CN1566145A - Polyvalent pneumococcal polysaccharide combination vaccine - Google Patents
Polyvalent pneumococcal polysaccharide combination vaccine Download PDFInfo
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Abstract
The invention provides a coupling substance of pneumococcoal polysaccharide carrier protein, vaccines containing the coupling substance and process for preparation, wherein the coupling substance has the structural formula of Pr-L-Ps, wherein Pr is carrier protein, Ps is pneumococcoal polysaccharide, L is covalent bond or connection group, and the mass ratio of Ps:Pr=0.5-3.5:1. The coupling substance can form protection to the homogeneous pneumococcosis protomer which produces capsular polysaccharide compositions.
Description
Technical field
The present invention relates to field of immunology, relate more specifically to the conjugate of streptococcus pneumoniae polysaccharides-carrier proteins, contain the vaccine and the method for making thereof of this conjugate.
Background technology
Pneumonia (pneumonia) is the acute inflammation of pulmonary parenchyma, for clinical modal infectious diseases, after the microbiotic widespread use, though its prognosis has remarkable improvement, but M ﹠ M does not have decline yet, and is higher in infant, the elderly and immunosuppressed patient especially.
Pneumonia can be categorized as Da Ye, lung section and a matter by anatomic distribution; For helping treatment.The many infectious pneumonia such as bacterium, virus, mycoplasma, fungi, rickettsia, chlamydozoan and protozoon that are categorized as by the cause of disease of current diagnosis.
Infantile pneumonia is common disease in children, frequently-occurring disease, is the principal disease that threatens children's's life and health.From cause of disease classification, it comprises bacillary (streptococcus pneumoniae, suis, streptococcus aureus, pneumobacillus, hemophilus influenza, Pseudomonas aeruginosa, intestinal bacteria etc.), viral (adenovirus, respiratory syncytial virus, influenza virus, parainfluenza virus etc.), pneumonia magma bacterium property (claiming mycoplasma pneumonia again, primary atypical pneumonia), mycotic, imbedibility, supersensitivity and hypostasis etc.Comprise Da Ye, segmental bronchus and a matter etc. by pathological classification.Can be divided into acute (in 1 month), delay property (1～3 month) and chronic (above 3 months) according to the course of disease, wherein bronchopneumonia is the maximum persons of morbidity in the pneumonia, and is constantly all the year, more with winter and spring especially.
Bronchopneumonia is infant's common disease in period, frequently-occurring disease, and 3 months～2 years old child sees more, all can fall ill throughout the year, but the highest with autumn and winter season sickness rate cold, climate variability, very big to the child health influence.Infant's age is little, and the body resistivity is poor, and cold is brought out flu easily, and flu back infringement tracheae, the through lung tissue of segmental bronchus cause pneumonia easily; Child's lung tissue elastic force is poor, and alveolar quantity is few, and circulation of blood is abundant, and pulmonary infection takes place easily.Treatment child pneumonia, the lighter gives the microbiotic intravenous drip and the relieving cough and reducing sputum medicine of capacity, and seriously ill person also will give cardiac drug, oxygen suction, the treatment of relievining asthma, and will fully recover in general 7～8 days.Treatment thoroughly will not change into the chronic persistent pneumonia.The pathogenic bacteria of infant's morbidity mostly is streptococcus pneumoniae.
Streptococcus pneumoniae extensively is distributed in all over the world, often colonizes in healthy people's pharynx nasalis, have the people of 40%-70% to carry disease germs approximately, but most had no pathogenicity or virulence is extremely low.Can streptococcus pneumoniae cause a disease, and depends primarily on invasive ability and the Abwehrkraft des Koepers size of bacterium to tissue.It also can cause pneumococcal septicemia, and bacterium also can enter blood during the pneumonia infection coccus, causes diseases such as meningitis, endocarditis then.Worldwide, streptococcus pneumoniae makes 1,000,000 death of child every year at least.Therefore, the best method of prevention pneumonia is inoculated Pnu-Imune 23 exactly.Besides because in recent years, because streptococcus pneumoniae produces in various degree resistance to microbiotic, the area that has reaches 50% unexpectedly, has brought difficulty for the treatment pneumonia.So should focus on prevention for pneumonia, the inoculation pneumovax is a kind of good method of prevention.
Pneumococcal infection, the one, carry disease germs from self, when resistibility reduced, streptococcus pneumoniae was invaded to lower respiratory tract and causes pneumonia; The 2nd, infected from germ-carrying others or patient, cause pneumonia.Everybody has the chance that this pneumonia takes place, but the high risk population of generation pneumonia comprises: weak children and grownup; The elderly more than 60 years old; The upper respiratory disease that shows effect repeatedly comprises the children and the grownup of sinusitis paranasal sinusitis, otitis media; Pulmonary disorder patient, heart disease patient, hepatic diseases patient, renal disease patient, diabetic subject, cancer patients, sicklemia patient, Hokdkin disease patient, the not normal person of function of immune system, splenectomy person (determine the patient that will carry out splenectomy, should at least in art vaccination in preceding 15 days); Determine to carry out the immunosuppressant therapy person; Hang up one's hat chronic disease nursing or long term care facilities person.Above crowd is the main object that should inoculate " polyvalent pneumococcal vaccine ".
