CN101785857B

CN101785857B - Novel pneumococcal conjugate vaccine and preparation method thereof

Info

Publication number: CN101785857B
Application number: CN2010101294042A
Authority: CN
Inventors: 王岩; 赵光辉; 郑玉清
Original assignee: ZHENG YVQING
Current assignee: Fosun Antekin (Chengdu) Biopharmaceutical Co.,Ltd.
Priority date: 2010-03-05
Filing date: 2010-03-05
Publication date: 2012-09-26
Anticipated expiration: 2030-03-05
Also published as: CN101785857A

Abstract

The invention relates to a novel pneumococcal conjugate vaccine and a preparation method thereof. The novel pneumococcal conjugate vaccine comprises a dual-valent serum type pneumococcal capsular sugar-protein combination and/or a multi-valent serum type pneumococcal capsular sugar-protein combination. The dual-valent serum type capsular sugar-protein combination is a combination formed by combining two different serum types of capsular sugar with a protein carrier through chemical bonds, and the two different serum types of capsular sugar form links in the structure according to the combination with the protein carrier through the chemical bonds. The multi-valent serum type capsular sugar-protein combination is a combination formed by combining two different serum types of capsular sugar with a protein carrier through chemical bonds, and the two different serum types of capsular sugar form links in the structure according to the combination with the protein carrier through the chemical bonds. In addition, the invention also provides a method of preparing the bacterial capsular sugar-protein conjugate vaccine.

Description

A kind of new pneumococcal conjugated vaccine and preparation method thereof

Technical field

The present invention relates to a kind of new pneumococcal conjugated vaccine.Particularly; The present invention relates to a plurality of serotype capsular saccharides of a kind of streptococcus pneumoniae and the bonded new pneumococcal conjugated vaccine of protein carrier; The capsular saccharides that derives from different serotypes through with the chemical bond of protein carrier after, be present in the vaccine with divalent serotype capsular saccharides-protein binding body and/or polyvalent serum type capsular saccharides-protein binding bodily form formula.The invention still further relates to the method for preparing of described new pneumococcal conjugated vaccine, and the method for preparing of new bacterial capsule glycoprotein combined vaccine.

Background technology

The disease that streptococcus pneumoniae (Streptococcus pneumoniae) causes is the especially main cause of disease of the child of developing country sickness rate and mortality rate of global child; Pneumococcal disease burden mainly comes from child below 2 years old and old people, and the streptococcus pneumoniae disease has become the serious public health problem in the whole world.The whole world annual about 1; The child suffers from the streptococcus pneumoniae disease below 0,600,000 5 years old; WHO estimates to have every year the 1600000 people coccus disease that dies of pneumonia; Comprise 70～child below 1,000,000 5 years old, wherein great majority are the children (http://www.who.int/biologicals/areas/vaccines/pneumo/en/) that live in developing country.It is preventible less than in 5 years old death of child disease that Global Link is crossed vaccination, and the streptococcus pneumoniae disease is the 1st, accounts for 28% (Yang Yonghong, Chinese vaccine and immunity, 2008; 14:4:376-377).Streptococcus pneumoniae disease death number has surpassed summation (Obaro, the Expert Rev Vaccines.2009 of AIDS, malaria and measles death toll; 8 (8): 1051-1061).Seasonal influenza virus, H1N1 influenza virus, HIV viral infection heighten the probability of suffering from pneumoccoccosis with other diseases relevant with immunodeficiency.U.S. CDC is announced each flu outbreak in last century; The streptococcus pneumoniae disease of secondary is the morbidity and the dead main cause of disease, also observes same situation (http://www.cdc.gov/h1n1flu/vaccination/provider/provider_pneumo coccal.htm) at the seasonal influenza epidemic period.

The serious disease that streptococcus pneumoniae is brought out comprises pneumonia, meningitis and septicemia, and otitis media, sinusitis and tracheitis also are common, but the clinical symptoms that infects is slightly light.After being exposed to streptococcus pneumoniae, temporary nasopharynx part bacteria planting often occurring but do not fall ill, when Abwehrkraft des Koepers descends, streptococcus pneumoniae can be caused the local infection of respiratory system, like acute otitis media, sinusitis etc.; Streptococcus pneumoniae gets into lower respiratory tract and causes pneumonia; Further; Formed infectious disease when streptococcus pneumoniae is invaded blood, cerebrospinal fluid or other originally aseptic positions; Be called aggressive streptococcus pneumoniae disease (Invasive Pneumococcal Disease; IPD), bacteremia property pneumonia, bacteremia heating, septicemia and meningitis are the main clinical manifestation of IPD.

The group of people at high risk of pneumococcal infection is＜5 years old child and >=65 years old old man, wherein＜child's sickness rate was the highest in 2 years old.The U.S. is before extensive use pneumococcal conjugated vaccine; The annual morbidity of IPD in≤12 monthly age childs is 1,67/,100,000; Sickness rate in 6～11 monthly age childs reaches 2,35/,100,000 peak; Sickness rate in 12～23 monthly age childs is 2,03/,100,000, and whole crowd's sickness rate has only 24/,100,000.In the Europe and the U.S., the annual morbidity of IPD is 10～100 examples/100,000.In the developing country below 5 years old among the child sickness rate of IPD than high quite a few times of industrialized country.In 1996～2000 years＜5 years old death of child analysis on monitoring result of China, pneumonia is the first reason (7,73/,100,000), accounts for 19% (Yang Yonghong, Chinese vaccine and immunity, 2008 of whole death; 14:4:376-377).

Streptococcus pneumoniae is Grain-positive, the diplococcus with pod membrane.(Polysaccharides PS) is its virulence factor that mainly causes a disease to capsular polysaccharide.According to the composition difference of capsular polysaccharide, streptococcus pneumoniae is distinguished 91 serotypes of 46 sero-groups.The capable serotype distribution profile of the predominant current of all ages and classes, time and geographic area is also different, but all discoveries to some extent in all parts of the world of common serotype, and the common pathogenic serotype in the whole world is consistent.In the world, surplus in the of about 20 kind of serotype to occur in the IPD of each age group relevant with surpassing 80%.The modal sero-group in the whole world is 6,14,19, yet in the child IPD of developing country, mainly is 1,5,8 sero-groups.

Streptococcus pneumoniae has become a phenomenon quite severe, that sharply develop to the drug resistance of antibiotic (like penicillin, cephalosporin, bactrim, Macrolide and FQNS) in the whole world.Along with the drug resistance of streptococcus pneumoniae to common antibiotics increases day by day, currently need effective vaccine badly and control streptococcus pneumoniae disease (WHO.Weekly Epidemiological Record.No.12,2007; 93-104).At present; There are 2 prevention streptococcus pneumoniae disease vaccines to go on the market; Be respectively to contain 23 the antigenic vaccine of serotype capsular polysaccharide (Pneumococcal Polysaccharide Vaccine; PPV23) and contain 7 serotype capsular saccharides and the bonded combined vaccine of protein carrier (Pneumococcal Conjugate Vaccine, PCV7).

Capsular saccharides (comprising capsular polysaccharide and pod membrane oligosaccharide) antigen right and wrong T cell dependent type antigen can not produce immunological memory reaction, in child below 2 years old a little less than the immunogenicity.Therefore PPV23 is mainly used in prevention adult and >=2 years old children Streptococcus coccus property disease.0.5 milliliter every dose of PPV23; Contain the purification capsular polysaccharide of 23 serotypes (1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F), each serotype capsular polysaccharide is 25 μ g.It is about 90% that these 23 serotypes cover most adult IPD in the U.S. and some other industrialized country, and PPV23 also comprises modal drug resistance serotype.Over more than 30 year, although carried out a plurality of researchs, PPV23 still is difficult to final conclusion in child and adult's effectiveness and effect, has many disputes.Effectiveness and effect to the PPV23 vaccine are carried out comprehensive meta analysis (comprising that WHO in 2007 entrusts the meta that carries out to analyze and the analysis-by-synthesis to RCTs) discovery; To IPD with entirely because of pneumonia has protective effect, in the old people, IPD at least also there is protection to a certain degree at the young philtrum PPV23 of health.But these tests do not confirm PPV23 excessive risk crowd (as suffering from adult and the child of the underlying diseases that can increase infection risk, the serious immunosuppressant person of all age brackets) to IPD with have protective effect (Moberley S et al.Cochrane Database ofSystematic Reviews, 2008:1 because of pneumonia entirely; CD000422, Huss A et al.Canadian Medical AssociationJournal, 2008.).Many industrialized countries recommend old man and other excessive risks crowd to inoculate PPV23; The WHO suggestion is in the country of resource-constrained; Existing evidence is not supported old man and the conventional inoculation of excessive risk crowd PPV23 (WHO.Weekly Epidemiological Record.No.42,2008; 373-584).

Capsular saccharides antigen and protein carrier change T cell dependent type antigen into after chemical bond combines, but the immunne response of inducing T cell mediation is characterized in that promoting the formation of protective immunity, especially in the baby, produce protection and induction of immunity memory.The PCV7 of every 0.5ml dosage contains the capsular polysaccharide of 2 μ g serotypes 4,9V, 14,19F and 23F; The oligosaccharide of 2 μ g serotype 18C; The polysaccharide of 4 μ g serotype 6B.Each serotype capsular saccharides all with avirulent diphtheria CRM.197 protein binding, and adsorb by the aluminum phosphate adjuvant.In the U.S., before this vaccine of introduction was carried out extensive immunity inoculation, the serotype that comprises in this vaccine accounted for 86% of child's aggressive streptococcus pneumoniae strain.The extensive field study of the U.S. shows, in having inoculated the child of at least 3 pins time, can reach 97.4% by PCV7 vaccine serotype inductive protective immunity to IPD, in inoculating the child of 1 pin time at least, can reach 93.9%.PCV7 being introduced national immunity inoculation planning after 1 year, reduced by 100% at all sickness rate among the inoculator below 1 years old by the IPD due to the streptococcus pneumoniae serotype that contains in this vaccine.Introducing this vaccine after 3 years, below 1 years old among the child sickness rate of all IPD reduced by 84.1%; Reduced by 52% at 20～39 years old among the adult, in individuality more than 60 years old, reduced by 27%.Follow-up monitoring shows, is introducing this vaccine after 2 years, and the sickness rate of IPD in child below 5 years old reduced by 75%.The decline of IPD sickness rate more than 5 years old, among the not vaccinated crowd possibly be because from below 5 years old, the spread of germs of vaccinated infant reduced promptly so-called " herd immunity " phenomenon.In the U.S., in the IPD that all are prevented from, have 68% can give the credit to this indirect effect approximately.PCV7 has been through having accepted check in the test of carrying out in global different regions, and has been proved to be effective, safe, better tolerance, even if also be like this in the HIV infant.WHO whole world vaccine safety Advisory Board assessed the safety of PCV7 in November, 2006; Conclusion is thought; About the evidence of the safety of PCV7 and other pneumococcal conjugated vaccines is compellent (WHO.Weekly Epidemiological Record.No.12,2007; 93-104).

Yet behind the PCV7 large-scale inoculation, variation has really appearred in streptococcus pneumoniae serotype epidemic diseases, PCV7 in 2000 behind U.S.'s large-scale inoculation, becoming maximum the growth, reach 10.8/10 ten thousand less than observing nonvaccine serotype IPD in 1 years old among the child.The IPD sickness rate that causes less than 19A serotype among 5 years old child from 1998～1999 years 2.6/10 ten thousand rise to 2005 9.3/10 ten thousand.2005 is being that nonvaccine serotype 19A causes less than 40% of the IPD among 5 years old child, and 19A becomes sickness rate and increases the highest serotype.Although continuing to descend less than PCV7 vaccine serotype disease among 5 years old child in 2005, the overall sickness rate of IPD but presented plateau (Reingold et al.Morbidity & Mortality WeeklyReport.2008 in 2002～2005 years; 57:6:144-148.).Another researcher also points out, 2002～2005 years less than 72～91% of annual IPD among 5 years old child, (the Pelton et al.Pediatr Infect DisJ.2007 that the nonvaccine serotype bacterial strain causes; 26 (6): 468-472.).

Because the huge public health that successful vaccine is brought the streptococcus pneumoniae disease influences, WHO thinks and answers preferential development safety, effective, moderate, and can the more Pnu-Imune 23 of broad spectrum protection be provided for the streptococcus pneumoniae disease.Need the vaccine of more effective combined vaccine or other types, these vaccines can be contained the most of streptococcus pneumoniae serotypes (often also being to the drug-fast serotype of antibacterials commonly used) that cause global big-age-child and adult's serious disease.WHO supports R&D work (WHO.Weekly Epidemiological Record.No.42,2008 of current this type vaccine product of carrying out; 373-584).Therefore, expansion PCV7 vaccine serotype just highlights and is instant task.

In recent years; In view of 1,3,5,6A, 7F and 19A serotype invasive pneumonia severity of disease and harm, the PCV13 that on PCV7 vaccine basis, increase serotype 1 and 5 PCV9, continue the PCV10 of increase serotype 7F, increase the PCV11 of serotype 3 again, further increases serotype 6A and 19A is under development in succession.PCV7 is respectively 80～90%, 60～80%, 40～80% in the coverage rate of the U.S., Europe, Asia and Latin America.In PCV7, increase by 6 type capsular glycoprotein conjugates, become 13 valency combined vaccines.13 valency combined vaccines all are promoted to more than 90% the coverage rate of US and European, and the coverage rate of Asia and Latin America also is promoted to 70～80%.Yet, compare the PPV23 vaccine the coverage rate in the whole world with consider the large-scale inoculation vaccine after the trend of the popular increase of nonvaccine serotype will appear, the PCV13 coverage rate still need continue to enlarge.

