CN106110316A - A kind of preparation method of streptococcus pneumoniae conjugate combination-vaccine - Google Patents
A kind of preparation method of streptococcus pneumoniae conjugate combination-vaccine Download PDFInfo
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Abstract
The present invention relates to a kind of streptococcus pneumoniae conjugate combination, by selecting the associated methods being suitable for different type polysaccharide to prepare high immune conjugate, and they are formed a combination, become the streptococcus pneumoniae conjugate combination of high immunogenicity, for the infectious disease preventing streptococcus pneumoniae to cause.Combination-vaccine of the present invention by selected from Pneumococcus serotypes 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20, the streptococcus pneumoniae conjugate combination of 22F, 23F, 33F is prepared from.
Description
Technical field:
The present invention relates to a kind of streptococcus pneumoniae conjugate combination, by selecting to be suitable for the associated methods system of different type polysaccharide
Standby high immune conjugate, and they are formed a combination, become the streptococcus pneumoniae conjugate combination of high immunogenicity, for pre-
The infectious disease that anti-streptococcus pneumoniae causes.
Background technology:
Streptococcus pneumoniae is to cause developing country and developed country each age group crowd's community acquired pneumonia
(community acquire pneumonia, CAP) most important pathogen, is also to cause otitis media, pneumonia, meninges simultaneously
Scorching and bacteremic the main pathogenic fungi.Worldwide, the death caused due to streptococcus pneumoniae infection every year accounts for total death
The 9% of number, every year by pneumonia streptococcus microbial meningitis 30,000 many cases, bacteremia 630,000 many cases, otitis media 14,000,000 example,
Pneumonia 20,000,000 example.
The biological product of prevention pneumococcal infection include polysaccharide vaccine and combined vaccine, and wherein polysaccharide vaccine can be used for 2
Year above crowd, be mainly suitable for the special population etc. that crowd is old people and some immunologic hypofunctions, and combined vaccine can
Using infant and above crowd, immunogenicity is more preferable, can induce immunity anamnesis reaction, remove Fit First etc., compare polysaccharide
Vaccine has obvious advantage, is a direction of Pnu-Imune 23 research, the pneumococcal conjugated vaccine the most listed
Including " 13 valent pneumococcal conjugate vaccine " and " 10 valent pneumococcal conjugate vaccine " of GlaxoSmithKline PLC company of Pfizer,
Pfizer's former listing kind " 7 valent pneumococcal conjugate vaccine " is the most gradually substituted by 13 valency vaccines.
The main antigenic component of combined vaccine is GL-PP conjugate, and the preparation method of GL-PP conjugate has many
Kind, conventional method includes:
1. directly in conjunction with method:
Utilize reactive group intrinsic on polysaccharide structures directly or by interval dose and carrier protein phase coupling.In Song
Containing carboxylic group in family name's dysentery polysaccharide structures, by the mediation of carbodiimide (EDC), be combined with adipyl dihydrazide, then with load
Carboxyl reaction on body protein, formed combine product (Taylor DN, TrofaAC, Sadoff J, et al.Synthesis,
Characterization, and clinical evaluation of conjugate vaccine composed of O-
Specific polysaccharides of Shigella dysenteriae type 1, shigella flexneri
Type 2a, and shigella sonnei (Plesiomonas shigelloides) bound to bacterial
Toxoids [J] .Infect Immun 1993,61:3678-3687.).
Carbodiimide, structure is as follows
Complete entitled 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, is usually used in the condensation of carboxyl and primary amine.
Carbodiimide can form, with carboxyl, O-acyl isourea (O-acylisourea) intermediate that can react with amino, is having amino
In the presence of, can be condensed.But if reaction system does not has amino, this intermediate can quickly hydrolyze and revert to carboxyl shape
Formula.If increasing N-hydroxy thiosuccinimide (N-hydroxysulfosuccinimide, Sulfo-in reaction system
NHS), then can form stable active fat intermediate, can be condensed with amino further.Carbodiimide condensation reaction scheme is as follows.
2. cyano group activation method:
By the adjacent hydroxyl on cyanogen root activated polysaccharide chain, then it is connected with six carbon interval dose adipyl dihydrazide (ADH), derives polysaccharide
Prepare derivative polysaccharide.Polysaccharide derivative and carrier protein are under the mediation of carbodiimide, by carbodiimide and carrier
Carboxyl on protein forms O-acyl isourea intermediate, then reacts formation proteinpolysaccharide conjugate with the amido on polysaccharide derivative.