At present, the Pnu-Imune 23 that China uses is " multivalent pneumococcal polysaccharide vaccine " (Pneumovax 23 23).It is U.S. Mo Shadong, the development and production of French Pasteur S.A., and approval is carried out in the whole nation through China Ministry of Health.This vaccine has comprised 23 kinds of streptococcus pneumoniae that mainly cause pneumonia and septicemia, can produce immunizing power to 90% streptococcus pneumoniae, so claim " multivalence ".After the shot, can produce protection antibody in 15 days, the protection period continues 5 years at least; In case of necessity, after a shot the 6th year again the injection once.The inoculation pneumovax does not generally have any reaction.Minor responses such as injection site pain, redness can appear in inoculation back a few peoples; Be less than 1% inoculator and low-heat can occur; Can in 2-3 days, recover voluntarily.Anaphylaxis is very rare.But the remarkable shortcoming of this polysaccharide vaccine is invalid to 3 months～2 years old infant.
84 serotypes of streptococcus pneumoniae are not that each type can both similarly be attacked human body.The streptococcus pneumoniae type that causes disease is different because of area, age and crowd often.The U.S. ratified first 14 valency pneumococcal polysaccharide vaccine in 1977 and puts on market.14 pneumonia that the serotype streptococcus pneumoniae causes in the vaccine account for 68% of aggressive pneumococcal pneumonia.Nineteen eighty-three, a kind of 23 valency vaccines of reformulating have replaced 14 valency vaccines, and these 23 serotype streptococcus pneumoniae cause that the aggressive more than 85% infects.
China is in May, 1981～1985 year July, from patient's different specimens, be divided into from 712 strain streptococcus pneumoniae, carried out somatotype by Denmark's method and standard that WHO recommends, wherein 5 types are maximum, next is 6,1,19,23,2,3 and 8 types totally 8 types (group), account for 63.6% of whole pneumococcal infections, these 8 types (group) all are included in the 23 valency pneumovaxs.
Relatively poor to the immunogenicity of the infant below 2 years old in view of 23 present valency pneumonia polysaccharide vaccines, therefore, this area presses for the new multivalence pneumovax that can effectively be applied to the infant below 2 years old of exploitation.
In addition, also to press for exploitation new for the pneumococcal pneumovax in Asia (especially China) in this area.
Summary of the invention
Purpose of the present invention just provide a kind of effectively be applied to below 2 years old the infant's and at the pneumococcal multivalence pneumovax in Asia (especially China).
In a first aspect of the present invention, a kind of streptococcus pneumoniae polysaccharides-carrier protein couplet thing is provided, it has following formula:
Pr-L-Ps
Wherein, Pr is a carrier proteins,
Ps is a streptococcus pneumoniae polysaccharides,
L is covalent linkage or linking group,
The mass ratio of and Ps: Pr is 0.5-3.5: 1.
In another preference, described carrier proteins is selected from: tetanus toxoid protein, diphtheria toxoid albumen or its mixture.
In another preference, described streptococcus pneumoniae polysaccharides is the polysaccharide from following streptococcus pneumoniae hypotype: hypotype 4,6B, 9V, 14,18C, 19F, 23F or its combination.
In another preference, described L is a covalent linkage.
In another preference, described Ps: Pr is that mass ratio is 0.5-3: 1.
In another preference, described streptococcus pneumoniae polysaccharides prepares with following step:
(b1) in the culture of streptococcus pneumonia thing, add Sodium desoxycholate, carry out sterilization;
(b2) centrifugal removal thalline is collected supernatant;
(b3) adding ethanol to volume final concentration is 70-90%;
(b4) centrifugal removal pigment, inorganic salt, collecting precipitation;
(b5) dissolution precipitation again;
(b6) adding phenol to volume final concentration is 72-76%, to remove albumen;
(b7) adding calcium chloride to weight final concentration is 1 ± 0.2M, and adding ethanol to volume final concentration is 5-35%;
(b8) the sedimentary nucleic acid impurity of centrifugal removal is got supernatant;
(b9) adding ethanol to volume final concentration is 60-95%;
(b10) centrifugal, collecting precipitation thing, the streptococcus pneumoniae polysaccharides of acquisition purifying.
In a second aspect of the present invention, a kind of pharmaceutical composition is provided, it contain the present invention above-mentioned streptococcus pneumoniae polysaccharides-carrier protein couplet thing and pharmaceutically acceptable carrier.
Preferably, the streptococcus pneumoniae polysaccharides in the described conjugate comprises the polysaccharide of following 7 kinds of streptococcus pneumoniae hypotypes: hypotype 4,6B, 9V, 14,18C, 19F and 23F.
In another preference, described pharmaceutical composition is a vaccine.
In a third aspect of the present invention, a kind of method for preparing streptococcus pneumoniae polysaccharides-carrier protein couplet thing is provided, it is characterized in that, may further comprise the steps:
(a) cultivate streptococcus pneumoniae, obtain the culture of streptococcus pneumonia thing;
(b) from streptococcus pneumoniae, extract polysaccharide;
(c) with the polysaccharide and the carrier protein couplet that extract, form streptococcus pneumoniae polysaccharides-carrier protein couplet thing, wherein said carrier proteins is tetanus toxoid protein, diphtheria toxoid albumen or its mixture.