Existing pneumococcal conjugated vaccine all be technically with the capsular saccharides of each serotype independent with protein carrier after chemical bond combines; Form monovalent serum type capsular saccharides-protein binding body; Again each monovalent serum type capsular saccharides-protein binding body is merged, and then preparation becomes the pneumococcal conjugated vaccine that contains a plurality of serotype capsular saccharides.Each capsular saccharides antigen independently exists with unit price capsular saccharides-protein binding body in the pneumococcal conjugated vaccine, does not have structural link each other.For example; PCV7 combines to form 7 unit price capsular saccharides-protein binding bodies with protein carrier CRM197 through chemical bond respectively with the different capsular saccharides that derive from 7 serotypes; Be 4PS-CRM197,9VPS-CRM197,14PS-CRM197,19F PS-CRM197,23F PS-CRM197,6B PS-CRM197 and 18C OS-CRM197, above-mentioned 7 unit price capsular saccharides-protein binding bodies merged in proportion be mixed with multivalence finished product vaccine again.Therefore; Utilize prior art to combine respectively with protein carrier above more serotype capsular saccharides of 13 serotypes; Form a plurality of monovalent serotype capsular saccharides-protein binding body; A plurality of monovalent serum type capsular saccharides-protein binding body are merged again, and process single bacterin preparation, (one Chinese patent application number 200680053139.6) technically still has difficulties.Simultaneously, existing P CV7 and the PCV13 that is about to listing compare with the Hib combined vaccine, are being equivalent to 7 and 13 Hib combined vaccines aspect production process and the production cost respectively.These effects limit further developing of pneumococcal conjugated vaccine.

Novel pneumococcal conjugated vaccine provided by the invention; System will derive from 2 different serotypes capsular saccharides antigens or a plurality of different serotypes capsular saccharides antigen; Carry out chemical bond with carrier protein, form divalent serotype capsular saccharides-protein binding body or polyvalent serum type capsular saccharides-protein binding body; Again with one or two or more than two such coalition be mixed with pneumococcal conjugated vaccine.Therefore, pneumococcal conjugated vaccine of the present invention has been inherited the safety of existing pneumococcal conjugated vaccine, good immunogenicity, the advantage of induction of immunity memory; Successfully solved again the included technical difficulty of more serotype capsular saccharides antigen, made the vaccine coverage wider; Further, significance ground has shortened the production of vaccine process and has reduced production cost.

Summary of the invention

One aspect of the present invention provides a kind of pneumococcal conjugated vaccine; The antigen of said vaccine comprises a plurality of serotype capsular saccharides of streptococcus pneumoniae; These capsular saccharides that derive from different serotypes carry out chemical bond with protein carrier and combine; Capsular saccharides is present in the vaccine with divalent serotype capsular saccharides-protein binding body and/or polyvalent serum type capsular saccharides-protein binding bodily form formula through combine to set up structural the link each other with other type capsular saccharides with the chemical bond of protein carrier.In other words; The present invention includes the capsular saccharides antigen that derives from different streptococcus pneumoniae serotypes and combine the pneumococcal conjugated vaccine formed with protein carrier, the form that said capsular saccharides is each other structurally set up link by protein carrier through chemical bond with divalent and/or multivalence is present in the vaccine.Pneumococcal conjugated vaccine provided by the invention is used for the disease that infant, child and adult's prevention pneumococcal infection cause.

Another aspect of the invention provides a kind of pneumococcal conjugated vaccine; The antigen of said vaccine comprises a plurality of serotype capsular polysaccharides of streptococcus pneumoniae; These capsular polysaccharides that derive from different serotypes carry out chemical bond with protein carrier and combine; Capsular polysaccharide is present in the vaccine with divalent serotype capsular polysaccharide-protein binding body and/or polyvalent serum type capsular polysaccharide-protein binding bodily form formula through combine to set up structural the link each other with other type capsular polysaccharide with the chemical bond of protein carrier.Therefore; The present invention includes the capsular polysaccharide antigen that derives from different streptococcus pneumoniae serotypes and combine the pneumococcal conjugated vaccine formed with protein carrier, the form that said capsular polysaccharide is each other structurally set up link by protein carrier through chemical bond with divalent and/or multivalence is present in the vaccine.

Another aspect of the invention provides a kind of pneumococcal conjugated vaccine; The antigen of said vaccine comprises a plurality of serotype pod membrane of streptococcus pneumoniae oligosaccharide; These pod membrane oligosaccharide that derive from different serotypes carry out chemical bond with protein carrier and combine; The pod membrane oligosaccharide is present in the vaccine with divalent serotype pod membrane oligosaccharide-protein binding body and/or polyvalent serum type pod membrane oligosaccharide-protein binding bodily form formula through combine to set up structural the link each other with other type pod membrane oligosaccharide with the chemical bond of protein carrier.Therefore; The present invention includes the pod membrane oligosaccharide antigen that derives from different streptococcus pneumoniae serotypes and combine the pneumococcal conjugated vaccine formed with protein carrier, the form that said pod membrane oligosaccharide is each other structurally set up link by protein carrier through chemical bond with divalent and/or multivalence is present in the vaccine.

Another aspect of the invention provides a kind of pneumococcal conjugated vaccine; The antigen of said vaccine comprises a plurality of serotype capsular polysaccharides of streptococcus pneumoniae and oligosaccharide; These capsular polysaccharide and oligosaccharide that derive from different serotypes carry out chemical bond with protein carrier and combine; Capsular polysaccharide and oligosaccharide be through combining to set up structural the link each other with other type capsular polysaccharide and oligosaccharide with the chemical bond of protein carrier, be present in the vaccine with divalent serotype capsular polysaccharide and oligosaccharide-protein binding body and/or polyvalent serum type capsular polysaccharide and oligosaccharide-protein binding bodily form formula.Therefore; The present invention includes the capsular polysaccharide that derives from different streptococcus pneumoniae serotypes and oligosaccharide antigen and combine the pneumococcal conjugated vaccine formed with protein carrier, the form that said capsular polysaccharide and oligosaccharide are each other structurally set up link by protein carrier through chemical bond with divalent and/or multivalence is present in the vaccine.

Another aspect of the invention provides a kind of pneumococcal conjugated vaccine; The antigen of said vaccine comprises a plurality of serotype capsular polysaccharides of streptococcus pneumoniae and oligosaccharide; These capsular polysaccharides that derive from one group of different serotypes carry out chemical bond with protein carrier and combine; This group capsular polysaccharide is present in the vaccine with divalent serotype capsular polysaccharide-protein binding body and/or polyvalent serum type capsular polysaccharide-protein binding bodily form formula through combine to set up each other structural the link with the chemical bond of protein carrier.These pod membrane oligosaccharide that derive from other one group different serotypes carry out chemical bond with protein carrier and combine; This group pod membrane oligosaccharide is present in the vaccine with divalent serotype pod membrane oligosaccharide-protein binding body and/or polyvalent serum type pod membrane oligosaccharide-protein binding bodily form formula through combine to set up each other structural the link with the chemical bond of protein carrier.Therefore; The present invention includes capsular polysaccharide that derives from one group of different streptococcus pneumoniae serotype and the pod membrane oligosaccharide antigen that derives from other one group of different serotypes and combine the pneumococcal conjugated vaccine formed respectively with protein carrier; The form that said various capsular polysaccharide is to each other structurally set up link by protein carrier through chemical bond with divalent and/or multivalence is present in the vaccine, and the form that said various pod membrane oligosaccharide is to each other structurally set up link by protein carrier through chemical bond with divalent and/or multivalence is present in the vaccine.Divalent or polyvalent serum type capsular polysaccharide-protein binding body and divalent or polyvalent serum type pod membrane oligosaccharide-protein binding body independently exist respectively in the vaccine, do not have structural link each other.

Another aspect of the invention provides a kind of pneumococcal conjugated vaccine; The antigen of said vaccine comprises a plurality of serotype capsular saccharides of streptococcus pneumoniae; These capsular saccharides that derive from different serotypes randomly carry out chemical bond with per 2～45 serotype capsular saccharides with protein carrier and combine; These 2～45 serotype capsular saccharides are through combining to set up each other structural the link with the chemical bond of protein carrier, with one or two or more than two 2～45 valency serotype capsular saccharides-protein binding bodily form formula be present in the vaccine.Therefore; The present invention includes the capsular saccharides antigen that derives from different streptococcus pneumoniae serotypes and combine the pneumococcal conjugated vaccine formed with protein carrier, said capsular saccharides randomly is present in the vaccine with the form that per 2～45 serotype capsular saccharides are structurally set up link by protein carrier through chemical bond to each other.The capsular saccharides that derives from different serotypes with one or two or more than two 2～45 valency serotype capsular saccharides-protein binding bodies independently exist in the vaccine; In other words, each 2～45 valency serotype capsular saccharides-protein binding body that comprises in the vaccine of the present invention do not have structural the link each other with other 2～45 valency serotype capsular saccharides-protein binding bodies.

Another aspect of the invention provides a kind of pneumococcal conjugated vaccine; The antigen of said vaccine comprises a plurality of serotype capsular saccharides of streptococcus pneumoniae; These capsular saccharides that derive from different serotypes randomly carry out chemical bond with per 2～45 serotype capsular saccharides with protein carrier and combine; These 2～45 serotype capsular saccharides are through combining to set up each other structural the link with the chemical bond of protein carrier; With one or two or more than two 2～45 valency serotype capsular saccharides-protein binding bodily form formula be present in the vaccine; Wherein, said two or more 2～45 valency serotype capsular saccharides-the protein binding body contains the serotype capsular saccharides of phase equivalent number respectively, and this valence mumber is all between 2～45.Therefore; The present invention includes the capsular saccharides antigen that derives from different streptococcus pneumoniae serotypes and combine the pneumococcal conjugated vaccine formed with protein carrier, said capsular saccharides randomly is present in the vaccine with the form that per 2～45 serotype capsular saccharides are structurally set up link by protein carrier through chemical bond to each other.The capsular saccharides that derives from different serotypes with one or two or more than two 2～45 valency serotype capsular saccharides-protein binding bodies independently exist in the vaccine; Wherein, Said two or more 2～45 valency serotype capsular saccharides-protein binding bodies independently are present in the vaccine with the serotype capsular saccharides-protein binding bodily form formula of phase equivalent number, and this phase equivalent number is all between 2～45.In other words, comprise the combination of the serotype capsular saccharides-protein binding body of phase equivalent number in the vaccine of the present invention, for example, comprise two above divalent serotype capsular saccharides-protein binding bodies; Or comprise two or more 3 valency serotype capsular saccharides-protein binding bodies; Or comprise two or more 4 valency serotype capsular saccharides-protein binding bodies; Or comprise two or more 5 valency serotype capsular saccharides-protein binding bodies; Or comprise one or two or 6 valency serotype capsular saccharides-protein binding bodies more than two; Or comprise one or two or 7 valency serotype capsular saccharides-protein binding bodies more than two; Or comprise one or two or 8 valency serotype capsular saccharides-protein binding bodies more than two; Or comprise one or two or 9 valency serotype capsular saccharides-protein binding bodies more than two; Or comprise one or two or 10 valency serotype capsular saccharides-protein binding bodies more than two; Or comprise one or two or 11 valency serotype capsular saccharides-protein binding bodies more than two; Or comprise one or two or 12 valency serotype capsular saccharides-protein binding bodies more than two; Or comprise one or two or 13 valency serotype capsular saccharides-protein binding bodies more than two; Or comprise one or two or 14 valency serotype capsular saccharides-protein binding bodies more than two; Or comprise one or two or 15 valency serotype capsular saccharides-protein binding bodies more than two; Or comprise one or two or 16 valency serotype capsular saccharides-protein binding bodies more than two; Or comprise one or two or 17 valency serotype capsular saccharides-protein binding bodies more than two; Or comprise one or two or 18 valency serotype capsular saccharides-protein binding bodies more than two; Or comprise one or two or 19 valency serotype capsular saccharides-protein binding bodies more than two; Or comprise one or two or 20 valency serotype capsular saccharides-protein binding bodies more than two; Or comprise one or two or 21 valency serotype capsular saccharides-protein binding bodies more than two; Or comprise one or two or 22 valency serotype capsular saccharides-protein binding bodies more than two; Or comprise one or two or 23 valency serotype capsular saccharides-protein binding bodies more than two; Or comprise one or two or 24 valency serotype capsular saccharides-protein binding bodies more than two; By that analogy, until comprising one or two 45 valency serotype capsular saccharides-protein binding body; And each 2～45 valency serotype capsular saccharides-protein binding body does not have structural the link each other with other 2～45 valency serotype capsular saccharides-protein binding bodies.