The cyanide reagent that this cyano group activation method uses is usually Bromine cyanide. (CNBr), 1-cyano group-4-dimethylamino
Base-pyridine tetrafluoride boron (CDAP) etc..Use interval dose include succinic acid hydrazide ii (Butanedihydrazide), oneself two
Two hydrazine class compounds such as acyl two hydrazine (ADH), also include diaminoethanes, diaminopropanes, diaminobutane, diamino hexane etc.
Diaminourea alkyl compound, also includes the ester type compounds such as adipic acid two succinimide ester (DEA), also includes containing reactive functional group
The interval dose that group is inconsistent, such as amineothiot.
When Chiayung Chu etc. prepares combined vaccine, just using cyanogen bromide-activated polysaccharide, adipyl dihydrazide derives as interval dose,
By EDAC mediation condensation, prepare conjugate (Chu Chiayung, R.Schneerson, JB Robbins, et al.Further
Studies on the Immunogenicity of Haemophilus influenzae Type b and Pneumococcal
Type 6A Polysaccharide Protein Conjugates [J] .Infect Immun 1983,40:245-256.).Also have
Do not use interval dose, such as Carine Capiau etc. with CDAP activated polysaccharide after, directly the amino with carrier protein reacts, and makes
For going out pneumonia and epidemic encephalitis combined vaccine (U.S.Pub.No.2003/0147922), " the 10 valency streptococcus pneumoniae knots of GlaxoSmithKline PLC listing
Close vaccine " just use CDAP activated polysaccharide, and conjugate is prepared in direct and carrier protein amido reaction.
3. Amine reduction:
Polysaccharide oxidized or hydrolysis after, can end generate aldehyde radical, aldehyde radical can with amino react formed schiff bases structure
(Schiffs ' base), then reduce through reducing agent, stable C-N structure can be formed, prepare conjugate reaction scheme as follows
Use sodium periodate method, periodic acid the most glycoxidative is a kind of oxidant more.Polysaccharide contains abundant glycol-based
(CHOH-CHOH), after periodate oxidation, two terminal aldehyde groups are broken to form.The feature of periodic acid is the most further to aoxidize
The aldehyde radical that the sixth of the twelve Earthly Branches generates.Such as following C group's epidemic encephalitis polysaccharide Malaprade reaction
Ammonia reducing process is a kind of proteinpolysaccharide conjugate preparation method that field of medicaments is most widely used.Jennings etc. adopt
Epidemic encephalitis combined vaccine (U.S.Pat.No.4,356,170) is prepared by ammonia reducing process.Anderson is also adopted by ammonia reducing process and prepares b
Type hemophilus influenza micromolecular polysaccharide and the conjugate (U.S.Pat.No.4,761,283) of protein.Pfizer
7 valent pneumococcal conjugate vaccines and 13 valent pneumococcal conjugate vaccines that (Hausdorff etc.) list are also adopted by Amine reduction
(U.S.Pub.No.2007/0184071A1).These are all the aldehyde radical utilizing how glycoxidative generation ammonia directly and on protein
Base reacts, and by the reduction of boron Cyanogran., prepares conjugate.Also there is scholar to utilize the principle of ammonia reducing process, first polysaccharide is produced
Aldehyde radical be connected with the interval dose containing amino, then be connected with carrier protein by interval dose.Jessouroun etc. are by albumen
Matter is modified with diazanyl, then the polysaccharide containing aldehyde radical after oxidation is combined (U.S.Pub.No.2007/0110762A1) with ammonia reducing process.
