CN108079286A - A kind of 13 valency pneumococcal polysaccharide-protein combination compositions and its preparation method and application - Google Patents

A kind of 13 valency pneumococcal polysaccharide-protein combination compositions and its preparation method and application Download PDF

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CN108079286A
CN108079286A CN201810053602.1A CN201810053602A CN108079286A CN 108079286 A CN108079286 A CN 108079286A CN 201810053602 A CN201810053602 A CN 201810053602A CN 108079286 A CN108079286 A CN 108079286A
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polysaccharide
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protein
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pneumococcal
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CN108079286B (en
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黄镇
向左云
施競
吴凯
袁琳
方国良
陈玉秋
钱雯
王铭
左智洁
陈南萍
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YUNNAN WOSEN BIOTECHNOLOGY CO Ltd
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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
    • 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
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • 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
    • A61K2039/70—Multivalent vaccine

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Abstract

The invention discloses a kind of 13 valency pneumococal polysaccharide protein binding compositions and its preparation method and application.The composition includes 13 kinds of different pneumococal polysaccharide protein conjugates, wherein each pneumococal polysaccharide protein conjugates includes capsular polysaccharide and carrier protein from different Pneumococcus serotypes, the capsular polysaccharide derives from 1 type, 3 types, 4 types, 5 types, 6A types, 6B types, 7F types, 9V types, 14 types, 18C types, 19A types, 19F types and 23F type pneumococcus, and carrier protein is the TT of monomer purity >=90%.Composition provided by the invention, which is verified to can induce in 5 one full year of life of 2 monthly age crowd through the clinical research of three phases, generates the special IgG antibody of serotype, wherein at 26 monthly age of primary target population after 3 pin fundamental immunities, the ratio of the μ g/ml of all serotype specificity IgG concentration >=0.35 >=85%, it is reinforced it is immune after ratio >=95%.

Description

A kind of 13 valency pneumococcal polysaccharide-protein combination compositions and preparation method thereof with Using
Technical field
The invention belongs to pneumococal polysaccharide protein conjugates technical field more particularly to a kind of 13 valency pneumococcus are more Sugar-protein binding compositions and its preparation method and application.
Background technology
Pneumococcus (Streptococcus pneumoniae) is meningitis, pneumonia and bad attack in global infant Property disease Etiological, according to the World Health Organization issue《Pnu-Imune 23 position paper》, the WHO predictions whole world is every year Less than 5 years old death of child 8,800,000, wherein because death of child caused by pneumococcal infection is 47.6 ten thousand.Developing country's lung The morbidity and mortality of scorching coccus disease are above developed country, and death mainly appears on Africa, Asia.China is every The die of pneumonia number of coccus disease of year 5 years old Infants Below is more than 30,000 people.
Pneumococcus can be divided into 90 serotypes according to capsular polysaccharide difference, the age, symptom, disease severity, Manage the distribution of the factors influences Pneumococcus serotypes such as position and pneumovax service condition.
Research shows that pneumococcal capsular polysaccharide is the main protection antigen of pneumococcus.1977, U.S.'s approval 14 Valency pneumococcal polysaccharide vaccine listing (1,2,3,4,6A, 7F, 8,9N, 12F, 14,18C, 19F, 23F, 25F), later again into 23 valency pneumococcal polysaccharide vaccines, 23 valency pneumonia ball of nineteen eighty-three are had developed on the basis of one step monitoring pneumococcus pathogenic strain Granulose vaccine goes through to list, and 23 valency pneumococcal polysaccharide vaccines 2 years old or more children and adolescents, adult are equal in the elderly Show the good result of prevention pneumococcal disease.But since pneumococal polysaccharide belongs to T cell-independent antigen, Infant is very weak for the immune response of pneumococal polysaccharide, and Wyeth's (being now subsidiary of Pfizer) successfully has developed the whole world the One pneumococcal conjugated vaccine --- 7 valent pneumococcal conjugate vaccines (Prevnar), and ratified to list through FDA in 2000.
7 valent pneumococcal conjugate vaccines only contain from serotype 4,6B, 9V, 14, the capsular polysaccharide of 18C, 19F and 23F, Each polysaccharide and CRM197(Cross reacting material 197) carrier protein combines, since the vaccine includes serotype Not less and its serotype type selecting is based primarily upon the U.S., European pneumococcal disease epidemic data, in covering for the U.S. Lid rate is 86%, and the coverage rate in Europe is 71%, and only has about 38% coverage rate in Asia.
After the U.S., European infant widely use 7 valent pneumococcal conjugate vaccine immunity inoculations, by 4,6B, 9V, 14, Pneumococcal disease is greatly lowered caused by 18C, 19F and 23F, and nonvaccine serotype causes pneumococcal disease then to present Certain rising.To improve vaccine protective rate, GlaxoSmithKline PLC is developed and has listed 10 valent pneumococcal conjugate vaccines (Synflorix), wherein add 1 compared with 7 valent pneumococcal conjugate vaccines, 5,7F；Hui Shi is developed and is had listed 13 valency pneumonia balls Bacterium combined vaccine (Prevnar 13), wherein add 1 compared with 7 valent pneumococcal conjugate vaccines, 3,5,6A, 7F, 19A.13 valency lungs Coverage rate of the scorching coccus combined vaccine in the U.S. is 92%, and European coverage rate is 89%, and in Asia, coverage rate is 74%.
Chinese patent application CN200680017776.8 " multivalent pneumococcal polysaccharide-protein conjugate composition ", it is public Opened a kind of method for preparing 13 valent pneumococcal conjugate vaccines, used in carrier protein for CRM197, and using reduction amine Chemical industry skill is combined object preparation." multivalent pneumococcal polysaccharide-protein is conjugated Chinese patent application CN200780051661.5 Compositions " prepare 3 types in the presence of bivalent cation after disclosing a kind of utilization sour water solution with oxidant reaction activated polysaccharide The method of pneumococal polysaccharide conjugate.
2000-2004, An Wante-Pasteur (existing Sai Nuofei-Pasteur) carry out malicious with tetanus in Philippine Element, 11 valency pneumococcal Polysaccharide Conjugate Vaccine, the 3 phase clinical research that diphtheria toxoid is carrier protein, because of not up to Major Clinical Terminal and (the Marilla G.Lucero et.al Efficacy of an 11-Valent Pneumococcal that fail Conjugate Vaccine Against Radiologically Confirmed Pneumonia Among Children Less Than 2Years of Age in the Philippines:ARandomized,Double-Blind,Placebo- Controlled Trial, The Pediatric Infectious Disease Journal.28 (6):455-462,JUN 2009).11 valency pneumococcal Polysaccharide Conjugate Vaccines of GlaxoSmithKline PLC research and development, are respectively adopted diphtheria toxoid, indecomposable form stream Haemophilus influenza protein D, tetanus toxoid are as carrier protein, but it not up to protects 3 type of pneumonia in clinical test Effect, the Synflorix finally listed are 10 valent pneumococcal conjugate vaccines for not containing 3 type of pneumococcus.This all fully says The technical difficulty of bright polyvalent pneumococcal polysaccharide combination vaccine research and development is very high, and so far, the whole world only has 13 valency lungs of Pfizer The 10 granted listings of valent pneumococcal conjugate vaccine Synflorix of scorching coccus combined vaccine Prevenar 13 and GlaxoSmithKline PLC Sale.
