19F, 22F, 23F and 33F; thereby develop the multivalent pneumococcal capsular polysaccharide-protein conjugation compositions designing for the pneumococcal popularity of the peculiar serotype of China; thereby at strict control carrier protein content, reduce under the prerequisite of immunosuppressive action, protection is to greatest extent provided.
Multivalent pneumococcal capsular polysaccharide-protein conjugation the compositions that adopts the inventive method to prepare, its adsorption effect and stability are better.This conjugation compositions has multiple immunogenicity and the protective value for the streptococcus pneumoniae invasion and attack of above-mentioned 14 kinds of serotypes, the low price that is better than having gone on the market time pneumonia compositions, and immunne response is higher than unconjugated compositions.Inoculate polyvalent pneumococcal capsular polysaccharide conjugate vaccine of the present invention and can reduce Inoculating needle time, simplify immune programme for children, can effectively prevent people and animal because of the disease due to above-mentioned 14 kinds of serotype streptococcus pneumoniae simultaneously, cover more extensively, immune effect is better.In addition, the polysaccharide vaccine that this multivalence combined vaccine immunogenicity effect is inferior far above similar multivalence, can effectively activate 2 years old following child B cell, thereby produce antibody, and activate memory B cell, brings out immunological memory reaction, can play better protection effect.
Polyvalent pneumococcal capsular polysaccharide conjugate vaccine of the present invention can be by the affecting conditions due to the above-mentioned 14 kinds of serotype streptococcus pneumoniae of prevention, convenient multiple-effect, and cost is lower.The production of above-mentioned 14 kinds of serotype capsular polysaccharides, all adopt the mode of the rear purification of fermentation to be prepared, there is similar production technology, be convenient to large-scale industrialization and produce, and by the capsular polysaccharide of purifying after deactivation, make vaccine there is higher safety and good immunogenicity, simultaneously, described multivalent pneumococcal capsular polysaccharide-protein conjugation composition adsorbs is complete, and physicochemical property is stable, noiseless mutually.
Zoopery shows, polyvalent pneumococcal capsular polysaccharide conjugate vaccine of the present invention can effectively induce body to produce high titre, the specific antibody for above-mentioned 14 kinds of serotype capsular polysaccharides, experiment in vitro shows, it is active with neutralization that the antibody of being induced by this multivalence combined vaccine has obvious reactivity; Particularly in compositions, the immune effect of each serotype capsular polysaccharide is not subject to impact and the interference of another component, and adsorption rate is greater than 75%, and immunogenicity is not less than unit price conjugate.This multivalence combined vaccine has with each serotype unit price in conjunction with the identical immunogenicity of stock solution, and combination inoculation can greatly reduce inoculation times on the basis not affecting the treatment.
The specific embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art, the raw materials used commercial goods that is.
Embodiment 1 pneumococcal capsular polysaccharide stock solution preparation
1,3,4,5,6A, 6B, 9V, 14,18C, 19A, 19F, 22F, 23F and 33F serotype streptococcus pneumoniae (Streptococcus pneumoniae) bacterial strain all derive from Nat'l Pharmaceutical & Biological Products Control Institute.Above-mentioned strain is gone down to posterity and sets up original species word bank, main seed bank and work seed bank, and generation is respectively: primordial seed is criticized as 1st generation, and main seed lot was the 4th generation, and work seed lot was the 8th generation.After work seed lot breakdown, to inoculation fermentation tank, cultivate, go down to posterity and should be no more than for 5 generations.Per generation seed employing milk powder is cooked freeze drying protectant, lyophilizing preservation.Get work seed at soy peptone solid medium line picking strain, be inoculated in the liquid synthetic medium of 5ml, after 35 ℃ ± 2 ℃ cultivations, transferred species is expanded to 10L culture fluid and 50L culture fluid through cultivating after the culture fluid of 500ml again.In the later stage of exponential phase or the early stage of resting stage, end to cultivate, pure bacterium inspection is carried out in sampling, and after pure bacterium passed examination, in the culture fluid of results, adding aseptic sodium deoxycholate to the final concentration of appropriate amount is that 1% sterilization two hours is with cracking bacterial cell and discharge capsular polysaccharide.To after the medium centrifugal having sterilized, collect supernatant, supernatant is with ultrafilter membrane bag ultrafiltration and concentration 3-10 times of 100KD molecular cut off.