Amino group is connected to reduce on oligosaccharide by Porro with ammonia reducing process, then be connected with carrier protein (U.S.Pat.No.5,
306,492)。
4. other associated methods:
Including oxime chemical reaction, diazo-reaction, disulfide bond coupled reaction etc., but it is not the widest for using.N-succinyl is sub-
Amine-3-dithiopyridines propanoic acid (SPDP) is conventional disulfide bond coupled reaction reagent, and SPDP is first connected to the group containing amino
On Fen, with the component generation sulfydryl exchange reaction containing sulfydryl, form the conjugate that disulfide bond connects.Kossaczka etc. use should
Method is prepared for typhoid fever combined vaccine, but human trial data proving effect undesirable (KossaczkaZuzana, FY Lin,
VAHo, et al.Safety and Immunogenicity of Vi Conjugate Vaccines for Typhoid
Fever in Adults, Teenagers, and 2-to 4-Year-Old Children in Vietnam [J] .Infect
Immun 1999,67:5806-5810.).Below for the association reaction of SPDP mediation.
Streptococcus pneumoniae has more than 90 serotype, the exploitation more options initiative and advantages of the localities serotype of vaccine, and therefore, vaccine is all many
Valency time, as 10 valencys listed, 13 valencys and Merck & Co., Inc. enter 15 clinical valencys.At present, each company exploitation multivalence pneumonia
During coccus combined vaccine, being mostly to utilize respective technical characterstic and advantage to prepare conjugate, the preparation of each valency time conjugate is all adopted
Using the cyano group activation method of CDAP mediation to prepare 10 kinds of conjugates by same process, such as GlaxoSmithKline PLC, Pfizer uses
Amine reduction prepares its 7 valent pneumococcal conjugate vaccine and 13 valent pneumococcal conjugate vaccines, 15 valency pneumonia balls of Merck & Co., Inc.
Bacterium combined vaccine is also adopted by Amine reduction and prepares conjugate (the raw thinking brought by combined vaccine product Historic Evolution of Du Lin Jiang Ren
" microbiology immunology progress " 2013,41 (6): 60-65.).It is a discovery of the invention that with the pneumococal polysaccharide of identical type with
When identical carrier protein prepares conjugate, the conjugate that different associated methods obtains has difference in immunogenicity,
And difference depends primarily on the type of polysaccharide, rather than the other polysaccharide of associated methods, i.e. different shaped has the conjugate being more suitable for it
Preparation method, is just substantially better than amine reduction as research report uses CDAP method to prepare 19F type streptococcus pneumoniae conjugate immune effect
Method (Jan Poolman, Carl Frasch, Anu Nurkka, et al.Impact of the Conjugation Method
on the Immunogenicity of Streptococcus pneumoniae Serotype 19F
Polysaccharidein Conjugate Vaccines.Clin Vaccine Immunol.2011,18 (2): 327-
336.), the present invention is directed to pneumococcal different type, select the method being suitable for prepare various conjugate, then conjugate is joined
Be made as the multivalent pneumococcal conjugate of a price time, described conjugate preparation method be two kinds or more than.
Summary of the invention:
The present invention relates to the preparation method of a kind of streptococcus pneumoniae conjugate combination-vaccine, described combination-vaccine is by multiple lung
Scorching coccus conjugate combination is prepared from, and described streptococcus pneumoniae conjugate is corresponding Pneumococcus serotypes capsular polysaccharide and physiology
The high immune polysaccharide albumen that upper acceptable carrier protein is prepared by selecting the associated methods being suitable for different type polysaccharide is tied
Compound, the preparation method of described conjugate uses two or more associated methods, including directly in conjunction with method, cyano group activation
Method, Amine reduction and other associated methods.
Preferably, described combination-vaccine by selected from Pneumococcus serotypes 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A,
11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20, the combination of the streptococcus pneumoniae conjugate of 22F, 23F, 33F is prepared from.
Most preferably, described combination-vaccine by selected from Pneumococcus serotypes 1,2,3,4,5,6A, 6B, 7F, 9V, 12F,
14, the streptococcus pneumoniae conjugate combination of 18C, 19A, 19F, 23F is prepared from.
The most preferred combination-vaccine of the present invention, preparation method is as follows:
Wherein 19A and 19F use CDAP mediation cyano group activation method prepare conjugate, 1,2,3,4,5,6A, 6B, 7F, 9V,
12F, 14,18C, 23F use Amine reduction prepare conjugate.