Since pneumococcus causes the principal causative serotype of disease more, it is necessary to contain enough pneumonia balls in vaccine Granulose type, vaccine just have enough clinical protection effects, according to the 7 valency pneumococcal Polysaccharide Conjugate Vaccines listed and 13 The immunogenicity data documents and materials of valency pneumococcal Polysaccharide Conjugate Vaccine, by institute in 13 valency pneumococcal Polysaccharide Conjugate Vaccines The Serotypes contained are more, due to pneumococal polysaccharide conjugate antigenic competition antigen presenting cell (antigen- Presenting cell, APC) factor, the immunogenicity in overall 13 valency pneumococcal Polysaccharide Conjugate Vaccines is than 7 valency pneumonia The immunogenicity of Streptococcus polysaccharides combined vaccine is slightly lower.Hui Shi is by the pneumococal polysaccharide in 13 valency pneumococcal Polysaccharide Conjugate Vaccines Content improves 10% to the polyoses content of corresponding serotype in 7 valency pneumococcal Polysaccharide Conjugate Vaccines, but still is ground in clinic Study carefully 13 valency pneumococcal Polysaccharide Conjugate Vaccine immunogenicities of middle presentation and be slightly less than 7 valency pneumococcal Polysaccharide Conjugate Vaccines.
According to the WHO pneumococcal conjugated vaccine manufactures issued and vertification regulation, according to 7 valency pneumococal polysaccharide combination epidemic diseases The clinical protection data of 33 clinical trial phases of independence of seedling, obtain the threshold value of 0.35 μ g/ml of serotype Specific IgG antibody, >= The special IgG concentration of serotype of 0.35 μ g/ml represents that inoculator has immunity to the serotype.Serotype is special after vaccine inoculation The ratio (reactivity) of the μ g/ml of IgG concentration >=0.35 is to evaluate the key index of pneumococcal Polysaccharide Conjugate Vaccine immunogenicity.
13 valency pneumococcal Polysaccharide Conjugate Vaccines (Prevenar13) of Wyeth are first by pneumococal polysaccharide with high iodine Hydrochlorate oxidation generates terminal aldehyde groups, pneumococcus oligosaccharides is then combined preparation with CRM197 using reduction amination method, at it The material that FDA biological products evaluate center is submitted to show, in its 6096A1-004 keys three phases clinical research, for 2-6 Month infant, after every dose of interval is immunized at the beginning of completing 3 doses within 2 months, there are three serotype (being respectively 6A types, 9V types, 3 types) is special The reactivity of the μ g/ml of IgG >=0.35 is not up to the non-bad effect with 7 valent pneumococcal conjugate vaccines, the reactivity of three serotype 95% lower limit of confidence interval of difference is respectively -10.9%, -12.4%, -36.2%, wherein special to 3 type polysaccharide of pneumococcus The reactivity of the μ g/ml of IgG >=0.35 is only 63.5%.
3 pin Post-immunisation serum type IgG >=0.35 of 13 valent pneumococcal conjugate vaccines of Hui Shi and 7 valent pneumococcal conjugate vaccine The reactivity of μ g/mL compares as shown in Table I.
Table I
N：The serotype has the quantity of IgG concentration measurements
n：The quantity of the μ g/mL of serotype IgG >=0.35
Reactivity=n/N × 100%
Rate is poor=13 valency combined vaccine reactivity-PCV7 correspond to serotype reactivity, for increasing serotype newly, using PCV7 The minimum 6B of middle reactivity is calculated
4 pin Post-immunisation serum type IgG >=0.35 of 13 valent pneumococcal conjugate vaccines of Hui Shi and 7 valent pneumococcal conjugate vaccine The reactivity of μ g/mL compares as shown in Table II.
Table II
N：The serotype has the quantity of IgG concentration measurements
n：The quantity of the μ g/mL of serotype IgG >=0.35
Reactivity=n/N × 100%
Rate is poor=13 valency combined vaccine reactivity-PCV7 correspond to serotype reactivity, for increasing serotype newly, using PCV7 The 98.7% of the minimum 19F of middle reactivity calculates.
From Table I and Table II, 2-6 month infants are being inoculated with three doses of 13 valency pneumonia of Hui Shi according to 2,4,6 immune programme After Streptococcus polysaccharides combined vaccine, under 95% confidence interval of the reactivity difference of the μ g/ml of the IgG of 3 type of pneumococcus >=0.35 It is limited to -36.2%；After the 12-15 monthly ages carry out 1 dose of booster immunization (totally 4 doses), to the μ g/ml of the IgG of 3 type of pneumococcus >=0.35 Ratio still not up to non-bad effect with 7 valent pneumococcal conjugate vaccines, the 95% of the reactivity difference of the μ g/ml of IgG >=0.35 Lower limit of confidence interval is -- 12.8%.
In addition, the carrier protein tetanus toxoid (TT) that Pnu-Imune 23 uses passes through for clostridium tetanus After fermentation, extracted, refined, detoxification is prepared, according to《Chinese Pharmacopoeia》2015 editions, prepare the tetanus toxoid of vaccine Purity requirement is >=1500Lf/mg proteinic nitrogens, according to document and produces the experience of tetanus toxoid, produces the lockjaw of acquisition Toxoid purity is generally in 1500-3000Lf/mg proteinic nitrogens, and thus data can be seen that：The tetanus toxoid of different batches Purity has larger difference, the wherein no small foreign protein of content ratio.According to documents and materials, existing pharmacopeia technique is prepared broken Wind toxoid monomer purity therein is only in about 55-70%.And if used using the tetanus toxoid containing more foreign protein It is combined in pneumococal polysaccharide, the effect of carrier protein and the content of floating preteins in conjugate, and and then shadow certainly will be influenced Ring the immunogenicity and security of pneumococal polysaccharide conjugate.
The content of the invention
The present invention overcomes existing 13 valent pneumococcal conjugate vaccine in 2-6 month infants to the spy of 3 type of pneumococcus Different IgG concentration >=low weakness of 0.35 μ g/ml reactivities and 13 valent pneumococcal conjugate vaccine of Wyeth are only criticized in China The mutatis mutandis limitation in -6 month infant of 2 monthly age provides a kind of 13 valency pneumococcal polysaccharide-protein combination compositions and its system Preparation Method and application are suitable for -5 one full year of life of 2 monthly age infant and prevent pneumococcal disease.
The present invention is had developed comprising 1 type of pneumococcus, 3 types, 4 types, 5 types, 6A types, 6B types, 7F types, 9V types, 14 types, 18C Type, 19A types, 19F types and 23F type polysaccharide using tetanus toxoid as 13 valent pneumococcal conjugate vaccines of carrier protein, and 3 clinical trial phases through double-blind, randomized controlled clinical study prove can be in -5 years old 2 monthly ages blood serum induced type-specific IgG antibody, according to immune The ratio of the μ g/ml of the special IgG concentration of whole Pneumococcus serotypes >=0.35 is included after program inoculation to vaccine >=85%, Prevent pneumococcal disease available for -5 one full year of life of 2 monthly age infant.