In another preference, step (b) comprising:
(b1) in the culture of streptococcus pneumonia thing, add Sodium desoxycholate, carry out sterilization;
(b2) centrifugal removal thalline is collected supernatant;
(b3) adding ethanol to volume final concentration is 70-90%;
(b4) centrifugal removal pigment, inorganic salt, collecting precipitation;
(b5) dissolution precipitation again;
(b6) adding phenol to volume final concentration is 72-76%, to remove albumen;
(b7) adding calcium chloride to weight final concentration is 1 ± 0.2M, and adding ethanol to volume final concentration is 5-35%;
(b8) the sedimentary nucleic acid impurity of centrifugal removal is got supernatant;
(b9) adding ethanol to volume final concentration is 60-95%;
(b10) centrifugal, collecting precipitation thing, the streptococcus pneumoniae polysaccharides of acquisition purifying.
In another preference, adopt the cyanogen bromide-activated polysaccharide in the described coupling step (c) earlier, and then adopt carbodiimide activatory polysaccharide and albumen covalent coupling.
Embodiment
The present invention is through deeply and extensive studies, finds formed conjugate behind the polysaccharide of streptococcus pneumoniae and the suitable carriers albumen coupling can be excited the immunne response of youngling effectively, effectively prevents provide protection thereby produce.In addition, the inventor has also determined to be specially adapted to the polysaccharide type in Asia (especially China) crowd's the Pnu-Imune 23, is applicable to the infant's and at the pneumococcal multivalence pneumovax in Asia (especially China) thereby developed.
Term
As used herein, " streptococcus pneumoniae polysaccharides-carrier protein couplet thing " refers to be connected in the formed conjugate of carrier proteins by streptococcus pneumoniae polysaccharides by covalent linkage or linking group.In the present invention, described conjugate is called " conjugate of the present invention " sometimes for short.
As used herein, term " streptococcus pneumoniae polysaccharides " refers to the polysaccharide of the streptococcus pneumoniae pod membrane of originating.Described capsular polysaccharide can prepare with the method for this area routine.In another preference, described polysaccharide is to extract with the ad hoc approach that the inventor invents.
As used herein, term " carrier proteins " refers to that any can be the albumen of 10-200KD with streptococcus pneumoniae polysaccharides link coupled, molecular weight.Preferred carrier proteins comprises (but being not limited to): tetanus toxoid protein, diphtheria toxoid albumen (for example diphtheria toxin albumen of the avirulent diphtheria bacterial strain generation of CRM197) or other suitable carriers albumen or its mixture.A kind of particularly preferred carrier proteins is a tetanus toxoid protein, because toxoid albumen itself is a kind of immunization agent.
As used herein, term " pneumococcal Polysaccharide Conjugate Vaccine " refers to contain the vaccine composition of conjugate of the present invention.
Discovering, in the mankind's immunity system evolution, is progressively maturation to the immune response of thymus independent antigen, produces specific antibody.Promptly only thymus gland interdependence antigen such as proteantigen are produced antibody,, then produce the antibody of anti-dissimilar polysaccharide along with the growth of system the infant.This order is proved in sheep placenta at first: it produces immunne response to two kinds of proteantigens, and to Salmonellas or the no response of mycobacterium polysaccharide surface antigen.With bacillary polysaccharide vaccine inoculation infant's immunization experiment, disclosed before two years old answer-mode all to sheep placenta in similar.
Polyose belongs to thymus independent antigen (TI); in youngling and the particularly later more weak immune response of infant's generation of t helper cell growth of immunity system; can not form effective protection; and polysaccharide is with after carrier proteins combines; become thymus dependent antigen, become effective vaccine the infant.
In the present invention, polysaccharide is attached to the combined vaccine of making on the protein carrier and can overcomes the infant the unresponsive phenomenon of polysaccharide antigen.The effect of combined vaccine is: after making polysaccharide antigen be converted into thymus dependent antigen by thymus independent antigen (TI), the output of polyose antibody is increased, special inoculation child; Can produce immuno-potentiation when injecting repeatedly; The maturation of stimulating immune system, and produce the antibody response that is mainly IgG; In advance, or can stimulate the propagation of T cell when injecting carrier proteins simultaneously, thereby strengthen, promote combined vaccine and reach the highest immune response.
The present invention also provides the preparation method of streptococcus pneumoniae polysaccharides-carrier protein couplet thing.It comprises step:
A, microbial culture:
Culture technique of streptococcus pneumoniae and condition can adopt the ordinary skill in the art and condition.For example, from seeding is criticized, draw plate picking bacterial classification, be inoculated in 5 milliliters of nutrient solutions, expand in 500 milliliters of nutrient solutions, expand to once more in 5 liters of nutrient solutions.Culture condition is the general streptococcic condition of cultivating.
B, polysaccharide extract:
When microbial culture during to OD 〉=1.5, stop to continue to cultivate, add the Sodium desoxycholate sterilization.
Centrifugal, remove thalline, collect supernatant.
Adding ethanol to volume final concentration in supernatant liquor is 70-90%, the rough polysaccharide of centrifugal collecting precipitation.In the technology of other reports, do not have this step ethanol sedimentation, make a lot of impurity bring next step separation and purification into easily like this, finally be difficult to be removed, thereby influence the purity of polysaccharide.
After throw out dissolves again, add phenol and remove albumen.