The most preferred combination-vaccine of the present invention, preferred preparation method is as follows:
19A and 19F uses the cyano group activation method of CDAP mediation to prepare conjugate, and preparation method is as follows:
Dissolving pneumococal polysaccharide, after acetum hydrolyzes, adjust pH value to 6.4, add sodium periodate oxidation, glycerol is eventually
Small-molecule substance is removed in only reaction, dialysis or ultrafiltration, it is thus achieved that oxidation of polysaccharides.Oxidation of polysaccharides adds ADH and boron Cyanogran. to end
Concentration 40mg/ml and 2mg/ml, room temperature reaction 3 days, prepare derivant.Polysaccharide derivative and carrier protein press certain mass than mixed
Close, add EDAC to final concentration 0.02mol/L, maintain pH value 5.6 to react more than 150 minutes, prepare conjugate.Conjugate warp
Sepharose 4FF chromatography purification, collects V0Neighbouring eluting peak, it is thus achieved that conjugate stock solution；
1,2,3,4,5,6A, 6B, 7F, 9V, 12F, 14,18C, 23F use Amine reduction to prepare conjugate, preparation method is as follows:
Dissolving pneumococal polysaccharide, adjustment pH value is to about 9.0, and in mass ratio 1: 0.75 addition CDAP acetonitrile solution is (dense
Degree is 100mg/ml), maintaining pH value is 9.00-10.00 reaction, adds 2% triethylamine solution with CDAP equal-volume, and pH value is
10.00-11.00, react 5min；Adjusting pH value is about 8.60, adds albumen by GL-PP quality than 1: 1, maintains pH value
Reaction more than 120 minutes near 8.6, glycine obtains polysaccharide conjugate after terminating reaction, ultrafiltration or dialysis.Conjugate warp
Sepharose 4FF chromatography purification, collects V0Neighbouring eluting peak, it is thus achieved that conjugate stock solution.
Associated methods of the present invention includes directly in conjunction with method, cyano group activation method, Amine reduction and other associated methods,
Aldehyde radical, carboxyl or the amino etc. that obtain on capsular polysaccharide are utilized to be joined directly together with carrier protein, it is possible to by bridging agent and load
Body protein is connected, and described bridging agent includes, but are not limited to succinyl two hydrazine (SDH), adipyl dihydrazide (ADH), 3-(2-
Pyridine dimercapto) propanoic acid N-hydroxy-succinamide ester (SPDP) etc..In associated methods, 19 types are prepared the preferred cyano group of conjugate and are lived
Change method, is further the cyano group activation method of CDAP mediation, and the preparation of other type conjugates is preferably lived directly in conjunction with method, cyano group
Change method and Amine reduction, more preferably cyano group activation method and Amine reduction, most preferably Amine reduction.
Carrier protein of the present invention is physiologically acceptable carrier protein, including diphtheria toxoid (DT) and
Its cross reacting material (CRM), CRM is a class and diphtheria antitoxin forms the material of reaction, containing as CRM197, CRM9,
CRM173, CRM228 and CRM45；Including tetanus toxoid (TT), DT-Pa (PT), choleratoxin B subunit, no
Thermotolerant coliform enterotoxin (LT), thermotolerant coliform enterotoxin (ST), Pseudomonas aeruginosa Exotoxin A (EPA)；Including adventitia egg
White complex (OMPC), pneumolysin (PLY), Pneumococal surface protein A (PspA), pneumococcal adhesin protein A
(PsaA), Hinfluenzae protein D.Carrier protein preferred DT, TT and CRM197, most preferably DT.
In multivalent pneumococcal conjugate of the present invention combination, the content of various polysaccharide is 0.1-10 μ g；Preferred content is
0.5-5μg；Most preferred content is 1-4 μ g.In conjugate compositions, the content of various polysaccharide can be identical, it is also possible to different.
Multivalent pneumococcal conjugate of the present invention combination can comprise adjuvant and/or buffer, and described adjuvant is helped selected from aluminum
Agent, such as aluminium hydroxide, aluminum phosphate, aluminum sulfate etc..Oil-in-water or bacteria cell wall composition, such as MF59, SAE, RAS etc..Or soap
Angle class adjuvant, such as Quil A, QS-21.Or bacteria lipopolysaccharide or synthesis lipoid A adjuvant.Cytokine, as interleukin,
Interferon, colony stimulating factor, tumor necrosis factor, costimulation molecules B7-1, B7-2 etc..Or toxin subunit class adjuvant,
Such as cholera toxin and subunit, pertussis toxin, PT, E.coli LT (LT) etc..Described buffer is selected from phosphoric acid
Buffer or Tris buffer, it is also possible to be physiological sodium chloride solution.Buffer concentration can be 0.01-0.05M, preferably
0.01-0.02M, most preferably 0.01M.