Technical scheme is as follows：A kind of 13 valency pneumococcal polysaccharide-protein combination compositions, the composition Including 13 kinds of different pneumococcal polysaccharide-protein conjugates, each pneumococcal polysaccharide-protein conjugate is included from difference The capsular polysaccharide and carrier protein of Pneumococcus serotypes, the capsular polysaccharide are respectively derived from 1 type, 3 types, 4 types, 5 types, 6A Type, 6B types, 7F types, 9V types, 14 types, 18C types, 19A types, 19F types and 23F type pneumococcus, the carrier protein are that monomer is pure The tetanus toxoid carrier albumen of degree >=90%.
Further, for molecular size K is detected on Sepharose CL-4B columnsDThe pneumococcus pod of value ＜ 0.20 Polysaccharide molecule size is reduced to by film polysaccharide before activating, deriving using ultrasonic degradation or 70-85 DEG C of pyrohydrolysis technique K is detected on Sepharose CL-4B columnsDIt is worth between 0.20-0.50.
Further, 3 types, 5 types, 9V types, 14 types use cyanogen bromide-activated in the capsular polysaccharide；1 type, 4 types, 6A types, 6B types, 7F types, 18C types, 19A types, 19F types and 23F types are activated using CDAP, are carried out after activation with adipic dihydrazide (ADH) It is derivative.
Further, the mass ratio of Streptococcus polysaccharides-protein conjugates is as follows in the composition：1 type pneumococal polysaccharide Protein conjugates 0.15-0.60：3 type pneumococal polysaccharide protein conjugates 0.40-0.90：4 type pneumococal polysaccharide albumen knots Close object 0.30-1.10：5 type pneumococal polysaccharide protein conjugates 0.10-0.30：6A type pneumococal polysaccharide protein conjugates 0.10-0.50：6B type pneumococal polysaccharide protein conjugates 0.20-0.65：7F type pneumococal polysaccharide protein conjugates 0.15- 0.45：9V type pneumococal polysaccharide protein conjugates 0.10-0.50：14 type pneumococal polysaccharide protein conjugates 0.15-0.70： 18C type pneumococal polysaccharide protein conjugates 0.30-0.60：19A type pneumococal polysaccharide protein conjugates 0.10-0.65：19F Type pneumococal polysaccharide protein conjugates 0.10-0.40：23F type pneumococal polysaccharide protein conjugates 0.25-0.65.
Further, in the composition various pneumococcal polysaccharide-protein conjugate dissociation amylase content≤35%, Free protein content is ≤5%.
Second aspect of the present invention also provides the preparation method of the 13 valency pneumococcal polysaccharide-protein combination compositions, The preparation method comprises the following steps：
(1) pneumococcus is killed using NaTDC, centrifugation removes thalline, and pneumonia is extracted through ethanol precipitation method Coccus raw sugar；
(2) after pneumococcus raw sugar is dissolved with saturated acetic acid sodium solution, with phenol extraction, through ultrafiltration and it is classified ethanol precipitation Afterwards, precipitation is collected, after being washed with absolute ethyl alcohol, ether, acetone, vacuum is drained, and is pneumococcal capsular polysaccharide；
(3) to detecting K on Sepharose CL-4BDThe various pneumococcal capsular polysaccharide of value ＜ 0.2, it is molten with sodium chloride Xie Hou extremely detects main peak K with ultrasonic wave or 70-85 DEG C of pyrohydrolysis on Sepharose CL-4BDBe worth for 0.20-0.50 it Between, obtain the pneumococal polysaccharide of degradation；
(4) molecular weight detects K on Sepharose CL-4BDThe pneumococal polysaccharide being worth in 0.20-0.50 uses bromination It after cyanogen or CDAP activation, adds in ADH and derives, concentration is pneumococal polysaccharide derivative after ultrafiltration；
(5) tetanus toxoid is purified using Sephacryl S-300HR column chromatographies, after concentration aseptic filtration be Tetanus toxoid carrier albumen；
(6) in the presence of EDAC (carbodiimide), by pneumococal polysaccharide derivative and tetanus toxoid carrier albumen With reference to after dialysed overnight, upper Sepharose 4FF column purifications are collected from V0To KDConjugate component between 0.2, degerming It is pneumococcal polysaccharide-protein conjugate stoste after filter；
(7) Aluminium phosphate adjuvant will be added in after 13 type pneumococcal polysaccharide-protein conjugate stoste mixings to final concentration 1.0mg/ml adds in disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium chloride solution, adds in sterilized water for injection to required objective body It is long-pending, it is dispensed after mixing.
Third aspect present invention also provides a kind of 13 valency pneumococcal polysaccharide-protein combination compositions and is preparing induction To the purposes in the drug or vaccine of the humoral immune response of pneumococal polysaccharide.
Further, the drug or vaccine are suitable for -5 one full year of life of 2 monthly age crowd.
Further, wherein pneumococcal polysaccharide-protein combination compositions are single 0.5ml dosage, it is formulated into Every milliliter contains：In addition to pneumococcus 6B types polysaccharide is 8.8-13.2 μ g, remaining Pneumococcal serotype polysaccharide is 4.4- 6.6μg；And every milliliter of Aluminium phosphate adjuvant containing 0.25mg aluminium elements, 8.5mg sodium chloride, 88.7 μ g di(2-ethylhexyl)phosphates in composition Hydrogen sodium and 38.0 μ g disodium hydrogen phosphates.
Further, absorption of the various pneumococcal polysaccharide-protein conjugate on Aluminium phosphate adjuvant in the composition Rate is in 20-85%.
Various pneumococcal capsular polysaccharide is the long-chain molecule of repetitive unit composition, and molecular weight is larger so that pneumonia ball The pneumococcal polysaccharide-protein conjugate that the combining to form of granulose and carrier protein can prepare vaccine faces great technology and chooses War.After bacterial polysaccharides and carrier protein covalent bond, T cell dependence antigen is converted by T cell independent antigen, and The immunogenicity of bacterial polysaccharides-protein conjugates depends on the molecular size range, derivative combined process, carrier egg of bacterial polysaccharides In vain, the influence of the multiple factors such as conjugate purification process, the quality control standard of conjugate and adjuvant.And between these factors It is to be mutually related, while 13 Pneumococcal serotype polysaccharide have entirely different polysaccharide repeat unit, polysaccharide repeats single Substituent group in member also has larger difference, this causes the technical study of 13 valency pneumococcal Polysaccharide Conjugate Vaccines and quality mark The addition of the far from 13 type research work of technical difficulty of quasi- research.
Bacterial eapsular polysaccharide is the long-chain molecule by glycosidic bond links by polysaccharide repeat unit, and research shows bacterial capsule The immunogenicity of polysaccharide is directly related with its molecular size, for meningococcal polysaccharide vaccine, Typhoid Vi Polysaccharide Vaccine, pneumonia Streptococcus polysaccharides vaccine is using polysaccharide molecule size as crucial Con trolling index, and formulated the lower limit of molecular size to ensure polysaccharide The immunogenicity of vaccine.In the World Health Organization《Pneumococcal polysaccharide vaccine regulation》And European Pharmacopoeia《Pneumococal polysaccharide Vaccine》In define the molecular size standard of pneumococal polysaccharide, wherein 1 type, 3 types, 4 types, 18C types, 23F types are required to The K detected on Sepharose CL-4BDThe K that≤0.15,7F of value types, the requirement of 19F types detect on Sepharose CL-4BDValue ≤ 0.20,14 types, the K of 19A typesDValue respectively no higher than 0.30 and 0.45, in addition 5 types, 6B, 9V need to use Sepharose The K detected on CL-2BDValue respectively no higher than 0.60,0.50,0.45.Simultaneously because the fermentation of pneumococcus and capsular polysaccharide production Life is influenced by conditions such as pH, temperature, culture medium, stirrings, the pneumococal polysaccharide molecule of the indirect fermentation purifying acquisition of every batch of There is also certain differences for amount.