In solution, add CaCl then
2To final concentration be the ethanol of 1 ± 0.2M and lower concentration, the ethanol of lower concentration can the sedimentation cell chip and some other impurity, but can not remove soluble impurity.Alcohol concn should not be too low, otherwise can not play sedimentation effect; Otherwise the too high meeting of concentration causes the co-precipitation of part capsular polysaccharide, influences yield.Concentration of ethanol should be in the 5%-35% scope; Preferable, at 10%-30%; Best, in the 20%-25% scope.Centrifugal, remove nucleic acid.Do not had impurity such as solid debris, nucleic acid, albumen in the solution this moment.
Add the ethanol of high density in solution, alcohol concn is controlled at 60%-95%; Preferable, 65%-90%; Best, the 70%-80% scope can be got off polysaccharide precipitation, and centrifugal collecting precipitate is refining polysaccharide.
Because the degree that the component of the oligose repeating unit of certain capsule polysaccharide serotype and ring connect has difference, so the performance on chemical property also can be different, adjust concentration of ethanol, centrifugal time and speed can obtain better separating effect.
The coupling of C, polysaccharide and carrier proteins
Coupling step can adopt the coupling technology and the condition of this area routine.For example, add cyanogen bromide-activated in polysaccharide, add adipic dihydrazide again and handle, the derivation rate is about 1-3%.Add Toxoid,tetanus and carbodiimide, reaction obtains the chemical covalent coupling thing of pneumococcal capsular polysaccharide and tetanus toxoid protein, removes micromolecular compound through separation and purification, obtains final conjugate product.
In a preference, the inventive method adopts cyanogen bromide-activated and carbodiimide stepwise reaction, has improved polysaccharide and proteic joint efficiency (improving 30% approximately), and the method for the simple employing cyanogen bromide-activated of this external report is far better.
Polysaccharide for some hypotype can adopt the additive method preparation to raise the efficiency.For example add sodium periodate oxidation in polysaccharide, detect aldehyde radical, molecular weight size and sugared content add the reaction of Toxoid,tetanus and boron hydrocyanation sodium again and obtain the polysaccharide-protein binding substances.
These two kinds of association reactions all can obtain polysaccharide-carrier proteins binding substances, but because the structure of different subtype bacterial strain capsular polysaccharide and composition are different, the efficient of two kinds of reactions also exists significant difference.Therefore should select different combining method for use according to homopolysaccharide not.Preferably, 9V, 14,18C, 19F, 23F type polysaccharide adopt cyanogen bromide-activated, carbodiimide combined techniques; 4,6B type polysaccharide should adopt sodium periodate oxidation, the preparation of boron hydrocyanation sodium combined techniques.
The method of separation coupling thing has ethanol precipitation, gel filtration method, gradient centrifugation or the like, and these methods are all this area professional and know.In a preference, adopt the DEAE ion exchange column to carry out chromatographic separation.The polysaccharide-protein binding substances is suspended in the damping fluid, and damping fluid can use 100mM Tris, pH8.6.
Through the initial gross separation product of above-mentioned processing, separate through molecular sieve column again, remove unconjugated albumen.Solution after the separation is the work in-process of polysaccharide conjugate vaccine after Sterile Filtration, can be by certain dosage than the multivalent vaccine composition that is mixed with effective dose.
The present invention also provides a kind of pharmaceutical composition (comprising vaccine composition), and it contains polysaccharide of the present invention-carrier protein couplet thing and pharmaceutically acceptable carrier.Suitable carriers comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical composition of the present invention also can various this areas adjuvant commonly used.
Pharmaceutical composition of the present invention can be made into various preparations such as injection, tablet and capsule, wherein contain immune effective amount of actives (being polysaccharide of the present invention-carrier protein couplet thing), for example every day about 1 microgram/kg body weight-Yue 10 mg/kg body weight.
Pharmaceutical composition of the present invention can be by conventional administering mode administration, comprising (but being not limited to): oral, intramuscular, intraperitoneal, intravenously, subcutaneous or intradermal administration.
When making pharmaceutical composition, be that polysaccharide of the present invention-carrier protein couplet thing with safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 10 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, and these all are within the skilled practitioners skill.
Major advantage of the present invention is:
(1) the streptococcus pneumoniae hypotype of Cai Yonging is to separate the local bacterial strain that obtains in China, and the pneumococcal capsular polysaccharide that these bacterial strains produce is the main pathogen that causes symptoms of pneumonia in China within the border.Therefore the capsular polysaccharide that adopts local pathogenic strains generation is as immunogen therapeutic action more targetedly.
(2) technology of the preparation pneumococcal polysaccharide of the present invention's employing is the technology through optimizing, and simple to operate, cost is low, and is easy to control.
(3) the chemical coupling method of the preparation polysaccharide conjugate vaccine of the present invention's employing is to adopt cyanogen bromide-activated or two kinds of different coupling modes of reductive amination method to combine, and has improved polysaccharide and protein bound efficient, has also improved the stability of combined vaccine.
(4) of the present invention conjugated protein be tetanus toxoid protein, be different from other albumen of external report.Adopt the advantage of such toxin protein to be: such toxin protein itself is a kind of immunization agent, and the security of its clinical application is verified; This albumen low price, convenient for production; This albumen and polysaccharide bonded efficient higher (can reach the level more than 70%).