Another aspect of the invention provides a kind of pneumococcal conjugated vaccine; The antigen of said vaccine comprises a plurality of serotype capsular saccharides of streptococcus pneumoniae; These capsular saccharides that derive from different serotypes randomly carry out chemical bond with per 2～45 serotype capsular saccharides with protein carrier and combine; These 2～45 serotype capsular saccharides are through combining to set up each other structural the link with the chemical bond of protein carrier; With one or two or more than two 2～45 valency serotype capsular saccharides-protein binding bodily form formula be present in the vaccine; Wherein, said two or more 2～45 valency serotype capsular saccharides-protein binding bodies are present in the vaccine with the serotype capsular saccharides-protein binding bodily form formula of unequal valence mumber, and this unequal valence mumber is between 2～45.Therefore; The present invention includes the capsular saccharides antigen that derives from different streptococcus pneumoniae serotypes and combine the pneumococcal conjugated vaccine formed with protein carrier, said capsular saccharides randomly is present in the vaccine with the form that per 2～45 serotype capsular saccharides are structurally set up link by protein carrier through chemical bond to each other.The capsular saccharides that derives from different serotypes with one or two or more than two 2～45 valency serotype capsular saccharides-protein binding bodies independently exist in the vaccine; Wherein, Said two or more 2～45 valency serotype capsular saccharides-protein binding bodies independently are present in the vaccine with the serotype capsular saccharides-protein binding bodily form formula of unequal valence mumber, and this unequal valence mumber is between 2～45.In other words; Comprise the combination of the serotype capsular saccharides-protein binding body of unequal valence mumber in the vaccine of the present invention simultaneously; Both can comprise divalent serotype capsular saccharides-protein binding body, also comprise 3 valency serotype capsular saccharides-protein binding bodies, 4 valency serotype capsular saccharides-protein binding bodies, 5 valency serotype capsular saccharides-protein binding bodies, until the optional combination of 45 valency serotype capsular saccharides-protein binding bodies.And each 2～45 valency serotype capsular saccharides-protein binding body does not have structural the link each other with other 2～45 valency serotype capsular saccharides-protein binding bodies.

Another aspect of the invention provides a kind of pneumococcal conjugated vaccine; The antigen of said vaccine comprises a plurality of serotype capsular saccharides of streptococcus pneumoniae; These capsular saccharides that derive from different serotypes randomly carry out chemical bond with per 2～45 serotype capsular saccharides with protein carrier and combine; These 2～45 serotype capsular saccharides are through combining to set up each other structural the link with the chemical bond of protein carrier; With one or two or more than two 2～45 valency serotype capsular saccharides-protein binding bodily form formula be present in the vaccine; Wherein, said more than two 2～45 valency serotype capsular saccharides-protein binding bodies be present in the vaccine with the serotype capsular saccharides-protein binding bodily form formula of part phase equivalent number, and this phase equivalent number is between 2～45.Therefore; The present invention includes the capsular saccharides antigen that derives from different streptococcus pneumoniae serotypes and combine the pneumococcal conjugated vaccine formed with protein carrier, said capsular saccharides randomly is present in the vaccine with the form that per 2～45 serotype capsular saccharides are structurally set up link by protein carrier through chemical bond to each other.The capsular saccharides that derives from different serotypes with one or two or more than two 2～45 valency serotype capsular saccharides-protein binding bodies independently exist in the vaccine; Wherein, Said more than two 2～45 valency serotype capsular saccharides-protein binding bodies independently be present in the vaccine with the serotype capsular saccharides-protein binding bodily form formula of part phase equivalent number, and this phase equivalent number is between 2～45.In other words; Comprise the combination of serotype capsular saccharides-protein binding body of serotype capsular saccharides-protein binding body and the unequal valence mumber of phase equivalent number in the vaccine of the present invention simultaneously; And each 2～45 valency serotype capsular saccharides-protein binding body does not have structural the link each other with other 2～45 valency serotype capsular saccharides-protein binding bodies.

Another aspect of the invention provides a kind of streptococcus pneumoniae-meningococcus-b type hemophilus influenza combined vaccine; Said combined vaccine antigen comprises a plurality of serotype capsular saccharides of streptococcus pneumoniae, 4 groups of (A, C, W135, Y) capsular saccharides of meningococcus and b type hemophilus influenza capsular saccharides; These capsular saccharides that derive from different bacterium serotype (crowd) carry out chemical bond with protein carrier and combine; Various (crowd) capsular saccharides is present in the vaccine with divalent serotype (crowd) capsular saccharides-protein binding body and/or polyvalent serum type (crowd) capsular saccharides-protein binding bodily form formula through combine to set up each other structural the link with the chemical bond of protein carrier.Therefore; The capsular saccharides antigen that the present invention includes the different serotypes (crowd) that derives from a plurality of serotypes of streptococcus pneumoniae, meningococcus 4 groups (A, C, W135, Y) and the bloodthirsty coccus of b type influenza combines the combined vaccine formed with protein carrier, the form that said various (crowd) capsular saccharides is to each other structurally set up link by protein carrier through chemical bond with divalent and/or multivalence is present in the vaccine.

Another aspect of the invention provides a kind of streptococcus pneumoniae-meningococcus-b type hemophilus influenza combined vaccine; Said combined vaccine antigen comprises a plurality of serotype capsular saccharides of streptococcus pneumoniae, 2 groups of (A, C) capsular saccharides of meningococcus and b type hemophilus influenza capsular saccharides; These capsular saccharides that derive from different bacterium serotype (crowd) carry out chemical bond with protein carrier and combine; Various (crowd) capsular saccharides is present in the vaccine with divalent serotype (crowd) capsular saccharides-protein binding body and/or polyvalent serum type (crowd) capsular saccharides-protein binding bodily form formula through combine to set up each other structural the link with the chemical bond of protein carrier.Therefore; The capsular saccharides antigen that the present invention includes the different serotypes (crowd) that derives from a plurality of serotypes of streptococcus pneumoniae, meningococcus 2 groups (A, C) and the bloodthirsty coccus of b type influenza combines the combined vaccine formed with protein carrier, the form that said various (crowd) capsular saccharides is to each other structurally set up link by protein carrier through chemical bond with divalent and/or multivalence is present in the vaccine.

Another aspect of the invention provides a kind of streptococcus pneumoniae-meningococcus-b type hemophilus influenza combined vaccine; Said combined vaccine antigen comprises a plurality of serotype capsular saccharides of streptococcus pneumoniae, 4 groups of (A, C, W135, Y) capsular saccharides of meningococcus and b type hemophilus influenza capsular saccharides; These capsular saccharides that derive from the streptococcus pneumoniae different serotypes are present in the vaccine with divalent serotype capsular saccharides-protein binding body and/or polyvalent serum type capsular saccharides-protein binding bodily form formula through combine to form structural to each other the link with the chemical bond of protein carrier.These capsular saccharides that derive from the different sero-groups of meningococcus are present in the vaccine with 4 valency sero-group capsular saccharides-protein binding bodily form formula through combine to form structural to each other link of each group capsular saccharides with the chemical bond of protein carrier.The capsular saccharides that derives from b type hemophilus influenza is present in the vaccine with monovalent serum type capsular saccharides-protein binding bodily form formula through combining with the chemical bond of protein carrier.Therefore; The capsular saccharides antigen that the present invention includes the different serotypes (crowd) that derives from a plurality of serotypes of streptococcus pneumoniae, meningococcus 4 groups (A, C, W135, Y) and the bloodthirsty coccus of b type influenza combines the combined vaccine formed with protein carrier; The form that said streptococcus pneumoniae is to each other structurally set up link by protein carrier through chemical bond with every divalent and/or polyvalent serum type capsular saccharides is present in the vaccine; Said each group of meningococcus capsular saccharides is present in the vaccine with the form of structurally being set up link to each other through chemical bond by protein carrier, and said b type hemophilus influenza capsular saccharides is to be present in the vaccine by the bonded form of protein carrier.Streptococcus pneumoniae capsular saccharides, meningococcal capsular saccharides, b type hemophilus influenza capsular saccharides do not have structural link each other.

Another aspect of the invention provides a kind of streptococcus pneumoniae-meningococcus-b type hemophilus influenza combined vaccine; Said combined vaccine antigen comprises a plurality of serotype capsular saccharides of streptococcus pneumoniae, 2 groups of (A, C) capsular saccharides of meningococcus and b type hemophilus influenza capsular saccharides; These capsular saccharides that derive from the streptococcus pneumoniae different serotypes are present in the vaccine with divalent serotype capsular saccharides-protein binding body and/or polyvalent serum type capsular saccharides-protein binding bodily form formula through combine to form structural to each other the link with the chemical bond of protein carrier.These capsular saccharides that derive from the different sero-groups of meningococcus are present in the vaccine with divalent sero-group capsular saccharides-protein binding bodily form formula through combine to form structural to each other link of each group capsular saccharides with the chemical bond of protein carrier.The capsular saccharides that derives from b type hemophilus influenza is present in the vaccine with monovalent serum type capsular saccharides-protein binding bodily form formula through combining with the chemical bond of protein carrier.Therefore; The capsular saccharides antigen that the present invention includes the different serotypes (crowd) that derives from a plurality of serotypes of streptococcus pneumoniae, meningococcus 2 groups (A, C) and the bloodthirsty coccus of b type influenza combines the combined vaccine formed with protein carrier; The form that said streptococcus pneumoniae is to each other structurally set up link by protein carrier through chemical bond with every divalent and/or polyvalent serum type capsular saccharides is present in the vaccine; Said each group of meningococcus capsular saccharides is present in the vaccine with the form of structurally being set up link to each other through chemical bond by protein carrier, and said b type hemophilus influenza capsular saccharides is to be present in the vaccine by the bonded form of protein carrier.Streptococcus pneumoniae capsular saccharides, meningococcal capsular saccharides, b type hemophilus influenza capsular saccharides do not have structural link each other.

Another aspect of the invention provides a kind of preparation to contain the method for the polyvalent bacterial capsular saccharides combined vaccine of divalent serotype (crowd) capsular saccharides-protein binding body and/or polyvalent serum type (crowd) capsular saccharides-protein binding body, said method comprising the steps of:

(a) from different serotypes (crowd) bacterial cultures, extract respectively, the purification capsular saccharides, process single serotype (crowd) capsular saccharides, i.e. monovalent serum type (crowd) capsular saccharides;

(b) monovalent serum type (crowd) capsular saccharides of step (a) the gained capsular saccharides in two or more serotypes (crowd) is merged according to ratio, become divalent or polyvalent serum type (crowd) capsular saccharides;

(c) divalent of step (b) or polyvalent serum type (crowd) capsular saccharides and protein carrier are carried out chemical bonded refractory and merge purification, process divalent serotype (crowd) capsular saccharides-protein binding isoantigen or polyvalent serum type (crowd) capsular saccharides-protein binding isoantigen;

(d) divalent serotype (crowd) capsular saccharides-protein binding isoantigen and/or polyvalent serum type (crowd) capsular saccharides-protein binding isoantigen with step (c) is mixed with vaccine.

In term of the present invention, capsular saccharides comprises capsular polysaccharide and pod membrane oligosaccharide; Said pod membrane oligosaccharide is the pod membrane oligosaccharide of capsular polysaccharide after the chemical method degradeds such as (the β method of elimination that comprises the mediation of alkali or lyases) of Hydrolyze method (comprising acid, alkali, enzyme hydrolysis), oxidizing process and method of elimination; Further, said pod membrane oligosaccharide also comprises the pod membrane oligosaccharide of capsular polysaccharide after the degraded of physical methods such as sonication and mechanical shearing.

In term of the present invention, monovalent serum type (crowd) capsular saccharides-protein binding body is meant a single serotype

(crowd) capsular saccharides and protein carrier combine the conjugate that forms through chemical bond, and this capsular saccharides does not have structural the link with other type (crowd) capsular saccharides.Divalent serotype (crowd) capsular saccharides-protein binding body is meant that 2 different serotypes (crowd) capsular saccharides combines to form conjugate with protein carrier through chemical bond, and this 2 types (crowd) capsular saccharides warp generates chemical bond with protein carrier and sets up structural the link to each other.In other words, protein carrier is linked at 2 different shaped (crowd) capsular saccharides together through chemical bond.Polyvalent serum type (crowd) capsular saccharides-protein binding body is meant that different serotypes (crowd) capsular saccharides more than 3 or 3 combines to form conjugate with protein carrier through chemical bond, and this type (crowd) capsular saccharides warp more than 3 or 3 generates chemical bond with protein carrier and sets up structural the link each other.In other words, protein carrier is linked at the different shaped more than 3 or 3 (crowd) capsular saccharides together through chemical bond.

The present invention provides the pneumococcal conjugated vaccine that derives from a plurality of serotypes of streptococcus pneumoniae and formed by the capsular saccharides antigen of protein carrier link.In the preferred embodiment of the invention; The streptococcus pneumoniae serotype of preparation capsular saccharides is selected from the whole serological type strains of streptococcus pneumoniae; Preferred 45 type bacterial strains; More preferably 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F type bacterial strain, most preferably 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F type bacterial strain.Further, the streptococcus pneumoniae serological type strain of preparation capsular saccharides is definite according to the popular advantage serological type strain of epidemiology.