And the present invention is proved by substantial amounts of technical study and the experiment of animal immune originality using ultrasonotomography or 70-85 DEG C High-temperature-hot-water solution by pneumococal polysaccharide Partial digestion to the Kd values detected on Sepharose CL-4B 0.20-0.50 it Afterwards, then carry out polysaccharide activation derive, not only increase the filtering feature of technique repetition stability and conjugate, and reduce column The dissociation amylase content in pneumococal polysaccharide protein conjugates after chromatographic purifying, while improve pneumococal polysaccharide albumen The immunogenicity of conjugate in animal body.
At development initial stage of the present invention, pneumococal polysaccharide has been carried out compared with the combined process of variety carrier albumen, respectively Using tetanus toxoid, the tetanus toxoid of monomer purity 90%, diphtheria toxoid, recombinant C RM197 and recombination hepatitis B table Face antigen (rHBsAg) is combined with various pneumococcus, and (the big ear of New Zealand is white) has carried out immunogene in animal body Property evaluation, the results showed that：Using tetanus toxoid, 90% tetanus toxoid of monomer purity, diphtheria toxoid, restructuring CRM197 can generate the special humoral immune response of serotype as carrier protein, but with broken using monomer purity 90% Wind toxoid is antibody titer GMT (geometric mean titer) highest of carrier protein, so screening monomer purity 90% is broken Carrier protein of the wind toxoid as 13 valency pneumococal polysaccharide protein conjugate vaccines.
But the vaccine prepared in the present invention is verified through phase iii clinical trial before obtaining result, and domestic and international bacterial polysaccharides combines Vaccine expert generally believes difficult as the sole support albumen of polyvalent pneumococcal polysaccharide combination vaccine using tetanus toxoid With successful, brainstrust is generally worried to include the absorption acellular hundred of National immunization programme for children at the 3-5 monthly ages (external 2-6 monthly ages) The anti-tetanus generated after the tetanus toxoid ingredient and inoculation absorption DTaP vaccine that contain in dtp vaccine Toxoid antibody will produce the immunogenicity using tetanus toxoid as the polyvalent pneumococcal polysaccharide combination vaccine of carrier protein Raw larger immune interference and inhibition, so as to be difficult to achieve the desired results.
In the pneumococcal Polysaccharide Conjugate Vaccine research of international vaccine producer, Hui Shi uses diphtheria toxin non-toxic mutant Body CRM197As carrier protein, Sai Nuofei-Pasteur is using tetanus toxoid, diphtheria toxoid as carrier protein (3 phases Clinical test does not succeed), GSK is made using tetanus toxoid, diphtheria toxoid, indecomposable form Hinfluenzae protein D For carrier protein, (wherein 3 types do not succeed in clinical test, release the 10 valency pneumococal polysaccharide combination epidemic diseases without 3 types Seedling).Show 3 doses, 4 doses of inoculation in the key clinical research carried out in 13 valent pneumococcal conjugate vaccines of Hui Shi at the 2-6 monthly ages Afterwards, the reactivity of the μ g/ml of the IgG special to 3 type of pneumococcus >=0.35 is not reaching to combines epidemic disease to 7 valency pneumococcus of Hui Shi The non-bad effect of seedling.
It is that multivalent pneumococcal combines to improve 3 type conjugate immunogenicity of pneumococcus in 2-6 monthly ages primary target population The key technical problems that vaccine is successfully researched and developed, the outstanding feature of pneumococal polysaccharide combination compositions of the invention use K on Sepharose CL4BDThe polysaccharide of 0.2-0.5 is activated respectively with cyanogen bromide and CDAP, ADH derive, then with monomer Purity is more than 90% tetanus toxoid carrier protein binding, and monovalent pneumococal polysaccharide protein binding is obtained through column chromatography Object, after various pneumococal polysaccharide protein conjugates are using appropriate proportioning mixing, add in Aluminium phosphate adjuvant and auxiliary material sodium chloride, After disodium hydrogen phosphate, sodium dihydrogen phosphate 13 valency pneumococcal Polysaccharide Conjugate Vaccine finished products are packed as according to every dose of 0.55ml.Through 3 phases Clinical test proves, in 2-6 month infants, after being spaced 1 month or being spaced bimestrial 3 pin immune programme, pneumococcus 3 The immunogenicity of type reaches the peer-level with other serotypes, and the μ g/ml's of the IgG special to 3 type of pneumococcus >=0.35 is anti- Should rate >=90%, it is reinforced it is immune after the μ g/ml of the special IgG of whole serotypes >=0.35 reactivity >=97%, all 13 A serotype reaches the effect same with 7 valent pneumococcal conjugate vaccines of Hui Shi, the brightness with the unique granted listing in the current whole world Auspicious 13 valency pneumococcal Polysaccharide Conjugate Vaccine, which is compared, has extremely significant clinical advantage.Meanwhile vaccine prepared by the present invention is through three Phase clinical verification is at the 7-11 monthly ages, and at the 12-23 monthly ages, 2-5 Sui can reach satisfied humoral immune response, and Hui Shi can be overcome public Take charge of the limitation that 13 valency pneumococcal polysaccharide vaccines are approved only for -6 month infant of 2 monthly age in China.
Compared with prior art, the invention has the advantages that：The present invention provides use Sephacryl S- 300HR column chromatographies are to meeting《Chinese Pharmacopoeia》The tetanus toxoid of 2015 editions requirements is further purified, and target is compared in removal Molecular weight of albumen bigger and smaller foreign protein obtain the tetanus toxoid carrier albumen that HPLC monomer purities are more than 90%, Tetanus toxoid using monomer purity more than 90% is combined as carrier protein with pneumococal polysaccharide, is conducive to improve lung The immunogenicity of scorching Streptococcus polysaccharides protein conjugates reduces floating preteins, dissociation amylase content in conjugate simultaneously.
13 valency pneumococal polysaccharide combination compositions prepared by the present invention, prove in 3 clinical trial phases, for 2-6 No matter month infant using 2,4,6 first exempts from program or 3,4,5 first exempts from program, 3 pins be immunized after to all serotype IgG The reactivity of >=0.35 μ g/ml >=90%, after 12-15 monthly age booster immunizations, to the μ g/ of all serotype IgG >=0.35 >=97%, and all serotypes reach and the same effect of 7 valent pneumococcal conjugate vaccines for the reactivity of ml.
Specific embodiment
Technical scheme is described in further details with reference to specific embodiment, but the present invention does not limit to In following technical scheme.
The preparation of 1 tetanus toxoid carrier albumen of embodiment
Bacterium numbering is the clostridium tetanus of CMCC 64008, from Products in China calibrating institute medicine Bacterium preservation administrative center.Original species word bank, main generation seed bank and work seed bank are established with above-mentioned strain.Primordial seed is criticized In 0th generation, passed 2 on behalf of main generation seed lot from primordial seed batch, 2 is passed on behalf of working seed lots from main generation seed lot.Open work kind Son batch strain, is inoculated in semisolid culturemedium, 35 DEG C, 48 it is small when amplification cultivation, production seed is made through passing on for 3 generations.By table 1 Middle recipe configuration lockjaw toxin producing medium, uses after high pressure steam sterilization.