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The selection of embodiment 1 streptococcus pneumoniae hypotype
China in May, 1981-1985 year July, from patient's different specimens, separate 712 strain streptococcus pneumoniae, carried out somatotype by Denmark's method and standard that WHO recommends, be divided into 42 types (group), do not find 40,42 and 47 types, wherein 5 types are maximum, secondly are 6,1,19,23,2,3 and 8 totally 8 types (group) totally 453 strains, account for 63.6%.Different sick main serotype differences of planting, pneumonia is mainly: 1,5,14 and 6 types; Meningitis is based on 2,5,6,1,27 and 14 types; Otitis media is common with 19,6,5,23,3 and 14 types then.Transition take place in pneumococcal main popular bacterial type sometimes, and therefore, the bacterial type monitoring is a crucial job, and it is the combination of determining Pnu-Imune 23 serotype, the important scientific basis of active prevention pneumococcal infection.
In actual applications, select the polyvalent vaccine of several hypotypes can cover immunoprotection basically for other whole hypotypes.This is because exist the reason of cross immunity between each hypotype polysaccharide.
The inventor screens at several main hypotype that causes the Chinese children cause of disease, pick out 10 kinds of hypotypes as the multivalence polysaccharide immunogens, and wherein seven kinds of hypotypes is main, and they are hypotype 4,6B, 9V, 14,18C, 19F and 23F.These hypotypes are to separate during China's prevailing disease is observed to obtain, and have more specific aim at Chinese pneumonia cause of disease.
The preparation of embodiment 2 streptococcus pneumoniae capsular polysaccharides
A, microbial culture:
Select 4 respectively for use, the streptococcus pneumoniae of 6B, 9V, 14,18C, 19F and 23F hypotype.From seeding is criticized, draw plate picking bacterial classification, be inoculated in 5 milliliters of nutrient solutions, expand in 500 milliliters of nutrient solutions, expand to once more in 5 liters of nutrient solutions.Culture condition is the general streptococcic condition of cultivating.
B, polysaccharide extract:
(b1) when microbial culture during, stop to continue to cultivate, add the Sodium desoxycholate sterilization to OD 〉=1.5.
(b2) 14000g is centrifugal, removes thalline, collects supernatant.
(b3) in supernatant liquor, add ethanol to concentration and be respectively 70%, 80% and 90%.
(b4) impurity such as centrifugal removal pigment, inorganic salt, the rough polysaccharide of collecting precipitation.
(b5) add distilled water, throw out is dissolved again.
(b6) adding phenol to volume final concentration is 72-76%, thereby removes albumen.
(b7) in solution, add CaCl then
2Ethanol to the volume final concentration that to final concentration is 1M and lower concentration is respectively 10%, 20% and 30%.
(b8) centrifugal removal nucleic acid is got supernatant.Do not had impurity such as solid debris, nucleic acid, albumen in the supernatant solution this moment.
(b9) ethanol to the volume final concentration of adding high density is respectively 65%, 75%, 85% and 95% in solution, respectively polysaccharide precipitation is got off.
(b10) centrifugal collecting precipitate is the purified streptococcus pneumoniae polysaccharides.
The polysaccharide that above-mentioned different condition obtained is detected, find that quality is close substantially.
Embodiment 3
The quality evalution of streptococcus pneumoniae polysaccharides
Purified polysaccharide quality is very big for the influence of association reaction, if the polysaccharide sample is impure, can influence the efficient of association reaction, combination rate is descended greatly, the ratio of polysaccharide and polysaccharide protein binding substances changes in the finished product thereby make, the minimizing of polysaccharide protein binding substances ratio in the finished product can reduce the effect of T cytositimulation, and the quality of therefore controlling the polysaccharide intermediates seems particularly crucial.
The inventor is through repetition test repeatedly, the streptococcus pneumoniae polysaccharides of different subtype summed up down column data, as the control criterion to the polysaccharide quality.Certainly this is not to be sole criterion.
| Hypotype | Nucleic acid | Albumen | Total nitrogen | Total phosphorus | Molecular size (Kd) | Uronic acid | Hexosamine | Methylpentose |
| ??4 | ??≤2 | ??≤3 | ??≤2 | ??4-6 | ??≤0.15 | ??≥40 | ||
| ??6B | ??≤2 | ??≤2 | ??≤2 | ??0-2 | ??≤0.50 | ??≥15 | ||
| ??9V | ??≤1 | ??≤2 | ??≤2 | ??0.5-3 | ??≤0.45 | ??≥15 | ??≥13 | |
| ??14 | ??≤2 | ??≤5 | ??≤2 | ??1.5-4 | ??≤0.30 | ??≥20 | ||
| ??18C | ??≤2 | ??≤3 | ??≤2 | ??0-1 | ??≤0.15 | ??≥14 | ||
| ??19F | ??≤2 | ??≤3 | ??≤2 | ??1.4-3.5 | ??≤0.20 | ??≥12.5 | ??≥20 | |
| ??23F | ??≤2 | ??≤2 | ??≤2 | ??0-1 | ??≤0.15 | ??≥37 |
Annotate: the detection of above every index, can be according to general chemical examination criteria as reference.These methods are that the professional of this area and chemical field is known.
Embodiment 4 polysaccharide and proteic coupling
In the present embodiment, 9V, 14,18C, 19F, 23F type polysaccharide are adopted cyanogen bromide-activated, the preparation of carbodiimide combined techniques, to 4,6B type polysaccharide adopts sodium periodate oxidation, the preparation of boron hydrocyanation sodium combined techniques.