With the phosphate buffer of about pH7.0, ultrafiltrate is adopted and carried out ultrafiltration and change liquid, then add dehydrated alcohol, making ethanol final concentration is 25% ~ 45%, centrifugal removal nucleic acid after fully stirring.
In supernatant, add ethanol to make its final concentration reach 55% ~ 80%, after stirring, centrifugal, remove supernatant collecting precipitation.
The precipitation that is dissolved in water, adds the phenol of 1-3 times of liquor capacity to carry out repeatedly extracting, removes albumen.
In supernatant, add ethanol, make its final concentration reach 55% ~ 80%, centrifugal rear collecting precipitation, to precipitate and adopt injection water to redissolve, after the sterile filters aseptic filtration of 0.2 μ m, obtain 1,3,4,5,6A, 6B, 9V, 14,18C, 19A, 19F, 22F, 23F and the 33F pneumococcal capsular polysaccharide of totally 14 kinds of purified blood serum types.
The preparation of embodiment 2 multivalent pneumococcal capsular polysaccharide-protein conjugation compositionss
By 14 kinds of pneumococcal capsular polysaccharide stock solutions of preparation in embodiment 1, adopt hydrochloric acid to carry out acidified degradation, then in polysaccharide, add sodium periodate oxidation, after oxidation, detect the activation grade of polysaccharide, then according to polysaccharide: the weight ratio of protein=1:2 ~ 2:1 adds the diphtheria toxoid CRM197 protein after purification to carry out combination, in conjunction with after add sodium borohydride to stop association reaction, prepare pneumococcal capsular polysaccharide conjugate, conjugate adopts ultrafiltration dialysis, remove unconjugated protein and polysaccharide, after purification, adopt the filter aseptic filtration of 0.2 μ m, be pneumococcal capsular polysaccharide-protein unit price conjugate.
By above-mentioned preparation 1,3,4,5, pneumococcal capsular polysaccharide-protein unit price conjugate of 6A, 9V, 14,18C, 19A, 19F, 22F, 23F and 33F serotype is according to polyoses content 4 μ g/ml, 6B conjugate mixes according to polyoses content 8 μ g/ml, obtains pneumococcal capsular polysaccharide-protein conjugation compositions of 14 valencys.
The preparation of embodiment 3 polyvalent pneumococcal capsular polysaccharide conjugate vaccines
By embodiment 2 preparation 1,3,4,5, pneumococcal capsular polysaccharide-protein unit price conjugate of 6A, 9V, 14,18C, 19A, 19F, 22F, 23F and 33F serotype is according to polysaccharide final concentration 4 μ g/ml, 6B conjugate mixes according to polysaccharide final concentration 8 μ g/ml, add aluminum phosphate adjuvant, make 14 valency pneumococcal capsular polysaccharide combined vaccines, aluminum content is 0.25mg/ml.
By 14 valency pneumococcal capsular polysaccharide combined vaccines of above-mentioned preparation, adopt the mode of cold drying to make freeze-dried formulation, before using, adopt normal saline to redissolve.
The aluminum absorption of embodiment 4 multivalent pneumococcal capsular polysaccharide-protein conjugation compositionss
Adopt in two ways multivalent pneumococcal capsular polysaccharide-protein conjugation compositions is adsorbed and prepared vaccine.
(1) pneumococcal capsular polysaccharide-protein unit price conjugate of 1 after purification, 3,4,5,6A, 6B, 9V, 14,18C, 19A, 19F, 22F, 23F and 33F serotype is first mixed, then mix with the aluminum phosphate adjuvant of 0.5 μ g/ml, make polyoses content be: 6B Serotype8 μ g/ml μ, 1,3,4,5,6A, 9V, 14,18C, 19A, 19F, 22F, 23F and 33F serotype are respectively 4 μ g/ml μ, make 14 valency pneumococcal capsular polysaccharide combined vaccines (14vPnC), aluminium adjuvant final concentration is 0.25mg/ml.