The present invention provides the pneumococcal conjugated vaccine that derives from a plurality of serotypes of streptococcus pneumoniae and formed by the capsular saccharides antigen of protein carrier link.In the preferred embodiment of the invention, the capsular saccharides antigen that derives from a plurality of serological type strains randomly is present in the vaccine with divalent serotype capsular saccharides-protein binding body and/or polyvalent serum type capsular saccharides-protein binding bodily form formula.Preferred 45 valency serotype capsular saccharides-protein binding bodies; More preferably 24 valency serotype capsular saccharides-protein binding bodies; 14 valency serotype capsular saccharides-protein binding bodies most preferably; Further, preferred 12 valency serotype capsular saccharides-protein binding bodies; Preferred 11 valency serotype capsular saccharides-protein binding bodies; Preferred 10 valency serotype capsular saccharides-protein binding bodies; Preferred 9 valency serotype capsular saccharides-protein binding bodies; Preferred 8 valency serotype capsular saccharides-protein binding bodies; Preferred 7 valency serotype capsular saccharides-protein binding bodies; Preferred 6 valency serotype capsular saccharides-protein binding bodies; Preferred 5 valency serotype capsular saccharides-protein binding bodies; Preferred 4 valency serotype capsular saccharides-protein binding bodies; Preferred 3 valency serotype capsular saccharides-protein binding bodies; Preferred divalent serotype capsular saccharides-protein binding body.In other words; For example, derive from 1,2,3,4,5, the capsular saccharides of 6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F type bacterial strain preferably exists with 24 valency serotype capsular saccharides-protein binding bodily form formula; More preferably exist with 12 valency serotype capsular saccharides-protein binding bodily form formula; Most preferably exist with 8 valency serotype capsular saccharides-protein binding bodily form formula; Further, preferably exist with 6 valency serotype capsular saccharides-protein binding bodily form formula; Preferably exist with 4 valency serotype capsular saccharides-protein binding bodily form formula; Preferably exist with 3 valency serotype capsular saccharides-protein binding bodily form formula; Preferably exist with divalent serotype capsular saccharides-protein binding bodily form formula.Saidly derive from 1,2,3,4,5, the capsular saccharides of 6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F type bacterial strain is to be divided into optional approach: 24 valencys (1 group); 12 valencys (2 groups); 8 valencys (3 groups); 6 valencys (4 groups); 4 valencys (6 groups); 3 valencys (8 groups); Divalent (12 groups).Every group of capsular saccharides combines the back remix to form vaccine of the present invention with protein carrier.Therefore, 24 valency serotype capsular saccharides-protein binding body vaccine is made up of 1 24 valency serotype capsular saccharides-protein binding body; 12 valency serotype capsular saccharides-protein binding body vaccine is made up of 2 12 different valency serotype capsular saccharides-protein binding bodies; 8 valency serotype capsular saccharides-protein binding body vaccine is made up of 38 different valency serotype capsular saccharides-protein binding bodies; 6 valency serotype capsular saccharides-protein binding body vaccine is made up of 46 different valency serotype capsular saccharides-protein binding bodies; 4 valency serotype capsular saccharides-protein binding body vaccine is made up of 64 different valency serotype capsular saccharides-protein binding bodies; 3 valency serotype capsular saccharides-protein binding body vaccine is made up of 83 different valency serotype capsular saccharides-protein binding bodies; Divalent serotype capsular saccharides-protein binding body vaccine is made up of 12 different divalent serotype capsular saccharides-protein binding bodies.

The present invention provides the pneumococcal conjugated vaccine that derives from a plurality of serotypes of streptococcus pneumoniae and formed by the capsular saccharides antigen of protein carrier link.In the preferred embodiment of the invention, the capsular saccharides antigen that derives from a plurality of serological type strains randomly is present in the vaccine with divalent serotype capsular saccharides-protein binding body and/or polyvalent serum type capsular saccharides-protein binding bodily form formula and with compound mode.In vaccine of the present invention, preferred 2～45 valency serotype capsular saccharides-protein binding bodies of antigen make up; More preferably 2～24 valency serotype capsular saccharides-protein binding bodies make up; Most preferably 2～14 valency serotype capsular saccharides-protein binding bodies make up; Further, preferred 2～12 valency serotype capsular saccharides-protein binding bodies make up; Preferred 2～11 valency serotype capsular saccharides-protein binding bodies make up; Preferred 2～10 valency serotype capsular saccharides-protein binding bodies make up; Preferred 2～9 valency serotype capsular saccharides-protein binding bodies make up; Preferred 2～8 valency serotype capsular saccharides-protein binding bodies make up; Preferred 2～7 valency serotype capsular saccharides-protein binding bodies make up; Preferred 2～6 valency serotype capsular saccharides-protein binding bodies make up; Preferred 2～5 valency serotype capsular saccharides-protein binding bodies make up; Preferred 2～4 valency serotype capsular saccharides-protein binding bodies make up; Preferred 2～3 valency serotype capsular saccharides-protein binding bodies make up.In other words; For example, derive from 1,2,3,4,5, the capsular saccharides of 6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F type bacterial strain preferably exists with the combining form of 2～24 valency serotype capsular saccharides-protein binding bodies; More preferably the combining form with 2～14 valency serotype capsular saccharides-protein binding bodies exists; Most preferably the combining form with 2～12 valency serotype capsular saccharides-protein binding bodies exists; Further, preferably exist with the combining form of 2～11 valency serotype capsular saccharides-protein binding bodies; Preferably the combining form with 2～10 valency serotype capsular saccharides-protein binding bodies exists; Preferably the combining form with 2～9 valency serotype capsular saccharides-protein binding bodies exists; Preferably the combining form with 2～8 valency serotype capsular saccharides-protein binding bodies exists; Preferably the combining form with 2～7 valency serotype capsular saccharides-protein binding bodies exists; Preferably the combining form with 2～6 valency serotype capsular saccharides-protein binding bodies exists; Preferably the combining form with 2～5 valency serotype capsular saccharides-protein binding bodies exists; Preferably the combining form with 2～4 valency serotype capsular saccharides-protein binding bodies exists; Preferably the combining form with 2～3 valency serotype capsular saccharides-protein binding bodies exists.Divalent serotype capsular saccharides-protein binding body and polyvalent serum type capsular saccharides-protein binding body mixes the back and forms vaccine of the present invention.Therefore, vaccine of the present invention can comprise divalent serotype capsular saccharides-protein binding body and polyvalent serum type capsular saccharides-protein binding body simultaneously.

The present invention provides the pneumococcal conjugated vaccine of forming by divalent serotype streptococcus pneumoniae capsular saccharides-protein binding body and/or polyvalent serum type streptococcus pneumoniae capsular saccharides-protein binding body; In the preferred embodiment of the invention; In advance the streptococcus pneumoniae capsular saccharides is divided into 2 and/or a plurality of groups, each group comprises 2 serotype capsular saccharides or a plurality of serotype capsular saccharides.The contained serotype capsular saccharides of each group carries out chemical bond with protein carrier again and combines after merging, form divalent serotype capsular saccharides-protein binding body or polyvalent serum type capsular saccharides-protein binding body, and then be mixed with the vaccine finished product.Therefore, the serotype capsular saccharides is divided quantity in groups and confirm that every group comprises 2 serotype capsular saccharides or a plurality of serotype capsular saccharides is according to capsular saccharides molecular weight size, total nitrogen, phosphorus, alduronic acid, hexosamine, methylpentose, the division of O-acetyl content.Further, optional randomly serotype capsular saccharides makes up.

The present invention provides the pneumococcal conjugated vaccine that derives from a plurality of serotypes of streptococcus pneumoniae and formed by the capsular saccharides antigen of protein carrier link, and capsular saccharides antigen randomly is present in the vaccine with divalent serotype capsular saccharides-protein binding body and/or polyvalent serum type capsular saccharides-protein binding bodily form formula.Wherein, the preparation of described divalent serotype capsular saccharides-protein binding body is to derive from the different serotypes capsular saccharides with 2 in advance to merge, and then carries out chemical bond with protein carrier, forms divalent serotype capsular saccharides-protein binding body.2 serotype capsular saccharides all produce chemical bond linkage with carrier protein, and carrier protein structurally forms link according to chemical bond with 2 capsular saccharides again.The preparation of described polyvalent serum type capsular saccharides-protein binding body is in advance a plurality of different serotypes capsular saccharides that derive to be merged, and then carries out chemical bond with protein carrier, forms polyvalent serum type capsular saccharides-protein binding body.A plurality of serotype capsular saccharides all produce chemical bond linkage with carrier protein, and carrier protein structurally forms link according to chemical bond with a plurality of capsular saccharides again.Therefore; Deriving from the mutual ratio that the different serotypes capsular saccharides merges in advance is one of essential condition of confirming various antigenic content in the combined vaccine finished product of the present invention, and capsular saccharides merges ratio to be changed according to factors such as capsular saccharides kind (polysaccharide or oligosaccharide), capsular saccharides architectural feature, capsular saccharides-protein binding body valence mumber, associated methods and process conditions.In the preferred embodiment of the invention; The mutual combined wt ratio of capsular saccharides is preferably 1: 0.05～and (w/w according to the invention means weight ratio to 20 (w/w); μ g/ μ g for example) ratio merges; Preferred combined wt ratio is 1: 0.1～10 (w/w), and most preferred combined wt ratio is 1: 0.2～5 (w/w).Capsular saccharides is associated with multiple other weight ratio, and said weight ratio is only used for explaining the present invention.So, can adopt the various capsular saccharides combined wts that are equal to than realizing divalent serotype capsular saccharides of the present invention-protein binding body or polyvalent serum type capsular saccharides-protein binding body.

The present invention provides the pneumococcal conjugated vaccine that derives from a plurality of serotypes of streptococcus pneumoniae and formed by the capsular saccharides antigen of protein carrier link.In the preferred embodiment of the invention, said protein carrier is selected from tetanus toxoid (TT), diphtheria toxoid (DT), the nontoxic variant of diphtheria toxin, diphtherotoxin (CRM197), B group meningitis cocci outer membrane protein (OMP), streptococcus pneumoniae surface protein A (PspA), PsaA pneumococcal surface adhesion A (PsaA), pneumolysin (Ply), hemophilus influenza D albumen (PD), pertussis toxin, PT (PT), cholera toxin (CT), purified protein derivative (PPD) of tuberculin (PPD), Pseudomonas aeruginosa exotoxin A (PEA), keyhole limpet hemocyanin (KLH).More preferably TT, DT, CRM197 and OMP.Most preferably DT and CRM197.In a word; Selecting above-mentioned albumen for use is to well known to a person skilled in the art scheme as modal carrier and antibacterial class capsular saccharides combination preparation combined vaccine; For example select for use these protein carriers to prepare b type hemophilus influenza combined vaccine, preparation pneumococcal conjugated vaccine, preparation meningococcus combined vaccine (Lee; People Infect Med19 (3): 127-133 such as Chi-Jen, 2002).In the preferred embodiment of the invention, most preferably be DT in conjunction with the protein carrier of pneumococal polysaccharide.If satisfy the demand, also can adopt other protein carrier, the protein carrier that here provides only is for illustrative the present invention.

The another scheme of pneumococcal conjugated vaccine of the present invention, will derive from merging such as pneumococcal albumen PspA, PsaA, Ply becomes the pneumoprotein complex, this albumen composition is combined with capsular saccharides as protein carrier again.On the one hand, the effect of pneumoprotein complex performance protein carrier becomes T cell dependent type antigen with capsular saccharides by T cell independent form antigenic shift, produces the immunological memory reaction.On the other hand, the pneumoprotein complex is participated in immune response as proteantigen again, and has enlarged combined vaccine cross immunity protection domain, strengthens the immune efficacy of vaccine.

In the present invention, the method for the preparation of streptococcus pneumoniae capsular saccharides and quality control and standard are known in those skilled in the art.In the preferred embodiment of the invention; The technical staff can be according to the pneumococcal conjugated vaccine manufacturing and vertification regulation (the WHO Technical Report Series of the formal promulgation of World Health Organization (WHO); No.927,2005) and three technological documents such as (versions in 2005) of Pharmacopoeia of People's Republic of China prepare capsular saccharides and carry out quality arbitration.

In the present invention, the bonded method of unit price capsular saccharides and protein carrier and be (Pawlowski et al.Vaccine 2000 known in those skilled in the art to method and standard that unit price capsular saccharides-protein binding body carries out quality control; 18:1873-1885; Wessels et al.J.Clin.Invest.1990; 86:1428-1433; WHOTechnical Report Series.No.897.2000; WHO Technical Report Series.No.924.2004; WHO Technical Report Series.No.927.2005; Robinson etc., the detailed annotation of vaccine key technology.Chemical Industry Press, 2006, the 165-178 page or leaf; Zhang Yanling etc., vaccinology.Science Press, 2004,506-517,652-653,679-681,699-700,896-902,918-925,943-945,961-964,968-978 page or leaf; ).In the preferred embodiment of the invention; Divalent or multivalence capsular saccharides and protein carrier associated methods can select for use any known unit price capsular saccharides and the bonded method of protein carrier to prepare; Preferred autoreduction amine method, amide condensed method, thioether condensation method, curing reaction method, thiocarbamoyl reaction method, O-alkyl isourea reaction method, diazonium association reaction method; More preferably reduce amine method and amide condensed method, most preferably the Combined application of 2 kinds of methods or several different methods.The technical staff can prepare divalent or multivalence capsular saccharides-protein binding body and carry out quality arbitration with reference to the pneumococcal conjugated vaccine manufacturing and the vertification regulation (WHO Technical Report Series, No.927,2005) of the formal promulgation of World Health Organization (WHO).In the preferred embodiment of the present invention; Earlier with Bromine cyanide. (CNBr) or cyanidization agent (1-cyanic acid-4-dimethylamino-pyridinium tetrafluoroborate; CDAP) divalent capsular saccharides or multivalence capsular saccharides are carried out activation processing; Be that difunctional nucleophilic spacer adds in the activatory capsular saccharides with AH (ADH) again, form capsular saccharides-ADH derivant, pass through the condensation and the protein carrier covalent bond of carbodiimide (EDAC) mediation again; Form divalent capsular saccharides-protein binding body or multivalence capsular saccharides-protein binding body, purified again acquisition is used to prepare the antigen component of pneumococcal conjugated vaccine.Capsular saccharides and proteic is combined with multiple known method, and said associated methods is only used for explaining the present invention.So, can adopt the various methods that are equal to realize combination of the present invention, therefore any capsular saccharides and bonded method of protein carrier and technology or process of being used for all might be used for embodiment of the present invention.