The multivalent pneumococcal conjugate combination of the present invention also can comprise stabilizer and/or dispersant, and stabilizer is selected from ammonia
Base acid, aminoacid can be histidine, preferably L-Histidine, concentration 1-100mM, preferably 2-50mM, more preferably 3-10mM,
Most preferably 5mM.Histidine can occur with the form of histidine buffering liquid.Dispersant is selected from many polysorbate, preferably Tween
80, concentration 0.01-1%, more preferably 0.01-0.1%, most preferably 0.02-0.05%.
The multivalent pneumococcal conjugate combination of the present invention can be made for clinical various dosage forms, described system
Agent is selected from liquid dosage form, freeze-dried formulation, preferred liquid dosage form.
The multivalent pneumococcal conjugate combination of the present invention, its route of inoculation includes intramuscular injection, subcutaneous injection, Intradermal note
Penetrate approach.
Beneficial effects of the present invention is further illustrated below by way of experimental data:
The most single associated methods is prepared conjugate combination and is prepared conjugate with multiple associated methods and combine Mouse immunogenicity and compare
The present invention, than right 3 conjugates combination, is that 6 valence group close, containing Pneumococcus serotypes 6B, 9V, 14,18C,
19F, 23F, carrier protein is DT, and prepared by the cyano group activation method that wherein combination 1 all conjugates all use CDAP to mediate, group
Closing 2 all conjugates all uses Amine reduction to prepare, and in the conjugate of combination 3,19F uses the cyano group activation legal system of CDAP mediation
Standby, other all use Amine reduction to prepare.By 3 conjugate combination subcutaneous inoculation NIH mices respectively, in conjugate combination, 6B is dense
Degree is 10 μ g/ml, and remaining various conjugate concentration is 5 μ g/ml, every mouse immune 0.1ml, altogether immune 3 pins, every pin interval 2
In week, after final immunization 4 weeks, blood sampling measured each type-specific IgG antibody titre, take 2 be the end logarithm be analyzed, result is shown in
Table 1, finds that the Specific antibody titre of the induction of combination 3 is better than combining 1 and combining 2.
The specific IgG antibodies level of table 1. different conjugate combination immune mouse
| 6B | 9V | 14 | 18C | 19F | 23F | |
| Combination 1 | 9.04 | 13.24 | 9.14 | 11.94 | 13.84 | 9.24 |
| Combination 2 | 10.44 | 13.04 | 10.64 | 12.44 | 9.64 | 10.74 |
| Combination 3 | 13.24 | 13.34 | 14.34 | 13.45 | 13.94 | 13.54 |
2. the present invention prepares 15 valency streptococcus pneumoniae conjugate combination mouse immunity experiments
In 15 valency streptococcus pneumoniae conjugate combinations of experiment, the preparation of 19A and 19F conjugate uses the cyano group of CDAP mediation to live
Change method, other types use Amine reduction, have employed 2 kind of 15 valency streptococcus pneumoniae conjugate altogether, a kind of containing Aluminium phosphate adjuvant, and one
Kind without any adjuvant, in every milliliter of combination containing 1,2,3,4,5,6A, 7F, 9V, 12F, 14,18C, 19A, 19F and 23F polysaccharide each
4 μ g, containing 6B polysaccharide 8 μ g.Experiment uses NIH mice, subcutaneous inoculation 2 pin, is spaced January, and dosage is 0.1ml/ time.Latter 14 days of immunity
Blood sampling, separates serum, measures 15 species specificity IgG antibody titers.Add 2 times of SD using negative control meansigma methods to sentence as cutoff value
Determine serum antibody titer, antibody titer take the logarithm post analysis process；Increase evaluation Conversion rate for more than 4 times with antibody, the results are shown in Table
2.Com-parison and analysis, containing adjuvant with without the situation between adjuvant combination induction of antibodies titre, does not finds notable difference.