(2) by 1,3,4,5, pneumococcal capsular polysaccharide-protein unit price conjugate of 6A, 6B, 9V, 14,18C, 19A, 19F, 22F, 23F and 33F serotype mixes with the aluminum phosphate adjuvant of 0.5mg/ml respectively, make polyoses content be: 6B serotype 112 μ g/ml μ, all the other serotypes are 56 μ g/ml μ (all the other each serotypes are mixed by comparative example), aluminium adjuvant concentration is 0.25mg/ml, then the solution equal-volume after each absorption is mixed, make end-product.
Detect absorption completeness, the various polyoses content response rate of 14vPnC prepared by above-mentioned suction type.The adsorption effect of the various polysaccharide of 14vPnC and various polyoses content adopt immune turbidimetry to detect.
Polysaccharide absorption completeness and various polysaccharide recovery computational methods are as follows:
Absorption completeness:
Xn type polysaccharide is 1,3,4,5,6A, 6B, 9V, 14,18C, 19A, 19F, 22F, 23F or 33F serotype.
The content of the free various polysaccharide of not caught by aluminium adjuvant in the absorption complete response vaccine of vaccine accounts for the ratio of the various polyoses content of theory total in vaccine, and the adsorption effect of the higher demonstration vaccine of result is better.
The various polysaccharide recovery of vaccine:
After vaccine dissociates, detect respectively the content of various polysaccharide, the theoretical value of detected value and this serotype polyoses content contrasted, require the response rate be theoretical value 70% ~ 130% between.First mix adsorb afterwards proportioning mode testing result in Table 1, first respectively after absorption the testing result of mixing match mode in Table 2.
The various polysaccharide absorption completeness of table 1 14vPnC and various polyoses content result
The various polysaccharide absorption completeness of table 2 14vPnC and various polyoses content result
As can be seen from Table 1 and Table 2, the adsorption rate effect of the 14vPnC compositions (vaccine) that employing said method makes is better, all over 75%(detection limit); Various polyoses content after vaccine dissociates is between 70% ~ 130%.The content that shows vaccine is accurate, and adsorption efficiency is high.Two kinds of suction type zero differences.
Adopt aluminium hydroxide and aluminum sulfate, adjust the aluminum content final concentration of alumina gel adjuvant to 0.25mg/ml, 4.0mg/ml; Prepare multivalent pneumococcal conjugate compositions, aluminum content is 0.125mg/ml, 2.0mg/ml, also obtains similar result.
Embodiment 5 adopts the impact of different aluminum adjuvant on polyvalent pneumococcal capsular polysaccharide conjugate vaccine adsorption effect
By 1,3,4,5, pneumococcal capsular polysaccharide-protein unit price conjugate of 6A, 6B, 9V, 14,18C, 19A, 19F, 22F, 23F and 33F serotype is mixed into 6B polyoses content 16 μ g/ml, all the other each serotype polyoses content 8 μ g/ml, then respectively with the aluminum phosphate of 0.5mg/ml, the aluminum hydroxide adjuvant equal-volume mixing and absorption of the aluminum sulfate of 0.5mg/ml, 0.5mg/ml.After absorption, centrifugal adsorbed product, draws supernatant, adopts immune turbidimetry to detect the various polyoses content in supernatant, detects the adsorption effect (table 3) of each proportioning mode.
The adsorption effect of table 3 different aluminum adjuvant
As can be seen from Table 3,14 valency pneumococcal capsular polysaccharide-protein conjugation compositionss, through the absorption of different aluminium adjuvant, when same concentrations, after absorption not the various polyoses content of absorption all lower than the detection lower limit of detection method.This result shows, under this aluminium adjuvant concentration, three kinds of adjuvants are all better to the adsorption effect of multivalent pneumococcal capsular polysaccharide-protein conjugation compositions, and the adsorption effect of three kinds of adjuvants has no difference.
Embodiment 6 impacts of different carriers protein on polyvalent pneumococcal capsular polysaccharide conjugate vaccine
By 1, 3, 4, 5, 6A, 6B, 9V, 14, 18C, 19A, 19F, 22F, the pneumococcal capsular polysaccharide of 23F and 33F serotype respectively with diphtheria toxoid CRM197, diphtheria toxin, diphtherotoxin, tetanus toxin carries out coupling, then after the pneumococcal capsular polysaccharide-protein unit price conjugate each coupling mode being obtained mixes respectively, mix with the aluminum hydroxide adjuvant of 0.5mg/ml, prepare 14 valency pneumococcal capsular polysaccharide combined vaccines, making 6B serotype pneumococcal capsular polysaccharide content is 8 μ g/ml, all the other serotype capsular polysaccharide content are respectively 4 μ g/ml, aluminium adjuvant final concentration 0.25mg/ml.Detect different carriers protein for the adsorption effect under identical suction type, and immunogenicity.