The present invention provides the method for preparing the bacterial capsule sugar combined vaccine that contains divalent serotype (crowd) capsular saccharides-protein binding body and/or polyvalent serum type (crowd) capsular saccharides-protein binding body.Wherein, said antibacterial includes but not limited to that streptococcus pneumoniae, meningococcus, b type hemophilus influenza, salmonella typhi, staphylococcus, B group streptococcus etc. have the antibacterial of capsular saccharides.In the preferred embodiment of the invention, the preferred streptococcus pneumoniae of antibacterial, meningococcus, b type hemophilus influenza.In the present invention, the bacteria culture of preparation capsular saccharides is available from Nat'l Pharmaceutical & Biological Products Control Institute medical science antibacterial preservation administrative center (CMCC) or the biological collecting center (ATCC) of Unite States Standard.

The present invention provides the method for preparing the bacterial capsule sugar combined vaccine that contains divalent serotype (crowd) capsular saccharides-protein binding body and/or polyvalent serum type (crowd) capsular saccharides-protein binding body.In the preferred embodiment of the present invention, after two or more serotypes (crowd) capsular saccharides merges according to weight ratio, it is carried out homogenize handle, benefit the comparatively equilibrium that combines of different serotypes (crowd) capsular saccharides and protein carrier.Preferred homogenize processing method is a supersound process; Preferred parameter is 25～75Hz 2～10 minutes.

The present invention provides the method for preparing the bacterial capsule sugar combined vaccine that contains divalent serotype (crowd) capsular saccharides-protein binding body and/or polyvalent serum type (crowd) capsular saccharides-protein binding body.In the preferred embodiment of the present invention; Divalent serotype (crowd) capsular saccharides-protein binding isoantigen or polyvalent serum type (crowd) capsular saccharides-protein binding isoantigen are carried out detoxification treatment, benefit and reduce the side reaction of polyvalent bacterial capsular saccharides combined vaccine after rolling up capsular saccharides antigen valence mumber.Detoxifying agent is preferably from formaldehyde, glutaraldehyde, hydrogen peroxide, more preferably glutaraldehyde.

Therefore, the present invention provides the method for preparing the bacterial capsule sugar combined vaccine that contains divalent serotype (crowd) capsular saccharides-protein binding body and/or polyvalent serum type (crowd) capsular saccharides-protein binding body.Said method further may further comprise the steps:

(a) from different serotypes (crowd) bacterial cultures, extract respectively, the purification capsular saccharides, process single serotype (crowd) capsular saccharides, i.e. monovalent serum type (crowd) capsular saccharides.Wherein, said antibacterial includes but not limited to that streptococcus pneumoniae, meningococcus, b type hemophilus influenza, salmonella typhi, staphylococcus, B group streptococcus etc. have the antibacterial of capsular saccharides;

(b) monovalent serum type (crowd) capsular saccharides of step (a) the gained capsular saccharides in two or more serotypes (crowd) is merged according to ratio, become divalent or polyvalent serum type (crowd) capsular saccharides.Wherein, said capsular saccharides is further handled through homogenize after merging;

(c) divalent of step (b) or polyvalent serum type (crowd) capsular saccharides and protein carrier are carried out chemical bond, process divalent serotype (crowd) capsular saccharides-protein binding isoantigen or polyvalent serum type (crowd) capsular saccharides-protein binding isoantigen;

(d) the product detoxification treatment and the purification of step (c) are processed divalent serotype (crowd) capsular saccharides-protein binding isoantigen or polyvalent serum type (crowd) capsular saccharides-protein binding isoantigen;

(e) divalent serotype (crowd) capsular saccharides-protein binding isoantigen and/or polyvalent serum type (crowd) capsular saccharides-protein binding isoantigen with step (d) is mixed with vaccine.

In pneumococcal conjugated vaccine provided by the invention and bacterial capsule sugar combined vaccine, vaccine dose is confirmed according to the known technical standard of pharmaceutical field those skilled in the art.For example, when confirming dosage, should be taken into account antigenic characteristic, adjuvant character, inoculator's factors such as age, vaccination approach.In the preferred embodiment of the invention; Preferred each serotype (crowd) capsular saccharides dosage of finished product vaccine is 0.1～50 μ g; Preferred each serotype (crowd) capsular saccharides dosage is 1～25 μ g, and most preferred each serotype (crowd) capsular saccharides dosage is 2～10 μ g.

In the preferred embodiment of the invention, pneumococcal conjugated vaccine and bacterial capsule sugar combined vaccine also can comprise this area adjuvant commonly used.Preferred adjuvants are aluminum adjuvant, calcium phosphate adjuvant, cholera toxin B subunit (CTB), saponin Q21, lipids, monophosphoryl lipid A (MPL), MF59, Escherichia coli heat-labile toxin ( LT), more preferably the adjuvant is an aluminum adjuvant such as aluminum hydroxide, aluminum phosphate or aluminum sulfate.

In pneumococcal conjugated vaccine provided by the invention and bacterial capsule sugar combined vaccine, the dosage form of vaccine and compatibility are confirmed according to the known technical standard of pharmaceutical field those skilled in the art.In the preferred embodiment of the invention, the vaccine dosage form is liquid dosage form, freeze-dried formulation, pill, tablet, capsule formulation preferably, is more preferably liquid dosage form or freeze-dried formulation, most preferably is liquid dosage form.

In the preferred embodiment of the invention, pneumococcal conjugated vaccine and bacterial capsule sugar combined vaccine can be the preservative free vaccines.In preferred another embodiment of the present invention, combined vaccine comprises antiseptic, and preferred antiseptic is thimerosal, 2-phenoxyethanol, benzyl alcohol.

Pneumococcal conjugated vaccine provided by the invention and bacterial capsule sugar combined vaccine, route of inoculation comprises intradermal injection, subcutaneous injection, intramuscular injection, lumbar injection, also comprises via intranasal application, oral cavity, anus, vagina, mucosa administration.

Pneumococcal conjugated vaccine of the present invention and bacterial capsule sugar combined vaccine method for preparing are compared with existing pneumococcal conjugated vaccine and preparation method thereof; Comprise and the for example comparison of PCV7 and PCV13 that is about to listing and preparation method thereof of the current pneumococcal conjugated vaccine that has gone on the market; Further with for example Chinese patent 200680017776.8,00807773.8,92100700.0,00807759.2,02827993.X, 01815748.3,200680052954.0,200680053139.6,200610054832.7 the comparison of the described pneumococcal conjugated vaccine of disclosed patent text and method for preparing; Further with the comparison of Chinese patent 03141407.9, its difference and beneficial effect are:

(1) the contained antigenic structure of vaccine of the present invention is different.Existing pneumococcal conjugated vaccine all be technically with the capsular saccharides that derives from each serotype independent with protein carrier after chemical bond; Form monovalent serotype capsular saccharides-protein binding body; Again each monovalent serum type capsular saccharides-protein binding body is mixed, and then preparation becomes the pneumococcal conjugated vaccine that contains many serotype capsular saccharides.Therefore, contained each serotype capsular saccharides antigen independently exists with unit price capsular saccharides-protein binding body in the existing pneumococcal conjugated vaccine, and each serotype capsular saccharides does not have structural link each other.Vaccine of the present invention be after the capsular saccharides with different serotypes merges by two or more serotype capsular saccharides in advance again with protein carrier after chemical bond combines; Form divalent serotype capsular saccharides-protein binding body or polyvalent serum type capsular saccharides-protein binding body; Again each divalent serotype capsular saccharides-protein binding body and/or polyvalent serum type capsular saccharides-protein binding body are mixed, and then preparation becomes the pneumococcal conjugated vaccine that contains many serotype capsular saccharides.Therefore; Contained each serotype capsular saccharides antigen exists with divalent serotype capsular saccharides-protein binding body and/or polyvalent serum type capsular saccharides-protein binding body in the vaccine of the present invention; And each serotype capsular saccharides has structural link each other in divalent serotype capsular saccharides-protein binding body and the polyvalent serum type capsular saccharides-protein binding body.

(2) the contained antigen molecular of vaccine of the present invention is bigger.Divalent serotype capsular saccharides-protein binding body that vaccine of the present invention is contained and/or polyvalent serum type capsular saccharides-protein binding body; Link because foundation combines divalent or multivalence capsular saccharides structurally to form each other with the chemical bond of protein carrier, the capsular saccharides antigen molecular is increased indirectly.Therefore, vaccine of the present invention has better immunogenicity than monovalent serum type capsular saccharides-protein binding body.

(3) vaccine of the present invention reduces protein carrier immunity competition.The contained antigen of vaccine of the present invention is divalent serotype capsular saccharides-protein binding body and/or polyvalent serum type capsular saccharides-protein binding body, and the chemical bonded refractory of different serotypes capsular saccharides foundation and protein carrier is combined in to form each other on the structure and links.Therefore; Than monovalent serum type capsular saccharides-protein binding body; Divalent serotype capsular saccharides-protein binding body that vaccine of the present invention is contained and/or the protein carrier total amount in polyvalent serum type capsular saccharides-protein binding isoantigen reduce; The B cell that causes replying with capsular saccharides is preponderated with the B cell that protein carrier is replied, and the more Th cells of competition participate in the process of B cell response capsular saccharides, thereby reduces the inductive epi-position depression effect of protein carrier.

(4) the contained antigen of vaccine of the present invention is more stable.Divalent serotype capsular saccharides-protein binding body that vaccine of the present invention is contained and/or polyvalent serum type capsular saccharides-protein binding body; Because combining divalent serotype capsular saccharides or polyvalent serum type capsular saccharides structurally to form each other with the chemical bond of protein carrier, foundation links; Therefore, the contained capsular saccharides antigen of vaccine of the present invention has more stability than the unit price capsular saccharides of prior art.

(5) the invention provides the method that a kind of preparation contains the bacterial capsule sugar combined vaccine of divalent serotype (crowd) capsular saccharides-protein binding body and/or polyvalent serum type (crowd) capsular saccharides-protein binding body, said method comprising the steps of:

(6) the present invention further provides the method for the bacterial capsule sugar combined vaccine that a kind of preparation contains divalent serotype (crowd) capsular saccharides-protein binding body and/or polyvalent serum type (crowd) capsular saccharides-protein binding body, said method comprising the steps of:

(c) divalent of step (b) or polyvalent serum type (crowd) capsular saccharides are carried out chemical bond with protein carrier and combine, process divalent serotype (crowd) capsular saccharides-protein binding isoantigen or polyvalent serum type (crowd) capsular saccharides-protein binding isoantigen;

The present invention passes through following embodiment, but unrestricted, further describes core content of the present invention for example, and said embodiment sets forth notion of the present invention and several embodiment preferred.Other embodiments of the invention are predicted by those skilled in the art under the prerequisite that does not deviate from core of the present invention.

The specific embodiment

The present invention can further understand through following embodiment, and said embodiment has selected several preferred embodiments, has been merely better displaying the present invention, but not summary of the invention is limited.

Embodiment

The preparation of embodiment 1 streptococcus pneumoniae fermentation culture and capsular polysaccharide

With the streptococcus pneumoniae freeze-drying lactobacillus breakdown of low-temperature preservation, after the physiological sodium chloride solution redissolution, be seeded to 5～10% SBA slant mediums, put 5～8%CO ₂In the environment, cultivated 16～24 hours in 35～37 ℃.Change and plant, cultivated 10～18 hours in 35～37 ℃ to streptococcus pneumoniae liquid base culture medium.Continue to change and kind to increase bacterium and cultivate, cultivated 10～18 hours in 35～37 ℃ to streptococcus pneumoniae liquid base culture medium.The streptococcus pneumoniae liquid culture that will increase behind the bacterium by 5～10% inoculum concentrations is seeded in the fermentation tank that adds the liquid base culture medium, stirs, aerobic culture, cultivates 8～12 hours in 35～37 ℃, and pH is controlled between 6.0～7.4.The monitoring bacterial concentration changes microscopy culture purity and morphological features during the fermentation culture.When fermentation culture finished, adding final concentration by the amount of culture medium in the fermentation tank was 0.05～0.20% sodium deoxycholate cracking somatic cells, stirs the termination fermentation culture 20～60 minutes.

With the centrifugal removal somatic cells of continuous flow centrifuge fragment, collect supernatant.Supernatant is carried out ultrafiltration and concentration, and the ultrafilter membrane specification is selected 50～100KD for use, 1/5～1/20 of cycles of concentration to original volume.The concentrated solution of putting after the ultrafiltration is dialysed in physiological sodium chloride solution.Concentrated solution after dialysis adds sodium acetate to final concentration 3～10%, is stirred to abundant dissolving, and pH transfers to 4.8～6.4.Continue in 2～8 ℃ of environment and slowly to add refrigerative in advance ethanol, stirred 15～45 minutes to final concentration 15～60%, through 12,000～16,000g, 2～8 ℃ of ultracentrifugations, collection supernatant.In supernatant, continue to add sodium acetate to final concentration 5～15%, be stirred to abundant dissolving, pH keeps 4.8～6.4.Continue in 2～8 ℃ of environment and slowly to add refrigerative in advance ethanol to final concentration 30～80%, stirred 15～45 minutes, through 2,200～2,800g, 2～8 ℃ are centrifugal, collection crude polysaccharides precipitate.Evacuation ,-20 ℃ of storages are waited to be further purified.