Table 2.15 valency streptococcus pneumoniae conjugate combination immunogenicity in mice
Through comparative experiments, the present invention is found surprisingly that, in the combinations of the invention, uses vaccine prepared by distinct methods
Being better than the vaccine using Same Way to prepare, especially 19A and 19F uses the cyano group activation method of CDAP mediation to prepare conjugate,
1,2,3,4,5,6A, 6B, 7F, 9V, 12F, 14,18C, 23F use Amine reduction to prepare conjugate.
Detailed description of the invention:
Below in conjunction with embodiment, the present invention will be further described, and protection scope of the present invention is not limited to the following stated:
Embodiment 1: streptococcus pneumoniae antibacterial culturing
After strain is opened, being forwarded in Columbia Blood Agar culture medium, 37 DEG C of cultivations, recovery grows bacterium colony, switching
To improvement pancreas soybean broth in a small amount, cultivate 8-12 hour, be forwarded to secondary improvement pancreas Semen sojae atricolor strain liquid culture medium
Middle cultivation, after proceed to the fermentation tank containing 35000ml fluid medium is cultivated, period maintain pH value about 7.0, raw to logarithm
Inactivation thalline during long-term latter stage.
Embodiment 2: pneumococcal capsular polysaccharide purification
After culture supernatant is centrifugal, 100K ultrafiltration through membranes concentrates, and in displacement buffer to 0.01mol/L phosphate buffer, adjusts
PH value, to 6.8～7.4, adds cetyl trimethylammonium bromide (CTAB) by 1% final concentration, stirs, placed
Night.Centrifugal collecting precipitation (supernatants collected by 7F, 14 types), adds the NaCl solution of 0.25mol/L and dissociates CTAB (3 types 0.35mol/L
NaCl solution), add the NaI of 0.5%, be sufficiently stirred for, centrifugal collect supernatant, use ultrafilter membrane ultrafiltration dialysis.Through hydroxy-apatite
Rock layers is analysed, and collects desired polysaccharide, and 75% ethanol precipitation, ethanol, washing with acetone, be purified polysaccharide after drying.
Embodiment 3: purified polysaccharide testing result
24 kinds of pneumococcal capsular polysaccharides are purified respectively, 3 batches, every type, the quality arbitration result of each batch such as table 3:
Table 3. pneumococcal capsular polysaccharide quality measurements
Embodiment 4: Amine reduction prepares conjugate
Dissolving pneumococal polysaccharide, after acetum hydrolyzes, adjust pH value to 6.4, add sodium periodate oxidation, glycerol is eventually
Small-molecule substance is removed in only reaction, dialysis or ultrafiltration, it is thus achieved that oxidation of polysaccharides.Oxidation of polysaccharides adds ADH and boron Cyanogran. to end
Concentration 40mg/ml and 2mg/ml, room temperature reaction 3 days, prepare derivant.Polysaccharide derivative and carrier protein press certain mass than mixed
Close, add EDAC to final concentration 0.02mol/L, maintain pH value 5.6 to react more than 150 minutes, prepare conjugate.Conjugate warp
Sepharose 4FF chromatography purification, collects V0Neighbouring eluting peak, it is thus achieved that conjugate stock solution.
The cyano group activation method of embodiment 5:CDAP mediation prepares conjugate
Dissolving pneumococal polysaccharide, adjustment pH value is to about 9.0, and in mass ratio 1: 0.75 addition CDAP acetonitrile solution is (dense
Degree is 100mg/ml), maintaining pH value is 9.00-10.00 reaction, adds 2% triethylamine solution with CDAP equal-volume, and pH value is
10.00-11.00, react 5min；Adjusting pH value is about 8.60, adds albumen by GL-PP quality than 1: 1, maintains pH value
Reaction more than 120 minutes near 8.6, glycine obtains polysaccharide conjugate after terminating reaction, ultrafiltration or dialysis.Conjugate warp
Sepharose 4FF chromatography purification, collects V0Neighbouring eluting peak, it is thus achieved that conjugate stock solution.