Result shows, adopt after different carrier protein coupling pneumococcal capsular polysaccharides, the absorption of employing aluminium hydroxide, after absorption, centrifugal adsorbent detects various polyoses content in supernatant, testing result is all less than and detects lower limit 1 μ g/ml, show that the adsorption effect after different carriers is for pneumococcal capsular polysaccharide combination is better, and without visible difference.
The unit price pneumococcal capsular polysaccharide of different carriers-protein conjugate is adopted separately respectively to aluminum hydroxide adjuvant absorption, and 6B serotype pneumococcal capsular polysaccharide content is 8 μ g/ml, and all the other serotypes are 4 μ g/ml.Then adopt various unit price conjugate and unconjugated various unit price pneumococcal capsular polysaccharide, unconjugated various unit price pneumococcal capsular polysaccharide aluminium adjuvant adsorbate, the BALB/c mouse of immune 18-22g of while, 0,14 days two pin peritoneal immunities, each 0.5ml, blood sampling in the 28th day, adopts ELISA method to detect the antibody titer of the anti-corresponding pneumococcal capsular polysaccharide of above-mentioned serum.Normal saline is as negative control.
Result shows, the unit price pneumococcal capsular polysaccharide of 14 kinds of serotypes and the pneumococcal capsular polysaccharide of 14 kinds of serotypes and the adsorbate of aluminium adjuvant, and after 2 pin immune mouses, the IgG antibody level of mice and negative control group there was no significant difference; After the conjugate immune mouse of various GL-PP and diphtheria toxoid CRM197, diphtheria toxin, diphtherotoxin, tetanus toxin, the anti-IgG of serum all has significance to improve than the aluminum adsorbate of the unit price polysaccharide of corresponding serotype and unit price polysaccharide and negative control, simultaneously, between former three, with respect to the antibody of the pneumococcal capsular polysaccharide of same serotype, while adopting tetanus toxoid carrier except 19F, antibody has remarkable rising.The horizontal there was no significant difference of other serotypes.Polysaccharide can produce higher levels of antibody after coupling protein matter, and meanwhile, three kinds of protein all can be used for the carrier of polysaccharide and protein bound in pneumococcal conjugated vaccine.
Embodiment 7 polyvalent pneumococcal capsular polysaccharide conjugate vaccine Analysis of Immunogenicities
With 14 valency pneumococcal capsular polysaccharide-protein conjugation compositionss of embodiment 2 preparations, 14 valency pneumococcal capsular polysaccharide combined vaccine and lyophilized preparation thereof of embodiment 3 preparations, and the unit price pneumococcal capsular polysaccharide of above-mentioned 14 kinds of serotypes is, unit price pneumococcal capsular polysaccharide-protein unit price conjugate of the pneumococcal capsular polysaccharide mixture of 14 kinds of serotypes, 14 kinds of serotypes, the BALB/c mouse of immunity 18-22g, the polyoses content of each immune material is 6B 8 μ g/ml, and all the other serotypes are respectively 4 μ g/ml.10 of every group of immune mouses.0,14 days two pin peritoneal immunities, each 0.5ml, blood sampling in the 28th day, adopts ELISA method to detect the antibody titer of the anti-above-mentioned all serotype pneumococcal capsular polysaccharides of serum.