(a) cyanogen bromide-activated, carbodiimide combined techniques
Add cyanogen bromide-activated (part by weight of polysaccharide and cyanogen bromide is 1: 0.5) in the polysaccharide of different subtypes such as 9V, 14, the 18C that embodiment 2 is made, 19F, 23F type, add adipic dihydrazide again and handle, the derivation rate is 1-3%.Add Toxoid,tetanus and carbodiimide (19.17mg/ml) again, reaction obtains the chemical covalent coupling thing of pneumococcal capsular polysaccharide and tetanus toxoid protein.Adopt the DEAE ion exchange column to carry out chromatographic separation, be about to the polysaccharide-protein binding substances and be suspended in the damping fluid, damping fluid can use 100mM Tris, and pH8.6 removes micromolecular compound, obtains the conjugate product of purifying.
This process using cyanogen bromide-activated and carbodiimide stepwise reaction have improved polysaccharide and proteic joint efficiency (improving 30% approximately), and the method for the simple employing cyanogen bromide-activated of external report is far better.
(b) sodium iodate activation, boron hydrocyanation sodium combined techniques
With embodiment 2 make 4, (polysaccharide is 1M with the ratio of sodium periodate: 1.2-1.8M) to add sodium periodate oxidation in the polysaccharide of different subtype such as 6B type, detect aldehyde radical, molecular weight size and sugared content, (polysaccharide is 1M with the ratio of boron hydrocyanation sodium: 1.2-1.8M), reaction obtains the polysaccharide-protein binding substances to add Toxoid,tetanus and boron hydrocyanation sodium again.
Adopt the ultra-fine filter purifying of 100KD, residual to remove, boron hydrocyanation sodium obtains the conjugate of preliminary purification.
(c) purifying once more
For (a) and (b) the initial gross separation products of two kinds of methods preparation, separate through molecular sieve column again, remove unconjugated albumen.Solution after the separation is the work in-process of polysaccharide conjugate vaccine after Sterile Filtration, can be by certain dosage than the multivalent vaccine composition that is mixed with effective dose.
The preparation of embodiment 5 multivalence polysaccharide conjugate vaccines
It is 10ug/ml that each polysaccharide conjugate vaccine (seven parts) of above-mentioned production is diluted to final concentration with sterile water for injection, get isopyknic various polysaccharide conjugate vaccine (seven parts) then, mix, be made into every milliliter and contain seven kinds of polysaccharide conjugate vaccine blended of 10ug multivalence polysaccharide conjugate vaccine.Can be diluted to every milliliter 0.1 to 10ug septivalency polysaccharide conjugate vaccine preparation according to the drug test effect.
The quality control of embodiment 6 unit prices pneumococcal polysaccharide-Toxoid,tetanus conjugate
Before producing preparation streptococcus pneumoniae polysaccharides multivalence combined vaccine, tackle the quality of each unit price pneumococcal polysaccharide-Toxoid,tetanus conjugate and carry out the strictness evaluation.
In the present embodiment, the combined vaccine to embodiment 4 and 5 preparations carries out quality examination.
One, testing method
(a) total sugar content is measured
1. sample determination
Accurately measure the 1.0ml liquid sample, it is even to add anthrone sulfuric acid mixture liquid 4.0ml, water-bath after 20 minutes on spectrophotometer the 620nm wavelength measure the A value.
2. standard curve determination
Accurately measuring 100ug/ml standard glucose liquid 0ml 0.2ml 0.4ml 0.6ml 0.8ml 1.0ml adds to 1.0ml with distilled water.The same product calibrating of all the other operations.Carry out regressing calculation with standard glucose concentration and corresponding A value, obtain regression equation.
3. the result calculates
With sample detection A value according to regression equation, calculation sample content.
(b) protein content determination
1. sample
Accurately measure certain volume sample (containing about protein 50ug) in test tube, add 5ml alkaline copper liquid, mixing, placed 10 minutes in room temperature, add the 0.5ml phenol reagent fast, mixing, placed 30 minutes in room temperature, with 650nm wavelength or Red lightscreening plate colorimetric (behind the colour generation, as finding muddiness) through 3000r/min colorimetric again after centrifugal 15 minutes.
2. typical curve
Accurately measure standard protein solution (100ug/ml) 0,0.2,0.4,0.6,0.8,1.0ml, place test tube respectively, adding distil water is mended to 1ml, and all the other operate same sample, can get a typical curve.
3. calculate
The ml that finds the suitable standard protein solution of sample from typical curve counts A:
Sample protein matter content (g/ml)=A * 0.01%/sample ml number
(c) capsular polysaccharide antigen molecule size measurement
1. the mensuration of polysaccharide antigen Kd value
(contain polysaccharide antigen 3～5mg) 1.1 get about 1ml sample.Be added on the sepharose 4B glue post of having demarcated, wash with 0.2mol/L sodium chloride solution stream, the flowing lotion consumption is 1.5 times of post bed cumulative volume, every pipe is collected 2～4ml, simultaneously in the monitoring automatically down of 206nm wavelength, record wash liquid collection of illustrative plates is played by application of sample that to wash volume to polysaccharide peak-to-peak top stream be Ve, calculation of distribution coefficient (Kd).