Embodiment 6: conjugate testing result
Prepare streptococcus pneumoniae 19A and 19F conjugate with the cyano group activation method of CDAP mediation, prepare other with Amine reduction
Type streptococcus pneumoniae conjugate, prepares 15 kinds of other conjugates of different shaped, each type 3 batches, the detection of each batch conjugate altogether
The results are shown in Table 4:
Embodiment 7: lyophilizing 15 valency streptococcus pneumoniae conjugate combines
15 kinds of streptococcus pneumoniae conjugate stock solutions are mixed by a certain percentage, is that (6B is 8 μ to 4 μ g/ml to various polyoses content
G/ml), addition lactose, to final concentration 0.5%, after 0.5ml/ props up subpackage, lyophilization, is prepared as 15 valency streptococcus pneumoniae conjugates
Combination.
Embodiment 8: adsorb 15 valency streptococcus pneumoniae conjugate combinations
15 kinds of streptococcus pneumoniae conjugate stock solutions are mixed by a certain percentage, adsorbs with appropriate Aluminium phosphate adjuvant, be formulated as lung
Scorching coccus various polyoses content 4 μ g/ml (6B is 8 μ g/ml), aluminium composition is not higher than the semi-finished product of 0.4mg/ml, 0.5ml/
Prop up subpackage to be prepared as adsorbing 15 valency streptococcus pneumoniae conjugates combinations.
Embodiment 9:15 valency pneumonia conjugate combination Mouse immunogenicity
Using NIH mice, subcutaneous inoculation 3 pin, every pin is spaced 2 weeks, and dosage is 0.1ml.Every pin immunity blood sampling in latter 14 days, point
From serum, measure specific serum titer.Add 2 times of SD using negative control meansigma methods and judge that serum antibody drips as cutoff value
Degree, laggard row data analysis of taking the logarithm, increase evaluation Conversion rate, the Conversion rate base of the different each pins of type time for more than 4 times with antibody
Originally 100% it is all up.
Table 5.15 valency streptococcus pneumoniae conjugate combines in mouse immune response situation
Claims (10)
1. a preparation method for streptococcus pneumoniae conjugate combination-vaccine, described combination-vaccine is by multiple streptococcus pneumoniae conjugate
Combination is prepared from, and described streptococcus pneumoniae conjugate is corresponding Pneumococcus serotypes capsular polysaccharide and physiologically acceptable load
The high immune polysaccharide protein conjugates that body protein is prepared by selecting the associated methods being suitable for different type polysaccharide, described combination
The preparation method of thing uses two or more associated methods, including directly in conjunction with method, cyano group activation method, Amine reduction and
Other associated methods.
Preparation method the most according to claim 1, it is characterised in that described combination-vaccine is by selected from Pneumococcus serotypes
1、2、3、4、5、6A、6B、7F、8、9N、9V、10A、11A、12F、14、15B、17F、18C、19A、19F、20、22F、23F、33F
Streptococcus pneumoniae conjugate combination be prepared from.
Preparation method the most according to claim 1, it is characterised in that described combination-vaccine is by selected from Pneumococcus serotypes
1,2,3,4,5,6A, 6B, 7F, 9V, 12F, 14, the streptococcus pneumoniae conjugate combination of 18C, 19A, 19F, 23F is prepared from.
Preparation method the most according to claim 3, it is characterised in that wherein 19A and 19F uses the cyano group of CDAP mediation to live
Change method prepares conjugate, 1,2,3,4,5,6A, 6B, 7F, 9V, 12F, 14,18C, 23F use Amine reduction prepare conjugate.
Preparation method the most according to claim 4, it is characterised in that step is as follows:
19A and 19F uses the cyano group activation method of CDAP mediation to prepare conjugate, and preparation method is as follows:
Dissolving pneumococal polysaccharide, after acetum hydrolyzes, adjust pH value to 6.4, add sodium periodate oxidation, glycerol terminates anti-
Should, small-molecule substance is removed in dialysis or ultrafiltration, it is thus achieved that oxidation of polysaccharides.Oxidation of polysaccharides adds ADH and boron Cyanogran. to final concentration
40mg/ml and 2mg/ml, room temperature reaction 3 days, prepare derivant.Polysaccharide derivative and carrier protein press certain mass than mixing,
Add EDAC to final concentration 0.02mol/L, maintain pH value 5.6 to react more than 150 minutes, prepare conjugate.Conjugate warp
Sepharose 4FF chromatography purification, collects V0Neighbouring eluting peak, it is thus achieved that conjugate stock solution；
1,2,3,4,5,6A, 6B, 7F, 9V, 12F, 14,18C, 23F use Amine reduction to prepare conjugate, preparation method is as follows:
Dissolving pneumococal polysaccharide, adjustment pH value is to about 9.0, and 1:0.75 in mass ratio adds CDAP acetonitrile solution, and (concentration is
100mg/ml), maintaining pH value is 9.00-10.00 reaction, adds 2% triethylamine solution with CDAP equal-volume, and pH value is 10.00-
11.00, react 5min；Adjusting pH value is about 8.60, adds albumen by GL-PP quality than 1:1, maintains near pH value 8.6
Reacting more than 120 minutes, glycine obtains polysaccharide conjugate after terminating reaction, ultrafiltration or dialysis.Conjugate is through sepharose
4FF chromatography purification, collects V0Neighbouring eluting peak, it is thus achieved that conjugate stock solution.