Crude polysaccharides is added in the sodium acetate solution of 0.3～0.6M, pH transfers to 6.5～7.2, in 2～8 ℃ of environment, is stirred to dissolving fully.Add cold phenol to final concentration 65～75% again and carry out extracting,, collect supernatant through 14,000～18,000 ultracentrifugation.Repeat cold phenol extraction steps 2～4 times.The supernatant of collecting is removed residual phenol in 2～8 ℃ of warp dialysis, and dialysis solution is a water for injection.In 2～8 ℃ of environment, slowly add refrigerative in advance ethanol to final concentration 40～90%, stirred 15～45 minutes, through 2,200～2,800g, 2～8 ℃ are centrifugal, collection polysaccharide precipitation thing.The polysaccharide precipitation thing soaks, washs each 2～4 times through dehydrated alcohol and acetone, and dry back is with the water for injection dissolving, and warp 0.22 μ m aseptic filtration obtains purified polysaccharide again, is stored in below-20 ℃ with liquid or lyophilized form.Pressing formal pneumococcal conjugated vaccine manufacturing of issuing of World Health Organization (WHO) and vertification regulation (WHO TechnicalReport Series, No.927,2005) prescriptive procedure and standard examines and determine purified polysaccharide.

Prepare streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F purification capsular polysaccharide according to the method described above respectively.

The preparation of embodiment 2 streptococcus pneumoniae pod membrane oligosaccharide

Purified polysaccharide is dissolved in 0.2～1.0M acetic acid solution to final concentration 1～4mg/ml, immersed in 70～100 ℃ of oil baths or the water-bath hydrolysis 6～40 hours.After the hydrolysis pH is transferred to 7.0, in 2～8 ℃ of dialysis.Through Sepharose 4FF chromatographic column purification,, collect oligosaccharide again with 15mM NaCl liquid eluting.Obtain the purification oligosaccharide through 0.22 μ m aseptic filtration, be stored in below-20 ℃ with liquid or lyophilized form.Pressing formal pneumococcal conjugated vaccine manufacturing of issuing of World Health Organization (WHO) and vertification regulation (WHO Technical Report Series, No.927,2005) prescriptive procedure and standard examines and determine the purification oligosaccharide.

Prepare streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F purification pod membrane oligosaccharide according to the method described above respectively.

The preparation of embodiment 3 meningococcal capsular saccharides and b type hemophilus influenza capsular saccharides

Prepare A crowd, C crowd, W135 crowd, Y group meningitis cocci capsular polysaccharide and b type hemophilus influenza capsular polysaccharide according to Chinese patent 200710007045.1 methods.

Prepare A crowd, C crowd, W135 crowd, Y group meningitis cocci capsular polysaccharide and b type hemophilus influenza pod membrane oligosaccharide according to the embodiment of the invention 2 methods again.

Meningococcal polysaccharide vaccine manufacturing and vertification regulation (WHOTechnical Report Series by the formal promulgation of World Health Organization (WHO); No.658; 1980) and b type hemophilus influenza combined vaccine is made and vertification regulation (WHO Technical Report Series; No.897,2000) prescriptive procedure and standard are examined and determine purified polysaccharide and oligosaccharide.

The preparation of embodiment 4 protein carriers-diphtheria toxoid (DT)

With the diphtheria corynebacterium freeze-drying lactobacillus breakdown of cryopreservation, with the physiological sodium chloride solution recovery, be seeded to the blood agar slant medium, cultivated 16～20 hours in 33～36 ℃.Change and plant, cultivated 16～20 hours in 33～36 ℃ to improveing the Lin Shi broth bouillon.Change again and plant, cultivated 16～20 hours in 33～36 ℃ to improveing the Lin Shi broth bouillon.The continuation commentaries on classics was planted to the seed bottle that contains improvement Lin Shi broth bouillon and is increased the bacterium cultivation, in 33～36 ℃ of cultivations 36～72 hours.Culture commentaries on classics in the seed bottle is planted to the fermentation tank that contains improvement Lin Shi broth bouillon, under ventilation, stirring, pH7.0～8.5 conditions, cultivated 40～56 hours in 33～36 ℃.Fermentation finishes, and adds the formaldehyde sterilization treatment of final concentration 0.1～0.5%, through centrifugal collection supernatant.Supernatant concentrates through 30～50KD membrane ultrafiltration through 0.22 μ m filter aseptic filtration again.Add NaHCO respectively ₃To final concentration 0.45～0.55%, activated carbon to final concentration 0.72～0.78%, (NH ₄) ₂SO ₄To final concentration 28.0～30.0%, stirred 30 minutes, put room temperature and left standstill 12～18 hours.Centrifugal collection supernatant.In the supernatant of collecting, add (NH ₄) ₂SO ₄To final concentration 42.0～45.0%, stirred 30 minutes, put room temperature and left standstill 4～8 hours.Centrifugal collecting precipitation also is dissolved in 0.1%NaHCO ₃In the solution.In 2～8 ℃, dialysis after 60～72 hours, again through 0.22 μ m filter aseptic filtration.Liquid after the aseptic filtration adds lysine to final concentration 1.0～2.0%, adds formaldehyde to final concentration 0.4～0.6% again, and pH transfers to 6.8～7.0, puts 37～40 ℃ of detoxifications 42～48 days.DT stock solution after the detoxification is pressed the Pharmacopoeia of the People's Republic of China (version in 2005) gainer and method calibrating, and it is subsequent use that stock solution is put 2～8 ℃ of preservations behind the assay approval.

The preparation of embodiment 5 capsular saccharides-protein binding vaccine

(1) preparation of streptococcus pneumoniae 24 valency polysaccharide conjugate vaccines (24 valency polysaccharide-protein coalition)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F purification capsular polysaccharide are merged with the water for injection dilution and by various polysaccharide 1: 1～5 (w/w) weight ratio, and the polysaccharide concentration after the merging is 2.0～10.0mg/ type polysaccharide/ml.Ultrasonic homogenize was handled 25～75Hz 2～10 minutes.Add the cyanogen bromide-activated polysaccharide to final concentration 1.0～4.0mg/mg total polysaccharides, regulate pH and keep pH9.5～11.5, stirred 30～60 minutes in 20～26 ℃ with NaOH.Add AH again to final concentration 12.0～18.0mg/ml, regulate and keep pH8.0～10.0, kept 15～30 minutes.In 20～26 ℃ of stirrings 30～60 minutes, put again at 2～8 ℃ and stirred 18～24 hours, form polysaccharide-AH derivant.Polysaccharide-AH derivant was dialysed warp 0.45 μ m filter aseptic filtration again 72～96 hours in 2～8 ℃.DT is diluted to 1.0～4.0mg/ml, is that 1: 0.5～2 ratios (v/v) add DT in polysaccharide-AH derivant in polysaccharide-AH derivant: DT, regulates and keep pH5.0～6.0.Add carbodiimide to final concentration 20～30mg/ml, regulate and keep pH5.0～6.0, keep 60～90 minutes after, regulate and keep pH6.5～7.5.Add glutaraldehyde to final concentration 0.05～0.15% detoxification treatment, kept 4～6 hours.Put 2～8 ℃ of dialysis 72～120 hours, through 100～300KD ultrafilter membrane ultrafiltration and concentration.With Sepharose 4FF chromatographic column purified polysaccharide-DT conjugate, collect polysaccharide-DT conjugate, through 0.22 μ m filter aseptic filtration, obtain 24 valencys pneumococal polysaccharide-DT coalition stock solution again.Dilute 24 valencys pneumococal polysaccharide-DT coalition stock solution, aluminum phosphate, sodium chloride, 2-phenoxyethanol with water for injection; Make each component content of finished product vaccine be respectively: combine each serotype polysaccharide 2～10 μ g/ml, aluminum phosphate 1.0～1.5mg/ml, sodium chloride 7.5～9.5mg/ml, the 2-phenoxyethanol 5.0～6.0mg/ml of DT, pH transfers to 5.8～7.2.By sucking-off amount >=0.5ml packing cillin bottle and seal.Pressing formal pneumococcal conjugated vaccine manufacturing of issuing of World Health Organization (WHO) and vertification regulation (WHO Technical Report Series, No.927,2005) prescriptive procedure and standard and examine and determine, is pneumococcal Polysaccharide Conjugate Vaccine behind the assay approval.

(2) preparation of streptococcus pneumoniae 24 valency oligosaccharide combined vaccines (24 valencys oligosaccharide-protein binding body)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F purification pod membrane oligosaccharide are merged with the water for injection dilution and by various oligosaccharide 1: 1～5 (w/w) weight ratio, and the oligosaccharide concentration after the merging is 2.0～10.0mg/ type oligosaccharide/ml.Add the cyanogen bromide-activated oligosaccharide to the total oligosaccharide of final concentration 1.0～4.0mg/mg, regulate pH and keep pH9.5～11.5, stirred 30～60 minutes in 20～26 ℃ with NaOH.Add AH again to final concentration 12.0～18.0mg/ml, regulate and keep pH8.0～10.0, kept 15～30 minutes.In 20～26 ℃ of stirrings 30～60 minutes, put again at 2～8 ℃ and stirred 18～24 hours, form oligosaccharide-AH derivant.Oligosaccharide-AH derivant was dialysed warp 0.45 μ m filter aseptic filtration again 72～96 hours in 2～8 ℃.DT is diluted to 1.0～4.0mg/ml, is that 1: 0.5～2 ratios (v/v) add DT in oligosaccharide-AH derivant in oligosaccharide-AH derivant: DT, regulates and keep pH5.0～6.0.Add carbodiimide to final concentration 20～30mg/ml, regulate and keep pH5.0～6.0, keep 60～90 minutes after, regulate and keep pH6.5～7.5.Put 2～8 ℃ of dialysis 72～120 hours, through 100～300KD ultrafilter membrane ultrafiltration and concentration.With Sepharose 4FF chromatographic column purification oligosaccharide-DT conjugate, collect polysaccharide-DT conjugate, through 0.22 μ m filter aseptic filtration, obtain 24 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution again.Dilute 24 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution, aluminum phosphate, sodium chloride, 2-phenoxyethanol with water for injection; Make each component content of finished product vaccine be respectively: combine each serotype oligosaccharide 2～10 μ g/ml, aluminum phosphate 1.0～1.5mg/ml, sodium chloride 7.5～9.5mg/ml, the 2-phenoxyethanol 5.0～6.0mg/ml of DT, pH transfers to 5.8～7.2.By sucking-off amount >=0.5ml packing cillin bottle and seal.Pressing formal pneumococcal conjugated vaccine manufacturing of issuing of World Health Organization (WHO) and vertification regulation (WHO Technical Report Series, No.927,2005) prescriptive procedure and standard and examine and determine, is streptococcus pneumoniae oligosaccharide combined vaccine behind the assay approval.

Present embodiment (1) method and present embodiment (2) method difference are that the latter does not have ultrasonic homogenize processing and two steps of glutaraldehyde detoxification treatment.

(3) preparation of streptococcus pneumoniae 24 valency polysaccharide conjugate vaccines (12 valency polysaccharide-protein coalitions+12 valency polysaccharide-protein coalitions)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F purification capsular polysaccharide are divided into 2 groups; The 1st group comprise 1,2,3,4,5,6B, 7F, 9V, 14,18C, 19F, 23F, the 2nd group comprises 6A, 8,9N, 10A, 11A, 12F, 15B, 17F, 19A, 20,22F, 33F.Merge with the water for injection dilution respectively and respectively organize polysaccharide; Prepare the 12 valencys pneumococal polysaccharide-DT coalition stock solution of 2 groups of different serotypes polysaccharide by present embodiment (1) method, again the 12 valencys pneumococal polysaccharide-DT coalition stock solution of these 2 groups of different serotypes polysaccharide is mixed with the vaccine finished product by present embodiment (1) method and examines and determine.

(4) preparation of streptococcus pneumoniae 24 valency oligosaccharide combined vaccines (12 valencys oligosaccharide-protein binding body+12 valencys oligosaccharide-protein binding body)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F purification pod membrane oligosaccharide are divided into 2 groups; The 1st group comprise 1,2,3,4,6A, 6B, 7F, 10A, 14,18C, 20,23F, the 2nd group comprise 5,8,9N, 9V, 11A, 12F, 15B, 17F, 19A, 19F, 22F, 33F.Merge with the water for injection dilution respectively and respectively organize oligosaccharide; Prepare the 12 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution of 2 groups of different serotypes oligosaccharide by present embodiment (1) method, again the 12 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution of these 2 groups of different serotypes oligosaccharide is mixed with the vaccine finished product by present embodiment (1) method and examines and determine.

(5) preparation of streptococcus pneumoniae 24 valency capsular saccharides combined vaccines (12 valency polysaccharide-protein coalitions+12 valencys oligosaccharide-protein binding body)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F purification capsular saccharides are divided into 2 groups; The 1st group comprise 1,2,3,4,5,7F, 10A, 11A, 14,15B, 20,23F polysaccharide, the 2nd group comprises 6A, 6B, 8,9N, 9V, 12F, 17F, 18C, 19A, 19F, 22F, 33F oligosaccharide.Merge with the water for injection dilution respectively and respectively organize polysaccharide and oligosaccharide; The 12 valencys pneumococal polysaccharide-DT coalition stock solution for preparing one group of different serotypes polysaccharide by present embodiment (1) method and the 12 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution of another group different serotypes oligosaccharide are mixed with the vaccine finished product with the 12 valencys pneumococal polysaccharide-DT coalition stock solution of these 2 groups of different serotypes polysaccharide and oligosaccharide and 12 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution by present embodiment (1) method again and examine and determine.