1 lockjaw toxin producing medium formula of table
Sequence number Name of material 100mL amount of preparation requirements
1 Beef peptic digest (total nitrogen) 1 gram
2 Cheese digestive juice (total nitrogen) 1 gram
3 Sodium acetate 4 grams
4 Disodium hydrogen phosphate 1 gram
5 Potassium dihydrogen phosphate 1 gram
6 Glycerine 60mL
7 Glucose 3 grams
8 Yeast extract 5 grams
9 Ferric trichloride 0.3 gram
Production is inoculated in seed in fermentation tank, under the conditions of 35 DEG C, when culture 65 is small in lockjaw toxin producing medium. Formalin is added in culture solution to final concentration of 0.35%, 15min is centrifuged under the conditions of 7000rpm, 8 DEG C, collects supernatant.
Supernatant is concentrated with 30kD ultrafiltration membranes.Ammonium sulfate is added in concentrate to final concentration of 16%, stand 46 it is small when.It is quiet It puts liquid and 20min is centrifuged under the conditions of 10000rpm, 8 DEG C, collect supernatant.Ammonium sulfate is added in supernatant to final concentration of 12%, it is quiet Put 20 it is small when.
It stands liquid and 15min collection precipitations is centrifuged under the conditions of 10000rpm, 8 DEG C.Precipitation is dissolved with water for injection.It precipitates molten Solution liquid centrifuges 15min under the conditions of 10000rpm, 8 DEG C and collects supernatant.The injection of supernatant 30kD ultrafiltration membranes, 50 times of volumes Water is concentrated by ultrafiltration.Ultrafiltration concentration liquid is tetanus toxin.
NaCl to final concentration of 0.85% is added in tetanus toxin, formalin is added in final concentration of 0.3%, in 37 36 days are stood under the conditions of DEG C and carries out detoxification.Tetanus toxin 30kD ultrafiltration membranes, the water for injection of 10 times of volumes after detoxification surpass Filter concentration, ultrafiltration concentration liquid are tetanus toxoid.
Tetanus toxoid through assay approval after the concentration of 30kD ultrafiltration membranes, is splined on 0.2mol/L NaCl balances 50/100 columns of Sephacryl S-300HR (95 centimetres of pillar height), loading volume 95ml, with 0.2mol/L NaCl elute, with Intravenous injection of immunoglobulin, which elutes, collects target peak at identical elution volume, after the concentration of 30kD ultrafiltration membranes, aseptic filtration, It is tetanus toxoid carrier albumen stoste after assay approval.With TSK gel G3000SWXL columns in efficient liquid phase system The monomer purity of tetanus toxoid carrier albumen is measured, mobile phase is 0.2mol/L Na2HPO4-NaH2PO4, pH7.0, flow velocity 1.0ml/min, Detection wavelength 280nm calculate lockjaw carrier protein monomer purity, as a result such as 2 institute of table by integrating peak areas method Show.
2 lockjaw carrier protein stoste testing result of table
The degradation of 2 pneumococcus of embodiment 1 type, 3 types, 4 types, 5 types, 6B types, 7F types, 18C types, 19A type polysaccharide
1 type of pneumococcus, 3 types, 4 types, 5 types, 6B types, 7F types, 18C types, 19A type polysaccharide, respectively with 0.2mol/L's NaCl is dissolved as 2-8mg/ml, is formulated as solution；It is respectively placed in condition of ice bath precooling；Under condition of ice bath, carry out at ultrasonic wave Reason, ultrasonic power are 350~400W, and ultrasound 3 seconds is 2-20 minutes ultrasonic under conditions of suspending 5 seconds.Various ultrasound condition is such as Shown in table 3.
3 polysaccharide ultrasonic technique parameter of table
Ultrasonic time
1 type polysaccharide 5
3 type polysaccharide 10
4 type polysaccharide 8
5 type polysaccharide 2
6B type polysaccharide 6
7F type polysaccharide 15
18C type polysaccharide 20
19A type polysaccharide 12
Polysaccharide after ultrasonotomography measures main peak K using Sepharose CL-4B XK16/100 columnsDValue, mobile phase are 0.9% sodium chloride, flow velocity 0.3ml/min collect 4.5ml/15min/ pipes.Polysaccharide, which is carried out, with corresponding serotype antiserum differentiates examination It tests, the results are shown in Table 4.
It is examined after 4 pneumococcus of table, 1 type, 3 types, 4 types, 5 types, 6B types, 7F types, 18C types, 19A type polysaccharide ultrasonotomographies
Survey result
The degradation of embodiment 3 pneumococcus 6A types, 9V types, 14 types, 19F types, 23F type polysaccharide
Pneumococcus 6A types, 9V types, 14 types, 19F types, 23F type polysaccharide, are dissolved as 2- with the NaCl of 0.2mol/L respectively 8mg/ml is formulated as solution；It is respectively placed in thermostatic equipment, controls -85 DEG C of temperature 70 C, it is cold to take out nature by time 0.5-2h But.Per shown in type hydrolysising condition table 5.
5 polysaccharide hydrolysis technological parameter of table
Control temperature (DEG C) Hydrolysis time (h)
6A type polysaccharide 70 1.5
9V type polysaccharide 80 1.0
14 type polysaccharide 85 0.5
19F type polysaccharide 75 1.0
23F type polysaccharide 70 2
Polysaccharide after high temperature degradation measures main peak K using Sepharose CL-4B XK16/100 columnsDValue, mobile phase are 0.9% sodium chloride, flow velocity 0.3ml/min collect 4.5ml/15min/ pipes.Polysaccharide, which is carried out, with corresponding serotype antiserum differentiates examination It tests, the results are shown in Table 6.
Testing result after 6 pneumococcus 6A types of table, 9V types, 14 types, 19F types, 23F type polysaccharide high temperature degradations
The Sepharose CL-4B molecular sizes of 4 pneumococal polysaccharide of embodiment and the pneumococal polysaccharide of Partial digestion It measures
Sepharose CL-4B gels are loaded on XK16/100 columns, and 90 centimetres of pillar height is made using 0.9% sodium chloride solution It is balanced for eluent with the flow velocity of 0.30ml/min.
After abundant balance, loading blue dextran 2000 and the biased sample of cromoci configuration, while start distribution Collector, collect 4.5ml/15min/ pipe, by measure often pipe 260nm and 370nm absorbance value measure chromatographic column V0 With Vi, first peak of 260nm corresponds to V0, the maximum absorption peak of 370nm corresponds to Vi。
The pneumococal polysaccharide loading 1.0ml of pneumococal polysaccharide or Partial digestion, while start distribution collector, 0.3ml/min is eluted, and collects 4.5ml/15min/ pipes.Polysaccharide concentration in often pipe is measured using anthrone method, draws elution curve, from Elution curve polysaccharide concentration top obtains elution volume Ve。
According to formula calculation of distribution coefficient KD；
KD=(Ve-V0)/(Vi-V0)。
53 type of embodiment, 5 types, 9V types, the activation of 14 type degradation of polysaccharide are derivative (cyanogen bromide-activated)
3 type of pneumococcus, 5 types, 9V types, 14 each 150mg of type degradation of polysaccharide are taken respectively, respectively by polysaccharide：Cyanogen bromide is 1: 0.5~1:1 mass ratio adds in cyanogen bromide, and control pH10.5 ± 0.5 reacts 10~30min, is then separately added into 800mg's again 1,6- adipic dihydrazide maintains pH8.5 ± 0.5 to react 30~60min, is concentrated by ultrafiltration with the ultrafiltration membrane of 10KD, i.e., lung is made respectively Scorching 3 type of coccus, 5 types, 9V types, 14 type polysaccharide-adipic dihydrazide derivative, cyanogen bromide-activated derive after testing result such as 7 institute of table Show.