Kd＝(Ve-Vo)/(Vi-Vo)
1.2 the calculating of Kd≤0.3 polysaccharide antigen rate of recovery
Distinguish the total area of the area of calculating K d value 0.3 former component peaks with planimeter, promptly get Kd≤0.3 polysaccharide antigen rate of recovery divided by all components peak.
(d) floating preteins
The carrier proteins that dissociates can be separated from conjugate with the HPLC exclusion chromatography.Big conjugate molecule is wash-out at first, is the floating preteins than small molecular weight subsequently.Detect the elution peak of time period to be measured with the 210nm UV-detector.
(e) free sugar
Sample is adsorbed onto (aluminium hydroxide is as sorbent material) behind the solid-phase matrix, free sugar is separated with the glycoprotein conjugate, free sugar appears in the elutriant.Detect the sugared content of elutriant and initial sample then with anthrone method, to determine the per-cent of free sugar in the stoste.Also need measure the elutriant protein content, conjugate all be removed to determine adsorption process with the Lowry method.
(f) animal experiment of binding substances
1. rabbit:
Can cause significant immune response behind the various conjugate injection rabbit of 2～4ug/0.5ml dosage.The mixture of polysaccharide that is closed after injection polysaccharide or the oxidation and carrier proteins does not produce immunne response or only has low antibody to increase separately.
2. mouse:
NIH or female BALb/c mouse, by negative control group, positive controls, each 20 of vaccine group, every 0.5ug/0.2ml, immunity on the 0th, 14,21, blood sampling (collection serum) on the 28th is respectively organized antibody titers with the detection of ELISA method.
(g) telling test
Two-way agar immunodiffusion: a specific antiserum(antisera) can be discerned its specific components specifically.The validity of experiment depends on every kind of antiserum(antisera) that each batch of type is specific.Every batch of antiserum(antisera) all needs to do the extent of dilution experiment with protein-carbohydrate conjugates before experiment.Can draw best weaker concn by this experiment.When its corresponding antiserum(antisera) of inspection product reacted, it is qualified that these inspection product are judged to, and each experiment all needs a positive and negative standard control.
Two, result
Every test-results is as shown in the table.
| Project | Method | ??4 | ??6B | ??9V | ??14 | ??18C | ??19F | ??23F |
| Molecular weight (kd≤0.3) | Chromatography | ??≥40％ | ??≥40％ | ??≥40％ | ??≥50％ | ??≥40％ | ??≥40％ | ??≥40％ |
| Sugar content (ug/ml) | Anthrone method | ??≥50 | ??≥50 | ??≥50 | ??≥50 | ??≥50 | ??≥50 | ??≥50 |
| Sugar/albumen | Sugar/protein mass ratio | ??0.7-2.2 | ??0.6-1.0 | ??0.8-2.8 | ??1.0-3.0 | ??0.6-2.0 | ??0.6-1.5 | ??0.5-1.5 |
| Telling test | Two expansion methods | Positive | Positive | Positive | Positive | Positive | Positive | Positive |
| Floating preteins (w/w) | Exclusion chromatography | ??≤1％ | ??≤1％ | ??≤1％ | ??≤1％ | ??≤1％ | ??≤1％ | ??≤1％ |
| Free sugar (w/w) | Anthrone method | ??≤25％ | ??≤25％ | ??≤25％ | ??≤25％ | ??≤25％ | ??≤25％ | ??≤25％ |
| Remaining cyano group (pg/ug) | The cyano group colorimetry | ??≤300 | ??≤300 | ??≤300 | ??≤300 | ??≤300 | ??≤300 | ??≤300 |
| Animal experiment | Rabbit or mice serum antibody | Positive | Positive | Positive | Positive | Positive | Positive | Positive |
These results show that multivalent pneumococcal polysaccharide combined vaccine of the present invention conforms to quality requirements, and can be used for the antibody that immune animal produces anti-streptococcus pneumoniae.
Formed conjugate makes polysaccharide change thymus dependent antigen into by thymus independent antigen after polysaccharide and the carrier proteins covalent attachment, is used in the infant and can produces immune response to polysaccharide antigen, forms effectively protection.To the control and the animal experiment of various physical and chemical indexs, it was safe and effective to have guaranteed that the conjugate combined vaccine uses in the infant during the polysaccharide-protein conjugate was made.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. streptococcus pneumoniae polysaccharides-carrier protein couplet thing is characterized in that it has following formula:
Pr-L-Ps
Wherein, Pr is a carrier proteins,
Ps is a streptococcus pneumoniae polysaccharides,
L is covalent linkage or linking group,
The mass ratio of and Ps: Pr is 0.5-3.5: 1.
2. conjugate as claimed in claim 1 is characterized in that, described carrier proteins is selected from: tetanus toxoid protein, diphtheria toxoid albumen or its mixture.
3. conjugate as claimed in claim 1 is characterized in that, described streptococcus pneumoniae polysaccharides is the polysaccharide from following streptococcus pneumoniae hypotype: hypotype 4,6B, 9V, 14,18C, 19F, 23F or its combination.
4. conjugate as claimed in claim 1 is characterized in that described L is a covalent linkage.
5. conjugate as claimed in claim 1 is characterized in that, described Ps: Pr is that mass ratio is 0.5-3: 1.