(6) preparation of streptococcus pneumoniae 24 valency polysaccharide conjugate vaccines (8 valency polysaccharide-protein coalitions+8 valency polysaccharide-protein coalitions+8 valency polysaccharide-protein coalitions)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F purification capsular polysaccharide are divided into 3 groups; The 1st group comprise 1,3,4,7F, 9V, 14,18C, 19F, the 2nd group comprises 6A, 8,9N, 12F, 15B, 17F, 19A, 22F, the 3rd group comprise 2,5,6B, 10A, 11A, 20,23F, 33F.Merge with the water for injection dilution respectively and respectively organize polysaccharide; Prepare the 8 valencys pneumococal polysaccharide-DT coalition stock solution of 3 groups of different serotypes polysaccharide by present embodiment (1) method, again the 8 valencys pneumococal polysaccharide-DT coalition stock solution of these 3 groups of different serotypes polysaccharide is mixed with the vaccine finished product by present embodiment (1) method and examines and determine.

(7) preparation of streptococcus pneumoniae 24 valency oligosaccharide combined vaccines (8 valencys oligosaccharide-protein binding body+8 valencys oligosaccharide-protein binding body+8 valencys oligosaccharide-protein binding body)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F purification pod membrane oligosaccharide are divided into 3 groups; The 1st group comprise 1,3,4,7F, 9N, 9V, 18C, 20, the 2 groups comprise 2,6A, 6B, 12F, 14,15B, 17F, 22F, the 3rd group comprise 5,8,10A, 11A, 19A, 19F, 23F, 33F.Merge with the water for injection dilution respectively and respectively organize oligosaccharide; Prepare the 8 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution of 3 groups of different serotypes oligosaccharide by present embodiment (1) method, again the 8 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution of these 3 groups of different serotypes oligosaccharide is mixed with the vaccine finished product by present embodiment (1) method and examines and determine.

(8) preparation of streptococcus pneumoniae 24 valency capsular saccharides combined vaccines (8 valency polysaccharide-protein coalitions+8 valencys oligosaccharide-protein binding body+8 valency polysaccharide-protein coalitions)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F purification capsular saccharides are divided into 3 groups; The 1st group comprise 1,3,4,7F, 10A, 11A, 14,20 polysaccharide, the 2nd group comprises 6A, 6B, 9N, 9V, 18C, 19A, 19F, 23F oligosaccharide, the 3rd group comprise 2,5,8,12F, 15B, 17F, 22F, 33F polysaccharide.Merge with the water for injection dilution respectively and respectively organize polysaccharide and oligosaccharide; The 8 valencys pneumococal polysaccharide-DT coalition stock solution for preparing 2 groups of different serotypes polysaccharide by present embodiment (1) method and the 8 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution of another group different serotypes oligosaccharide are mixed with the vaccine finished product with the 8 valencys pneumococal polysaccharide-DT coalition stock solution of these 3 groups of different serotypes polysaccharide and oligosaccharide and 8 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution by present embodiment (1) method again and examine and determine.

(9) preparation of streptococcus pneumoniae 24 valency polysaccharide conjugate vaccines (6 valency polysaccharide-protein coalitions+6 valency polysaccharide-protein coalitions+6 valency polysaccharide-protein coalitions+6 valency polysaccharide-protein coalitions)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F purification capsular polysaccharide are divided into 4 groups; The 1st group comprise 1,3,4,7F, 18C, 19F, the 2nd group comprises 6A, 8,9N, 12F, 17F, 19A, the 3rd group comprise 2,9V, 11A, 14,23F, 33F, the 4th group comprise 5,6B, 10A, 15B, 20,22F.Merge with the water for injection dilution respectively and respectively organize polysaccharide; Prepare the 6 valencys pneumococal polysaccharide-DT coalition stock solution of 4 groups of different serotypes polysaccharide by present embodiment (1) method, again the 6 valencys pneumococal polysaccharide-DT coalition stock solution of these 4 groups of different serotypes polysaccharide is mixed with the vaccine finished product by present embodiment (1) method and examines and determine.

(10) preparation of streptococcus pneumoniae 24 valency oligosaccharide combined vaccines (6 valencys oligosaccharide-protein binding body+6 valencys oligosaccharide-protein binding body+6 valencys oligosaccharide-protein binding body+6 valencys oligosaccharide-protein binding body)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F purification pod membrane oligosaccharide are divided into 4 groups; The 1st group comprise 3,4,7F, 9N, 12F, 18C, the 2nd group comprise 5,6A, 8,17F, 19A, 19F, the 3rd group comprise 2,9V, 10A, 11A, 22F, 23F, the 4th group comprise 1,6B, 14,15B, 20,33F.Merge with the water for injection dilution respectively and respectively organize oligosaccharide; Prepare the 6 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution of 4 groups of different serotypes polysaccharide by present embodiment (1) method, again the 6 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution of these 4 groups of different serotypes oligosaccharide is mixed with the vaccine finished product by present embodiment (1) method and examines and determine.

(11) preparation of streptococcus pneumoniae 24 valency capsular saccharides combined vaccines (6 valency polysaccharide-protein coalitions+6 valencys oligosaccharide-protein binding body+6 valency polysaccharide-protein coalitions+6 valencys oligosaccharide-protein binding body)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F purification capsular saccharides are divided into 4 groups; The 1st group comprise 1,3,4,11A, 20,33F polysaccharide, the 2nd group comprises 6A, 6B, 18C, 19A, 19F, 23F oligosaccharide, the 3rd group comprise 2,5,8,15B, 17F, 22F polysaccharide, the 4th group comprises 7F, 9N, 9V, 10A, 12F, 14 oligosaccharide.Merge with the water for injection dilution respectively and respectively organize polysaccharide and oligosaccharide; Prepare the 6 valencys pneumococal polysaccharide-DT coalition stock solution of 2 groups of different serotypes polysaccharide and the 6 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution of other 2 groups of different serotypes oligosaccharide by present embodiment (1) method, again the 6 valencys pneumococal polysaccharide-DT coalition stock solution of these 4 groups of different serotypes polysaccharide and oligosaccharide and 6 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution are mixed with the vaccine finished product by present embodiment (1) method and examine and determine.

(12) preparation of streptococcus pneumoniae 24 valency polysaccharide conjugate vaccines (12 valency polysaccharide-protein coalitions+6 valency polysaccharide-protein coalitions+4 valency polysaccharide-protein coalitions+divalent polysaccharide-protein coalition)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F purification capsular polysaccharide are divided into 4 groups; The 1st group comprise 1,2,3,4,6A, 6B, 7F, 10A, 14,18C, 20,23F, the 2nd group comprises 11A, 12F, 15B, 17F, 22F, 33F, the 3rd group comprise 5,8,9N, 9V, the 4th group comprises 19A, 19F.Merge with the water for injection dilution respectively and respectively organize polysaccharide; Prepare 12 valencys pneumococal polysaccharide-DT coalition stock solution, 6 valencys pneumococal polysaccharide-DT coalition stock solution, 4 valencys pneumococal polysaccharide-DT coalition stock solution and the divalent pneumococal polysaccharide-DT coalition stock solution of 4 groups of different serotypes polysaccharide by present embodiment (1) method, again 12 valencys pneumococal polysaccharide-DT coalition stock solution, 6 valencys pneumococal polysaccharide-DT coalition stock solution, 4 valencys pneumococal polysaccharide-DT coalition stock solution and the divalent pneumococal polysaccharide-DT coalition stock solution of these 4 groups of different serotypes polysaccharide is mixed with the vaccine finished product by present embodiment (1) method and examines and determine.

(13) preparation of streptococcus pneumoniae 14 valency polysaccharide conjugate vaccines (14 valency polysaccharide-protein coalition)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F purification capsular polysaccharide are merged with the water for injection dilution; Prepare 14 valencys pneumococal polysaccharide-DT coalition stock solution by present embodiment (1) method, again this 14 valency pneumococal polysaccharide-DT coalition stock solution is mixed with the vaccine finished product by present embodiment (1) method and examines and determine.

Present embodiment (1) method and present embodiment (12) method difference are, do not add the 2-phenoxyethanol during preparation of latter's finished product vaccine.Therefore do not contain any antiseptic in present embodiment (12) the finished product vaccine.

(14) preparation of streptococcus pneumoniae 14 valency oligosaccharide combined vaccines (14 valencys oligosaccharide-protein binding body)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F purification pod membrane oligosaccharide are merged with the water for injection dilution; Prepare 14 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution by present embodiment (1) method, again this 14 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution is mixed with the vaccine finished product by present embodiment (1) method and examines and determine.

Present embodiment (1) method and present embodiment (13) method difference are, do not add the 2-phenoxyethanol during preparation of latter's finished product vaccine.Therefore do not contain any antiseptic in present embodiment (13) the finished product vaccine.

(15) preparation of streptococcus pneumoniae 14 valency polysaccharide conjugate vaccines (7 valency polysaccharide-protein coalitions+7 valency polysaccharide-protein coalitions)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F purification capsular polysaccharide are divided into 2 groups; The 1st group comprise 1,2,3,4,5,9V, 23F polysaccharide, the 2nd group comprises 6A, 6B, 7F, 14,18C, 19A, 19F polysaccharide.Merge with the water for injection dilution respectively and respectively organize polysaccharide; Prepare the 7 valencys pneumococal polysaccharide-DT coalition stock solution of 2 groups of different serotypes polysaccharide by present embodiment (1) method, again the 7 valencys pneumococal polysaccharide-DT coalition stock solution of these 2 groups of different serotypes polysaccharide is mixed with the vaccine finished product by present embodiment (1) method and examines and determine.

(16) preparation of streptococcus pneumoniae 14 valency oligosaccharide combined vaccines (7 valencys oligosaccharide-protein binding body+7 valencys oligosaccharide-protein binding body)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F purification pod membrane oligosaccharide are divided into 2 groups; The 1st group comprise 4,5,7F, 9V, 14,19A, 19F oligosaccharide, the 2nd group comprise 1,2,3,6A, 6B, 18C, 23F oligosaccharide.Merge with the water for injection dilution respectively and respectively organize oligosaccharide; Prepare the 7 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution of 2 groups of different serotypes oligosaccharide by present embodiment (1) method, again the 7 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution of these 2 groups of different serotypes oligosaccharide is mixed with the vaccine finished product by present embodiment (1) method and examines and determine.

(17) preparation of streptococcus pneumoniae 14 valency capsular saccharides combined vaccines (7 valency polysaccharide-protein coalitions+7 valencys oligosaccharide-protein binding body)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F purification capsular saccharides are divided into 2 groups; The 1st group comprise 1,2,3,4,7F, 9V, 14 polysaccharide, the 2nd group comprise 5,6A, 6B, 18C, 19A, 19F, 23F oligosaccharide.Merge with the water for injection dilution respectively and respectively organize polysaccharide and oligosaccharide; The 7 valencys pneumococal polysaccharide-DT coalition stock solution for preparing one group of different serotypes polysaccharide by present embodiment (1) method and the 7 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution of another group different serotypes oligosaccharide are mixed with the vaccine finished product with the 7 valencys pneumococal polysaccharide-DT coalition stock solution of these 2 groups of different serotypes polysaccharide and oligosaccharide and 7 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution by present embodiment (1) method again and examine and determine.

(18) preparation of streptococcus pneumoniae 14 valency polysaccharide conjugate vaccines (4 valency polysaccharide-protein coalitions+4 valency polysaccharide-protein coalitions+4 valency polysaccharide-protein coalitions+divalent polysaccharide-protein coalition)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F purification capsular polysaccharide are divided into 4 groups; The 1st group comprise 1,7F, 9V, 18C polysaccharide, the 2nd group comprise 14,19A, 19F, 23F polysaccharide, the 3rd group comprises 2,3,4,5 polysaccharide, the 4th group comprises 6A, 6B polysaccharide.Merge with the water for injection dilution respectively and respectively organize polysaccharide; Prepare the 4 valencys pneumococal polysaccharide-DT coalition stock solution of 3 groups of different serotypes polysaccharide and the divalent pneumococal polysaccharide of 1 group of different serotypes polysaccharide-DT coalition stock solution by present embodiment (1) method, again the 4 valencys pneumococal polysaccharide-DT coalition stock solution of these 4 groups of different serotypes polysaccharide and divalent pneumococal polysaccharide-DT coalition stock solution are mixed with the vaccine finished product by present embodiment (1) method and examine and determine.

(19) preparation of streptococcus pneumoniae 14 valency oligosaccharide combined vaccines (4 valencys oligosaccharide-protein binding body+4 valencys oligosaccharide-protein binding body+4 valencys oligosaccharide-protein binding body+divalent oligosaccharide-protein binding body)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F purification pod membrane oligosaccharide are divided into 4 groups; The 1st group comprise 1,3,5, the 9V oligosaccharide, the 2nd group comprise 2,7F, 18C, 23F oligosaccharide, the 3rd group comprise 4,6A, 6B, 14 oligosaccharide, the 4th group comprises 19A, 19F oligosaccharide.Merge with the water for injection dilution respectively and respectively organize oligosaccharide; Prepare the 4 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solutions of 3 groups of different serotypes oligosaccharide and the divalent streptococcus pneumoniae oligosaccharide-DT coalition stock solution of 1 group of different serotypes oligosaccharide by present embodiment (1) method, again 4 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solutions of these 4 groups of different serotypes oligosaccharide and divalent streptococcus pneumoniae oligosaccharide-DT coalition stock solution are mixed with the vaccine finished product by present embodiment (1) method and examine and determine.