Preparation method the most according to claim 1, it is characterised in that wherein, described physiologically acceptable carrier protein
Matter is selected from: diphtheria toxoid (DT) and cross reacting material (CRM) thereof, tetanus toxoid (TT), DT-Pa (PT),
Choleratoxin B subunit, thermo-labile enterotoxins of Escherichia coli (LT), thermotolerant coliform enterotoxin (ST), the outer poison of bacillus pyocyaneus
Element A (EPA)；Outer membrane protein composite (OMPC), pneumolysin (PLY), Pneumococal surface protein A (PspA), lung
Scorching coccus attachment proteins A (PsaA), Hinfluenzae protein D.
Preparation method the most according to claim 1, it is characterised in that wherein, in the combination-vaccine of unit dose, various pod
The content of film polysaccharide is 0.1-10 μ g.
Preparation method the most according to claim 1, it is characterised in that wherein, in the combination-vaccine of unit dose, various pod
The content of film polysaccharide is 0.5-5 μ g.
Preparation method the most according to claim 1, it is characterised in that wherein, in the combination-vaccine of unit dose, various pod
The content of film polysaccharide is 1-4 μ g.
Preparation method the most according to claim 3, it is characterised in that wherein, in the combination-vaccine of unit dose, various
Capsular polysaccharide content is combined as 1,2,3,4,5,6A, 7F, 9V, 12F, 14, each 2 μ g of 18C, 19A, 19F and 23F, 6B is 4 μ g.
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| CN108079286A (en) * | 2018-01-19 | 2018-05-29 | 云南沃森生物技术股份有限公司 | A kind of 13 valency pneumococcal polysaccharide-protein combination compositions and its preparation method and application |
| CN108144056A (en) * | 2016-12-02 | 2018-06-12 | 武汉博沃生物科技有限公司 | Polyvalent pneumococcal polysaccharide combination vaccine and preparation method thereof |
| CN108245674A (en) * | 2018-01-18 | 2018-07-06 | 北京智飞绿竹生物制药有限公司 | A kind of prescription of multivalent pneumococcal conjugate combination-vaccine and preparation method thereof |
| CN109771640A (en) * | 2019-02-28 | 2019-05-21 | 北京智飞绿竹生物制药有限公司 | Multivalent pneumococcal combined vaccine |
| CN112646050A (en) * | 2021-01-09 | 2021-04-13 | 北京智飞绿竹生物制药有限公司 | Purification process of pneumococcal polysaccharide |
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| CN108144056A (en) * | 2016-12-02 | 2018-06-12 | 武汉博沃生物科技有限公司 | Polyvalent pneumococcal polysaccharide combination vaccine and preparation method thereof |
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| CN108079286A (en) * | 2018-01-19 | 2018-05-29 | 云南沃森生物技术股份有限公司 | A kind of 13 valency pneumococcal polysaccharide-protein combination compositions and its preparation method and application |
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| CN109771640A (en) * | 2019-02-28 | 2019-05-21 | 北京智飞绿竹生物制药有限公司 | Multivalent pneumococcal combined vaccine |
| CN112646050A (en) * | 2021-01-09 | 2021-04-13 | 北京智飞绿竹生物制药有限公司 | Purification process of pneumococcal polysaccharide |
| CN114106210A (en) * | 2021-11-09 | 2022-03-01 | 北京智飞绿竹生物制药有限公司 | Production process of 23-valent pneumococcal polysaccharide vaccine |
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