6. conjugate as claimed in claim 1 is characterized in that, described streptococcus pneumoniae polysaccharides prepares with following step:
(b1) in the culture of streptococcus pneumonia thing, add Sodium desoxycholate, carry out sterilization;
(b2) centrifugal removal thalline is collected supernatant;
(b3) adding ethanol to volume final concentration is 70-90%;
(b4) centrifugal removal pigment, inorganic salt, collecting precipitation;
(b5) dissolution precipitation again;
(b6) adding phenol to volume final concentration is 72-76%, to remove albumen;
(b7) adding calcium chloride to weight final concentration is 1 ± 0.2M, and adding ethanol to volume final concentration is 5-35%;
(b8) the sedimentary nucleic acid impurity of centrifugal removal is got supernatant;
(b9) adding ethanol to volume final concentration is 60-95%;
(b10) centrifugal, collecting precipitation thing, the streptococcus pneumoniae polysaccharides of acquisition purifying.
7. a pharmaceutical composition is characterized in that, it contains the described streptococcus pneumoniae polysaccharides of claim 1-carrier protein couplet thing and pharmaceutically acceptable carrier.
8. pharmaceutical composition as claimed in claim 7 is characterized in that it is a vaccine.
9. a method for preparing the described streptococcus pneumoniae polysaccharides of claim 1-carrier protein couplet thing is characterized in that, may further comprise the steps:
(a) cultivate streptococcus pneumoniae, obtain the culture of streptococcus pneumonia thing;
(b) from streptococcus pneumoniae, extract polysaccharide;
(c) with the polysaccharide and the carrier protein couplet that extract, form streptococcus pneumoniae polysaccharides-carrier protein couplet thing, wherein said carrier proteins is tetanus toxoid protein, diphtheria toxoid albumen or its mixture.
10. method as claimed in claim 9 is characterized in that, step (b) comprising:
(b1) in the culture of streptococcus pneumonia thing, add Sodium desoxycholate, carry out sterilization;
(b2) centrifugal removal thalline is collected supernatant;
(b3) adding ethanol to volume final concentration is 70-90%;
(b4) centrifugal removal pigment, inorganic salt, collecting precipitation;
(b5) dissolution precipitation again;
(b6) adding phenol to volume final concentration is 72-76%, to remove albumen;
(b7) adding calcium chloride to weight final concentration is 1 ± 0.2M, and adding ethanol to volume final concentration is 5-35%;
(b8) the sedimentary nucleic acid impurity of centrifugal removal is got supernatant;
(b9) adding ethanol to volume final concentration is 60-95%;
(b10) centrifugal, collecting precipitation thing, the streptococcus pneumoniae polysaccharides of acquisition purifying.
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| CN 03141407 CN1241937C (en) | 2003-07-04 | 2003-07-04 | Polyvalent pneumococcal polysaccharide combination vaccine |
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| CN 03141407 CN1241937C (en) | 2003-07-04 | 2003-07-04 | Polyvalent pneumococcal polysaccharide combination vaccine |
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| CN1241937C CN1241937C (en) | 2006-02-15 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102068690A (en) * | 2010-12-31 | 2011-05-25 | 北京民海生物科技有限公司 | Polyvalent pneumococcal capsular polysaccharide conjugate vaccine and preparation method thereof |
| CN101610785B (en) * | 2006-12-22 | 2013-11-27 | 惠氏公司 | Multivalent pneumococcal polysaccharide-protein conjugate composition |
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| CN105131139A (en) * | 2015-07-31 | 2015-12-09 | 兰州生物制品研究所有限责任公司 | Purification method of streptococcus pneumoniae capsular polysaccharide |
| CN106039300A (en) * | 2016-05-26 | 2016-10-26 | 北京民海生物科技有限公司 | Preparation method of streptococcus pneumoniae capsular polysaccharide protein conjugate |
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| CN1971275B (en) * | 2006-12-06 | 2012-07-04 | 云南沃森生物技术股份有限公司 | Method for detecting content of free polysaccharides in polysaccharides combo of pneumonia streptococcus 6B, 18C, 19F and 23F type |
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| CN113198013B (en) * | 2005-04-08 | 2024-02-20 | 惠氏有限责任公司 | Multivalent pneumococcal polysaccharide-protein conjugate composition |
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| CN103599529A (en) * | 2006-12-22 | 2014-02-26 | 惠氏公司 | Multivalent pneumococcal polysaccharide-protein conjugate composition |
| CN103599529B (en) * | 2006-12-22 | 2016-03-16 | 惠氏公司 | Multivalent pneumococcal polysaccharide-protein conjugate composition |
| CN102068690A (en) * | 2010-12-31 | 2011-05-25 | 北京民海生物科技有限公司 | Polyvalent pneumococcal capsular polysaccharide conjugate vaccine and preparation method thereof |
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| CN105131139A (en) * | 2015-07-31 | 2015-12-09 | 兰州生物制品研究所有限责任公司 | Purification method of streptococcus pneumoniae capsular polysaccharide |
| CN106039300A (en) * | 2016-05-26 | 2016-10-26 | 北京民海生物科技有限公司 | Preparation method of streptococcus pneumoniae capsular polysaccharide protein conjugate |
| CN106039300B (en) * | 2016-05-26 | 2019-05-24 | 北京民海生物科技有限公司 | A kind of preparation method of streptococcus pneumoniae capsular polysaccharide protein conjugates |
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