Result shows, after the unit price pneumococcal capsular polysaccharide of 14 kinds of serotypes and the pneumococcal capsular polysaccharide mixture of 14 kinds of serotypes 2 pin immune mouses, and the IgG antibody level of mice and negative control group zero difference; After the freeze-dried products of various GL-PP unit price conjugate, 14 valency pneumococcal capsular polysaccharide combined vaccines and 14 valencys pneumococcal capsular polysaccharide-protein conjugation compositions immune mouse, the anti-IgG of serum has significance to improve than negative control, meanwhile, between three with respect to the antibody horizontal there was no significant difference of the pneumococcal capsular polysaccharide of same serotype.The antibody horizontal of 14 valency pneumococcal capsular polysaccharide combined vaccines is than the remarkable rising of negative control, and higher than not containing 14 valency pneumococcal capsular polysaccharide-protein conjugation compositionss of adjuvant.Therefore, polysaccharide can produce higher levels of antibody after coupling carrier protein, meanwhile, is adsorbed onto after aluminium adjuvant antibody horizontal higher than the polysaccharide-protein conjugate of absorption not.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. multivalent pneumococcal capsular polysaccharide-protein conjugation compositions, is characterized in that, it is by 14 kinds of different serotypes streptococcus pneumoniae (Streptococcus pneumoniae) capsular polysaccharide and the covalently bound conjugation compositions forming of carrier protein; Wherein, described 14 kinds of different serotypes be 1,3,4,5,6A, 6B, 9V, 14,18C, 19A, 19F, 22F, 23F and 33F.
2. conjugation compositions according to claim 1, it is characterized in that, the mass ratio of capsular polysaccharide and carrier protein is 1:2-2:1, and the quality of each serotype capsular polysaccharide is serotype 1: serotype 3: serotype 4: serotype 5: serotype 6A: serotype 9V: serotype 14: serotype 18C: serotype 19A: serotype 19F: serotype 22F: serotype 23F: serotype 33F: serotype 6B=1:1:1:1:1:1:1:1:1:1:1:1:1:2.
3. conjugation compositions according to claim 1, is characterized in that, described carrier protein is diphtheria toxoid or tetanus toxoid.
4. conjugation compositions according to claim 3, is characterized in that, described carrier protein is diphtheria toxoid CRM197.
5. the method for preparing conjugation compositions described in claim 1-4 any one, it is characterized in that, respectively the capsular polysaccharide of 14 kinds of different serotypes streptococcus pneumoniae is carried out to separation and purification, then each serotype capsular polysaccharide is carried out covalently bound with carrier protein respectively, form unit price conjugate, finally various unit price conjugate is mixed, obtain multivalent pneumococcal capsular polysaccharide-protein conjugation compositions.
6. method according to claim 5, is characterized in that, the step of streptococcus pneumoniae capsular polysaccharide separation and purification is:
1) set up streptococcus pneumoniae strain library, adopt work seed to carry out streptococcus pneumoniae fermentation;
2) in streptococcus pneumoniae fermentation liquid, add appropriate sodium deoxycholate to sterilize and peel off pneumonia capsular polysaccharide;
3) fermentation liquid after sterilization is carried out centrifugal, remove thalline, collect centrifugal supernatant, then carry out ultrafiltration and concentration;
4) in concentrated solution, add dehydrated alcohol, making ethanol final concentration is 25% ~ 45%, centrifugal removal nucleic acid after stirring;
5) in supernatant, add ethanol to make its final concentration reach 55% ~ 80%, after stirring, centrifugal, remove supernatant collecting precipitation;
6) precipitation that is dissolved in water, adds the phenol of 1-3 times of liquor capacity to carry out extracting, removes albumen;
7) in supernatant, add ethanol, make its final concentration reach 55% ~ 80%, centrifugal rear collecting precipitation, obtains the pneumococcal capsular polysaccharide of purification.
7. the application of conjugation compositions in the medicine for the preparation of prevention or treatment streptococcus pneumoniae associated diseases described in claim 1-4 any one.
8. polyvalent pneumococcal capsular polysaccharide conjugate vaccine, it contains the conjugation compositions described in claim 1-4 any one, and optional adjuvant and/or excipient based on aluminum.
9. combined vaccine according to claim 8, is characterized in that, its final concentration of the adjuvant based on aluminum is 0.125-2.0mg/ml.
10. combined vaccine according to claim 8 or claim 9, is characterized in that, it is spray, injection, lyophilized preparation, capsule, tablet or pill.
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| CN107835821A (en) * | 2015-04-28 | 2018-03-23 | 生物E有限公司 | For the method from microbial capsular separation of polysaccharides protein and other impurities |
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| CN111757753A (en) * | 2018-02-05 | 2020-10-09 | 赛诺菲巴斯德有限公司 | Multivalent pneumococcal polysaccharide-protein conjugate composition |
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