Testing result after 7 cyanogen bromide-activated of table derivative
The activation of 61 type of embodiment, 4 types, 6A types, 6B types, 7F types, 18C types, 19A types, 19F types and 23F type degradation of polysaccharide Derivative (CDAP activation)
Each 150mg of pneumococcus is taken respectively, respectively by polysaccharide：CDAP is 1:0.2~1:1 mass ratio adds in CDAP, control PH9.5 processed ± 0.5 reacts 2~15min, is then separately added into 1, the 6- adipic dihydrazide of 800mg again, and pH8.5 ± 0.5 is maintained to react 30~60min is concentrated by ultrafiltration with the ultrafiltration membrane of 10KD, i.e., pneumococcus type, 4 types, 6A types, 6B types, 7F types, 18C is made respectively Type, 19A types, 19F types and 23F types polysaccharide-adipic dihydrazide derivative, testing result are as shown in table 8.
Testing result after table 8CDAP activation is derivative
Project Derivation rate (%) Derivative KDValue
1 type pneumococal polysaccharide derivative 4.85 0.24
4 type pneumococal polysaccharide derivatives 3.67 0.32
6A type pneumococal polysaccharide derivatives 3.07 0.32
6B type pneumococal polysaccharide derivatives 2.53 0.48
7F type pneumococal polysaccharide derivatives 4.16 0.36
18C type pneumococal polysaccharide derivatives 4.57 0.36
19A type pneumococal polysaccharide derivatives 6.55 0.40
19F type pneumococal polysaccharide derivatives 4.13 0.36
23F type pneumococal polysaccharide derivatives 2.03 0.40
It is prepared by 7 pneumococcal polysaccharide-protein conjugate of embodiment
Under ice-water bath, various pneumococal polysaccharide-AH derivatives are taken respectively, by polysaccharide-AH derivatives：Tetanus poison Element is 1：0.8-1：1.2 mass ratio adds in tetanus toxoid carrier albumen, adds in 0.2mol/L hydrochloric acid regulation systems pH and is 5.5 ± 0.5, EDAC to 0.01~0.1mg/ml of final concentration is added in, when reaction 2-4 is small.After reaction, dialysed overnight.Loading In Sepharose 4FF BPG200/950 columns, eluent is 0.2mol/L sodium chloride, collects 0.2 pervious components of Kd, warp It is pneumococcal polysaccharide-protein conjugate stoste after 0.22 micron of aseptic filtration, testing result is as shown in table 9.
9 various pneumococcal polysaccharide-protein conjugate stoste testing result of table
8 polysaccharide of type containing 6B of embodiment, 4.4 microgram, 13 valency pneumococal polysaccharide combination epidemic diseases of other various 2.2 micrograms of polysaccharide Seedling semi-finished product are prepared, packing
1st, prescription
In the every 0.5ml (agent) of this composition in addition to pneumococcus 6B types polysaccharide is 4.4 μ g, remaining Pneumococcal serotype Polysaccharide is 2.2 μ g；The Aluminium phosphate adjuvant of 0.125mg aluminium elements；4.25mg sodium chloride；44.35 μ g sodium dihydrogen phosphates；19.0μg Disodium hydrogen phosphate.
2nd, prepare
(1) prepare auxiliary material liquid storage and weigh medicinal grade sodium chloride in container by formula ratio, endotoxin is removed in dry roasting sterilizing；By with Side's amount weighs pharmaceutical grade phosphate (sodium dihydrogen phosphate, disodium hydrogen phosphate) in another container, and endotoxin is removed in dry roasting sterilizing；Xiang Rong Formula ratio water for injection, mixing are added in device, high pressure sterilization obtains auxiliary material liquid storage；2-8 DEG C is placed, for use；
(2) the various conjugate stoste of formula ratio is measured, and mixing obtains conjugate mixed liquor；
(3) by formula ratio Aluminium phosphate adjuvant, phosphate buffer liquid storage (pH6.5), sodium chloride liquid storage, water for injection, mixing Uniformly, auxiliary material mixed liquor is obtained；
(4) auxiliary material mixed liquor is pumped into during conjugate mixed liquor is stirred, and mixing is semi-finished product
3rd, dispense
Under aseptic condition cillin bottle or pre-encapsulated injector are filled to by every bottle of (branch) 0.55ml volume integral.
9 polysaccharide of type containing 6B of embodiment, 6.6 microgram, 13 valency pneumococal polysaccharides of other various 3.3 micrograms of polysaccharide combine Vaccine semi-finished product are prepared, packing
1st, prescription
In the every 0.5ml (agent) of this composition in addition to pneumococcus 6B types polysaccharide is 6.6 μ g, remaining Pneumococcal serotype Polysaccharide is 3.3 μ g；The Aluminium phosphate adjuvant of 0.125mg aluminium elements；4.25mg sodium chloride；44.35 μ g sodium dihydrogen phosphates；19.0μg Disodium hydrogen phosphate.
2nd, prepare
(1) prepare auxiliary material liquid storage and weigh medicinal grade sodium chloride in container by formula ratio, endotoxin is removed in dry roasting sterilizing；By with Side's amount weighs pharmaceutical grade phosphate (sodium dihydrogen phosphate, disodium hydrogen phosphate) in another container, and endotoxin is removed in dry roasting sterilizing；Xiang Rong Formula ratio water for injection, mixing are added in device, high pressure sterilization obtains auxiliary material liquid storage；2-8 DEG C is placed, for use；
(2) the various conjugate stoste of formula ratio is measured, and mixing obtains conjugate mixed liquor；
(3) by formula ratio Aluminium phosphate adjuvant, phosphate buffer liquid storage (pH6.5), sodium chloride liquid storage, water for injection, mixing Uniformly, auxiliary material mixed liquor is obtained；
(4) auxiliary material mixed liquor is pumped into during conjugate mixed liquor is stirred, and mixing is semi-finished product
3rd, dispense
Under aseptic condition cillin bottle or pre-encapsulated injector are filled to by every bottle of (branch) 0.55ml volume integral.
10 polysaccharide of type containing 6B of embodiment, 5.5 microgram, 13 valency pneumococal polysaccharide knots of other various 2.75 micrograms of polysaccharide Vaccine semi-finished product are closed to prepare, dispense
1st, prescription
In the every 0.5ml (agent) of this composition in addition to pneumococcus 6B types polysaccharide is 5.5 μ g, remaining Pneumococcal serotype Polysaccharide is 2.75 μ g；The Aluminium phosphate adjuvant of 0.125mg aluminium elements；4.25mg sodium chloride；44.35 μ g sodium dihydrogen phosphates；19.0μ G disodium hydrogen phosphates.
2nd, prepare
(1) prepare auxiliary material liquid storage and weigh medicinal grade sodium chloride in container by formula ratio, endotoxin is removed in dry roasting sterilizing；By with Side's amount weighs pharmaceutical grade phosphate (sodium dihydrogen phosphate, disodium hydrogen phosphate) in another container, and endotoxin is removed in dry roasting sterilizing；Xiang Rong Formula ratio water for injection, mixing are added in device, high pressure sterilization obtains auxiliary material liquid storage；2-8 DEG C is placed, for use；
(2) the various conjugate stoste of formula ratio is measured, and mixing obtains conjugate mixed liquor；
(3) by formula ratio Aluminium phosphate adjuvant, phosphate buffer liquid storage (pH6.5), sodium chloride liquid storage, water for injection, mixing Uniformly, auxiliary material mixed liquor is obtained；
(4) auxiliary material mixed liquor is pumped into during conjugate mixed liquor is stirred, and mixing is semi-finished product
3rd, dispense
Under aseptic condition cillin bottle or pre-encapsulated injector are filled to by every bottle of (branch) 0.55ml volume integral.
Various pneumococal polysaccharide conjugate adsorption rate in 11 13 valency pneumococcal Polysaccharide Conjugate Vaccine finished product of embodiment It measures
13 12 bottles of valency pneumococcal Polysaccharide Conjugate Vaccine finished products are taken, after merging, 8000g is centrifuged 5 minutes, takes supernatant, accurate 5.0ml is measured, adds in Beckman Coulter Inc. dilution 6ml, 2.2 times of extension rate is sample to be tested 1.
13 11 bottles of valency pneumococcal Polysaccharide Conjugate Vaccine finished products are taken, after merging, precision measures 5.0ml, adds in 0.25ml 0.3mol/LNaOH solution, mixing add in 0.25ml 0.1mol/L citric acid solutions, add Beckman Ku Er after 10 seconds Special company's dilution 5.5ml, 2.2 times of extension rate are sample to be tested 2.
Rate turbidimetry for Determination sample to be tested 1 and various polysaccharide in sample to be tested 2 are used using immunochemistry analysis system Content, each replication 3 times is averaged calculating.Pneumococal polysaccharide standard serum used is purchased from serum research institute of Denmark, Pneumococal polysaccharide standard items used are purchased from ATCC (American type culture collection).
The adsorption rate=(certain type pneumococal polysaccharide concentration in sample to be tested 2-of certain type pneumococal polysaccharide conjugate is treated Certain type pneumococal polysaccharide concentration in sample 1) certain type pneumococal polysaccharide concentration × 100% in/sample to be tested 2, inspection The results are shown in Table 10 for survey.
The adsorption rate detection of various pneumococal polysaccharide conjugate in 10 13 valency pneumococcal Polysaccharide Conjugate Vaccine finished product of table As a result
Type Adsorption rate (%)
1 type pneumococal polysaccharide conjugate 37.4%
3 type pneumococal polysaccharide conjugates 31.5%
4 type pneumococal polysaccharide conjugates 22.4%
5 type pneumococal polysaccharide conjugates 28.6%
6A type pneumococal polysaccharide conjugates 20.2%
6B type pneumococal polysaccharide conjugates 36.9%
7F type pneumococal polysaccharide conjugates 65.1%
9V type pneumococal polysaccharide conjugates 39.8%
14 type pneumococal polysaccharide conjugates 83.2%
18C type pneumococal polysaccharide conjugates 33.5%
19A type pneumococal polysaccharide conjugates 34.1%
19F type pneumococal polysaccharide conjugates 25.0%
23F type pneumococal polysaccharide conjugates 38.2%
The immunogenicity effect that 12 13 valency pneumococcal Polysaccharide Conjugate Vaccine of embodiment is immunized in -5 years old 2 monthly ages infant Fruit
The 7 valent pneumococcal conjugate vaccine Prevenar produced using Wyeth as control vaccine, by random, blind, Similar vaccine investigates the immunogene of 13 valency pneumococcal Polysaccharide Conjugate Vaccines prepared by the present invention to impinging upon -5 years old 2 monthly ages infant Property with security (clinical drug trial register and public notification of information platform clinic registration number：CTR20160188).
13 valency pneumococcal Polysaccharide Conjugate Vaccine, III clinical trial phase always enters group number 2760, wherein, 2 monthly ages (minimum 6 Week old) -6 monthly ages totally 1560, test group 1040, control group 520；The 7-11 monthly ages totally 400, test group is with compareing component It Wei not be 200；The 12-23 monthly ages totally 400, test group and control group are respectively 200；24-5 years old monthly ages totally 400, test group It it is respectively 200 with control group, immune programme is as shown in table 11.
11 clinical test of table grouping, inoculation number and immune programme
* wherein subject of the test group 2 not less than 10% can enter group a 1st dose of 13 valency pneumococal polysaccharide of inoculation in 6 week old Combined vaccine completes the inoculation of fundamental immunity program in the 14th, 22 week old, and booster immunization inoculation is completed in the 12-15 monthly ages.
Subject's venous blood is gathered respectively and is no less than 3.5ml within 30 days after first dose of immune preceding and fundamental immunity；It needs to strengthen Immune subject need to also be no less than 3.5ml by 30 days acquisition venous blood after booster immunization.By detecting 13 pneumococcus blood Rear each serotype specificity IgG antibody concentration >=0.35 is immunized in clear type-specific IgG antibody, comparative test vaccine and control vaccine Subject's ratio of μ g/ml.
Clinical test immunogenicity result is as follows：
(1) 1 immunogenicity comparative result of 2-6 monthly ages test group is as shown in table 12.
The reactivity of the μ g/ml of (fundamental immunity) IgG antibody concentration after 12 3,4,5 monthly age of table immune group, 3 pin is immunized >=0.35 Compare
As can be seen that after three pins are immunized by 3,4,5 monthly ages in 13 valent pneumococcal conjugate vaccines prepared by the present invention, all blood The reactivity of clear type is all higher than 90%.
The reactivity of the μ g/ml of (booster immunization) IgG antibody concentration after 13 3,4,5 monthly age of table immune group, 4 pin is immunized >=0.35 Compare
As can be seen from Table 13, three pins are immunized by 3,4,5 monthly ages in 13 valent pneumococcal conjugate vaccines that prepared by the present invention, The 12-15 monthly ages strengthen it is one after, the reactivity of all serotypes is all higher than 97%
(2) 2 immunogenicity comparative result of 2-6 monthly ages test group is as shown in table 14.
The reactivity of the μ g/ml of (fundamental immunity) IgG antibody concentration after 14 2,4,6 monthly age of table immune group, 3 pin is immunized >=0.35 Compare
As can be seen that after three pins are immunized by 2,4,6 monthly ages in 13 valent pneumococcal conjugate vaccines prepared by the present invention, all blood The reactivity of clear type is all higher than 90%.
The reactivity of the μ g/ml of (booster immunization) IgG antibody concentration after 15 2,4,6 monthly age of table immune group, 4 pin is immunized >=0.35 Compare
As can be seen that three pins are immunized by 2,4,6 monthly ages in 13 valent pneumococcal conjugate vaccines prepared by the present invention, in 12-15 After monthly age reinforcement is one, the reactivity of all serotypes is all higher than 97%.
(3) 7-11 monthly age immunogenicities comparative result
The reactivity of the μ g/ml of (fundamental immunity) IgG antibody concentration after 16 7-11 monthly ages of table, 2 pin is immunized >=0.35 compares
As can be seen that 13 valent pneumococcal conjugate vaccines prepared by the present invention after 2 pins are immunized in 7-11 month infants, own The reactivity of serotype is all higher than 90%.
The reactivity of the μ g/ml of (booster immunization) IgG antibody concentration after 17 7-11 monthly ages of table, 3 pin is immunized >=0.35 compares
As can be seen that 2 pins, 12-15 is immunized in 7-11 month infants in 13 valent pneumococcal conjugate vaccines prepared by the present invention After monthly age reinforcement is one, the reactivity of all serotypes is all higher than 93%.
(4) 12-23 monthly age immunogenicities compare
The reactivity that the μ g/ml of rear IgG antibody concentration >=0.35 are immunized in 18 12-23 monthly ages of table, 2 pin compares
As can be seen that 13 valent pneumococcal conjugate vaccines that prepare of the present invention are after 2 pins are immunized in 12-23 monthly age childs, institute The reactivity for having serotype is all higher than 96%.
(5) 24-5 years old monthly ages immunogenicities compare
The reactivity that the μ g/ml of rear IgG antibody concentration >=0.35 are immunized in 1 pin of -5 years old 19 24 monthly age of table compares
As can be seen that 13 valent pneumococcal conjugate vaccines that prepare of the present invention are after 24 monthly age -5 one full year of life immunization programs for children, 1 pin, The reactivity of all serotypes is all higher than 88%.

Claims (10)

1. a kind of 13 valency pneumococcal polysaccharide-protein combination compositions, which is characterized in that the composition includes 13 kinds of differences Pneumococcal polysaccharide-protein conjugate, each pneumococcal polysaccharide-protein conjugate included from different pneumococcus serum The capsular polysaccharide and carrier protein of type, the capsular polysaccharide be respectively derived from 1 type, 3 types, 4 types, 5 types, 6A types, 6B types, 7F types, 9V types, 14 types, 18C types, 19A types, 19F types and 23F type pneumococcus, the carrier protein are the broken of monomer purity >=90% Wind toxoid carrier protein.
2. 13 valency pneumococcal polysaccharide-protein combination compositions according to claim 1, which is characterized in that for dividing Sub- size detects K on Sepharose CL-4B columnsDThe various pneumococcal capsular polysaccharide of value ＜ 0.2 is adopted before activating, deriving Polysaccharide molecule size is reduced to ultrasonic degradation or 70-85 DEG C of pyrohydrolysis technique and is examined on Sepharose CL-4B columns Survey KDIt is worth for 0.20-0.50.
3. 13 valency pneumococcal polysaccharide-protein combination compositions according to claim 1, which is characterized in that the pod 3 types, 5 types, 9V types, 14 types use cyanogen bromide-activated in film polysaccharide；1 type, 4 types, 6A types, 6B types, 7F types, 18C types, 19A types, 19F types and 23F types are activated using CDAP, are derived after activation with adipic dihydrazide (ADH).
4. the 13 valency pneumococcal polysaccharide-protein combination compositions according to claims 1, which is characterized in that described The mass ratio of Streptococcus polysaccharides-protein conjugates is as follows in composition：1 type pneumococal polysaccharide protein conjugates 0.15-0.60：3 Type pneumococal polysaccharide protein conjugates 0.40-0.90：4 type pneumococal polysaccharide protein conjugates 0.30-1.10：5 type pneumonia Streptococcus polysaccharides protein conjugates 0.10-0.30：6A type pneumococal polysaccharide protein conjugates 0.10-0.50：6B type pneumococcus Polysaccharide protein conjugate 0.20-0.65：7F type pneumococal polysaccharide protein conjugates 0.15-0.45：9V type pneumococal polysaccharides Protein conjugates 0.10-0.50：14 type pneumococal polysaccharide protein conjugates 0.15-0.70：18C type pneumococal polysaccharide albumen Conjugate 0.30-0.60：19A type pneumococal polysaccharide protein conjugates 0.10-0.65：19F type pneumococal polysaccharide albumen knots Close object 0.10-0.40：23F type pneumococal polysaccharide protein conjugates 0.25-0.65.
5. the 13 valency pneumococcal polysaccharide-protein combination compositions according to claims 1, which is characterized in that described ≤ 35%, Free protein content is ≤5% for various pneumococcal polysaccharide-protein conjugate dissociation amylase content in composition.
6. the preparation method of the 13 valency pneumococcal polysaccharide-protein combination compositions as described in Claims 1 to 5 is any, It is characterized in that, the preparation method comprises the following steps：
(1) pneumococcus is killed using NaTDC, centrifugation removes thalline, and pneumococcus is extracted through ethanol precipitation method Raw sugar；
(2) after pneumococcus raw sugar is dissolved with saturated acetic acid sodium solution, with phenol extraction, after ultrafiltration and classification ethanol precipitation, Precipitation is collected, after being washed with absolute ethyl alcohol, ether, acetone, vacuum is drained, and is pneumococcal capsular polysaccharide；
(3) to detecting K on Sepharose CL-4BDThe various pneumococcal capsular polysaccharide of value ＜ 0.20, after being dissolved with sodium chloride, Main peak K is extremely detected on Sepharose CL-4B with ultrasonic wave or 70-85 DEG C of pyrohydrolysisDIt is worth between 0.20-0.50, obtains The pneumococal polysaccharide that must be degraded；
(4) molecular weight detects K on Sepharose CL-4BDBe worth the pneumococal polysaccharide in 0.20-0.50 using cyanogen bromide or It after CDAP activation, adds in ADH and derives, concentration is pneumococal polysaccharide derivative after ultrafiltration；
(5) tetanus toxoid is purified using Sephacryl S-300HR column chromatographies, aseptic filtration is broken after concentration Wind toxoid carrier protein；
(6) in the presence of EDAC, by pneumococal polysaccharide derivative and tetanus toxoid carrier protein binding, dialysed overnight Afterwards, upper Sepharose 4FF column purifications, are collected from V0To KDConjugate component between 0.2 is pneumococcus after aseptic filtration Polysaccharide-protein conjugate stoste；
(7) Aluminium phosphate adjuvant will be added in after 13 type pneumococcal polysaccharide-protein conjugate stoste mixings to final concentration 1.0mg/ Ml adds in disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium chloride solution, adds in sterilized water for injection to required target volume, mixing After dispense.
7. a kind of 13 valency pneumococcal polysaccharide-protein combination compositions are preparing humoral immunity of the induction to pneumococal polysaccharide Purposes in the drug or vaccine of response.
8. purposes as claimed in claim 7, which is characterized in that the drug or vaccine are suitable for -5 one full year of life of 2 monthly age crowd.
9. purposes as claimed in claim 7 or 8, which is characterized in that wherein pneumococcal polysaccharide-protein combination compositions are Single 0.5ml dosage, it is formulated to contain into every milliliter：In addition to pneumococcus 6B types polysaccharide is 8.8-13.2 μ g, remaining blood Clear type pneumococal polysaccharide is 4.4-6.6 μ g；And every milliliter of Aluminium phosphate adjuvant containing 0.25mg aluminium elements in composition, 8.5mg sodium chloride, 88.7 μ g sodium dihydrogen phosphates and 38.0 μ g disodium hydrogen phosphates.
10. the purposes described in claims 9, which is characterized in that various pneumococcal polysaccharide-protein combines in the composition Adsorption rate of the object on Aluminium phosphate adjuvant is in 20-85%.
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