(20) preparation of streptococcus pneumoniae 14 valency capsular saccharides combined vaccines (4 valency polysaccharide-protein coalitions+4 valencys oligosaccharide-protein binding body+4 valency polysaccharide-protein coalitions+divalent oligosaccharide-protein binding body)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F purification capsular saccharides are divided into 4 groups; The 1st group comprise 2,5,7F, 23F polysaccharide, the 2nd group comprise 4,6A, 6B, 18C oligosaccharide, the 3rd group comprise 1,3,9V, 14 polysaccharide, the 4th group comprises 19A, 19F oligosaccharide.Merge with the water for injection dilution respectively and respectively organize polysaccharide and oligosaccharide; Prepare the 4 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solutions of the 4 valencys pneumococal polysaccharide-DT coalition stock solution of 2 groups of different serotypes polysaccharide, 1 group of different serotypes oligosaccharide and the divalent streptococcus pneumoniae oligosaccharide-DT coalition stock solution of 1 group of different serotypes oligosaccharide by present embodiment (1) method, again 4 valencys pneumococal polysaccharide-DT coalition stock solution, 4 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solutions and the divalent streptococcus pneumoniae oligosaccharide-DT coalition stock solution of these 4 groups of different serotypes polysaccharide is mixed with the vaccine finished product by present embodiment (1) method and examines and determine.

(21) preparation of streptococcus pneumoniae 14 valency polysaccharide conjugate vaccines (divalent polysaccharide-protein coalition+divalent polysaccharide-protein coalition+divalent polysaccharide-protein coalition+divalent polysaccharide-protein coalition+divalent polysaccharide-protein coalition+divalent polysaccharide-protein coalition+divalent polysaccharide-protein coalition)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F purification capsular polysaccharide are divided into 7 groups; The 1st group comprise 1, the 9V polysaccharide, the 2nd group comprises 7F, 18C polysaccharide, the 3rd group comprise 14, the 23F polysaccharide, the 4th group comprises 19A, 19F polysaccharide, the 5th group comprises 2,3 polysaccharide, the 6th group comprises 4,5 polysaccharide, the 7th group comprises 6A, 6B polysaccharide.Merge with the water for injection dilution respectively and respectively organize polysaccharide; Prepare the divalent pneumococal polysaccharide-DT coalition stock solution of 7 groups of different serotypes polysaccharide by present embodiment (1) method, again the divalent pneumococal polysaccharide-DT coalition stock solution of these 7 groups of different serotypes polysaccharide is mixed with the vaccine finished product by present embodiment (1) method and examines and determine.

(22) preparation of streptococcus pneumoniae 14 valency oligosaccharide combined vaccines (divalent oligosaccharide-protein binding body+divalent oligosaccharide-protein binding body+divalent oligosaccharide-protein binding body+divalent oligosaccharide-protein binding body+divalent oligosaccharide-protein binding body+divalent oligosaccharide-protein binding body+divalent oligosaccharide-protein binding body)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F purification pod membrane oligosaccharide are divided into 7 groups; The 1st group comprises 1,3 oligosaccharide, the 2nd group comprise 5, the 9V oligosaccharide, the 3rd group comprise 2, the 23F oligosaccharide, the 4th group comprises 7F, 18C oligosaccharide, the 5th group comprises 6A, 6B oligosaccharide, the 6th group comprises 4,14 oligosaccharide, the 7th group comprises 19A, 19F oligosaccharide.Merge with the water for injection dilution respectively and respectively organize oligosaccharide; Prepare the divalent streptococcus pneumoniae oligosaccharide-DT coalition stock solution of 7 groups of different serotypes oligosaccharide by present embodiment (1) method, again the divalent streptococcus pneumoniae oligosaccharide-DT coalition stock solution of these 7 groups of different serotypes oligosaccharide is mixed with the vaccine finished product by present embodiment (1) method and examines and determine.

(23) preparation of streptococcus pneumoniae 14 valency capsular saccharides combined vaccines (divalent polysaccharide-protein coalition+divalent oligosaccharide-protein binding body+divalent polysaccharide-protein coalition+divalent oligosaccharide-protein binding body divalent polysaccharide-protein coalition+divalent oligosaccharide-protein binding body+divalent polysaccharide-protein coalition)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F purification capsular saccharides are divided into 7 groups; The 1st group comprise 2, the 23F polysaccharide, the 2nd group comprises 6A, 6B oligosaccharide, the 3rd group comprise 5, the 7F polysaccharide, the 4th group comprise 4, the 18C oligosaccharide, the 5th group comprises 9V, 14 polysaccharide, the 6th group comprises 19A, 19F oligosaccharide, the 7th group comprises 1,3 polysaccharide.Merge with the water for injection dilution respectively and respectively organize polysaccharide and oligosaccharide; Prepare the divalent pneumococal polysaccharide-DT coalition stock solution of 4 groups of different serotypes polysaccharide and the divalent streptococcus pneumoniae oligosaccharide of 3 groups of different serotypes oligosaccharide-DT coalition stock solution by present embodiment (1) method, again the divalent pneumococal polysaccharide-DT coalition stock solution of these 7 groups of different serotypes polysaccharide and divalent streptococcus pneumoniae oligosaccharide-DT coalition stock solution are mixed with the vaccine finished product by present embodiment (1) method and examine and determine.

(24) preparation of pneumonia meningitis combined vaccine (15 valency GL-PP coalition)

Streptococcus pneumoniae serotype 1,4,5,6A, 6B, 9V, 14,18C, 19A, 19F, 23F purification capsular polysaccharide and meningococcus A crowd, C crowd, W135 crowd, Y crowd's purification capsular polysaccharide are merged with the water for injection dilution; Prepare 15 valencys polysaccharide-DT coalition stock solution by present embodiment (1) method, again this 15 valency polysaccharide-DT coalition stock solution is mixed with the vaccine finished product by present embodiment (1) method.Pneumococcal conjugated vaccine manufacturing and vertification regulation (WHO Technical Report Series, No.927,2005) and manufacturing of meningococcus combined vaccine and vertification regulation (WHO BS 06.2041,2006 by the formal promulgation of World Health Organization (WHO); WHO TechnicalReport Series, No.924,2004) prescriptive procedure and standard examine and determine.

(25) preparation of pneumonia meningitis Hib combined vaccine (17 valency oligosaccharide protein binding body)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F purification pod membrane oligosaccharide, meningococcus A crowd purification pod membrane oligosaccharide, meningococcus C crowd purification pod membrane oligosaccharide, b type hemophilus influenza purification pod membrane oligosaccharide are merged with the water for injection dilution; Prepare 17 valencys oligosaccharide-DT coalition stock solution by present embodiment (1) method, again this 17 valency oligosaccharide-DT coalition stock solution is mixed with the vaccine finished product by present embodiment (1) method.Pneumococcal conjugated vaccine manufacturing and vertification regulation (WHO Technical Report Series, No.927,2005) and manufacturing of meningococcus combined vaccine and vertification regulation (WHO BS 06.2041,2006 by the formal promulgation of World Health Organization (WHO); WHO Technical Report Series, No.924,2004) and b type hemophilus influenza combined vaccine is made and vertification regulation (WHO Technical Report Series, No.897,2000) prescriptive procedure and standard are examined and determine.

(26) preparation of pneumonia meningitis Hib combined vaccine (7 valencys oligosaccharide-protein binding body+7 valencys oligosaccharide-protein binding body+5 valencys oligosaccharide-protein binding body)

Streptococcus pneumoniae serotype 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, 23F purification pod membrane oligosaccharide, meningococcus A crowd purification pod membrane oligosaccharide, meningococcus C crowd purification pod membrane oligosaccharide, meningococcus W135 crowd purification pod membrane oligosaccharide, meningococcus Y crowd purification pod membrane oligosaccharide, b type hemophilus influenza purification pod membrane oligosaccharide are divided into 3 groups; The 1st group comprise 4,5,7F, 9V, 14,19A, 19F oligosaccharide, the 2nd group comprise 1,2,3,6A, 6B, 18C, 23F oligosaccharide, the 3rd group comprises meningococcus A crowd, C crowd, W135 crowd, Y crowd's oligosaccharide and b type hemophilus influenza oligosaccharide.Merge with the water for injection dilution respectively and respectively organize oligosaccharide; Prepare the 7 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solution of 2 groups of different serotypes oligosaccharide and 5 valency meningitis Hib oligosaccharide-DT coalition stock solutions of 1 group of different blood group (crowd) by present embodiment (1) method, 7 valency streptococcus pneumoniae oligosaccharide-DT coalition stock solutions and the 5 valency meningitis Hib oligosaccharide-DT coalition stock solutions with these 3 groups of different serotypes oligosaccharide are mixed with the vaccine finished product by present embodiment (1) method again.Pneumococcal conjugated vaccine manufacturing and vertification regulation (WHO Technical Report Series, No.927,2005) and manufacturing of meningococcus combined vaccine and vertification regulation (WHO BS 06.2041,2006 by the formal promulgation of World Health Organization (WHO); WHO TechnicalReport Series, No.924,2004) and b type hemophilus influenza combined vaccine is made and vertification regulation (WHOTechnical Report Series, No.897,2000) prescriptive procedure and standard are examined and determine.

The inspection of embodiment 6 combined vaccine specificity toxicity

Take a sample respectively to embodiment 5 26 kinds of prepared vaccines that equivalent is merged into 26 duplicate samples, every duplicate samples is selected 4 of body weight 250～350g Cavia porcelluss for use, and every veutro subcutaneous injection sample 2.5ml observed 30 days, the record body weight change.The result shows that all samples injection site does not have necrosis, does not have decortication, do not have depilation, and no late period, paralysis symptom all had increase before every Cavia porcellus weight ratio is injected.After the employed diphtheria toxoid carrier of 26 kinds of combined vaccines that the present invention preparation is described is handled through combined process diphtheria toxin, diphtherotoxin specificity toxic reaction not taking place, the Poison Reverse phenomenon do not occur yet.

Embodiment 7 combined vaccine security inspections

To 26 kinds of prepared vaccines samplings of embodiment 5, every duplicate samples is selected 5 of body weight 18～22g mices and 2 of body weight 250～350g Cavia porcelluss respectively for use, every mouse peritoneal injected sample 0.5ml, and every guinea pig intraperitoneal injection 5.0ml observed 7 days, write down body weight change.The result shows all mices in the observation period, Cavia porcellus is homodesmic deposits, and no abnormal reaction, and every mice, Cavia porcellus weight ratio all have increase before injecting when expiring.Explain in 26 kinds of vaccines of the present invention preparation and do not pollute exogenous toxicant, also do not have the unexpected dangerous factor.

Embodiment 8 combined vaccine immunogenicity determinings

Get the NIH mice of 24 serotype polysaccharide of 26 kinds of vaccines, streptococcus pneumoniae, each sero-group polysaccharide of meningococcus, Hib polysaccharide and the physiological sodium chloride solution peritoneal immunity body weight 12～14g of embodiment 5 preparations, each sample is according to 10 mice 3 pins of 0,14,28 day program immunity.Combined vaccine group immunizing dose is contained each serotype (crowd) capsular saccharides 2～10 μ g/0.5ml/ time, contained each serotype of polysaccharide group immunizing dose and vaccine group (crowd) capsular saccharides equivalent.Just exempt from back blood sampling in the 35th day, separation of serum.Measure antibody GMT value with the ELISA method.Each serotype (crowd) glycocalyx in 96 hole ELISA Plates, is put 2～8 ℃ and spent the night.After the sealing, add the test serum sample of serial dilution, 37 ℃ were reacted 45 minutes.The sheep anti-mouse igg that adds horseradish peroxidase-labeled again adds the substrate tetramethyl benzidine at last in 37 ℃ of reactions 45 minutes, and colour developing is 15 minutes in the dark place, uses the stop buffer cessation reaction.On ELIASA, measure, calculate serum antibody GMT.The result shows that the independent polysaccharide GMT of contained each serotype of vaccine (crowd) capsular saccharides has significance to increase.Wherein, each serotype polysaccharide GMT of streptococcus pneumoniae is 23～75, and contained each serotype capsular saccharides GMT is 1864～8053 in the pneumococcal conjugated vaccine.Table 1, table 2 have been enumerated wherein several vaccine immunogenicity GMT determination datas.

In sum, the streptococcus pneumoniae capsular saccharides combined vaccine of being made up of divalent capsular saccharides-protein binding body and/or multivalence capsular saccharides-protein binding body of the present invention preparation and have good safety and immunogenicity with bacterium combined combined vaccine that meningococcus, Hib form is described.

Claims

1. pneumococcal conjugated vaccine; It is characterized in that; The antigen of said vaccine comprises divalent serotype streptococcus pneumoniae capsular saccharides-protein binding body and/or 3～24 valency serotype streptococcus pneumoniae capsular saccharides-protein binding bodies; Said divalent serotype streptococcus pneumoniae capsular saccharides-protein binding body is meant that two different serotype capsular saccharides and protein carrier combine the conjugate that forms through chemical bond; Protein carrier is linked at these two different serotypes capsular saccharides together through chemical bond, and the chemical bonded refractory of two different serotype capsular saccharides foundations and protein carrier is combined in to form on the structure and links; Said 3～24 valency serotype streptococcus pneumoniae capsular saccharides-protein binding bodies are meant that 3～24 different serotype capsular saccharides and protein carrier combine the conjugate that forms through chemical bond; Protein carrier is linked at these 3～24 different serotype capsular saccharides together through chemical bond, and the chemical bonded refractory of 3～24 different serotypes capsular saccharides foundations and protein carrier is combined in to form on the structure and links.

2. pneumococcal conjugated vaccine according to claim 1 is characterized in that, the antigen of said vaccine comprises one or more 2～24 valency serotype capsular saccharides-protein binding body.

3. pneumococcal conjugated vaccine according to claim 2; It is characterized in that; Said more than two 2～24 valency serotype capsular saccharides-protein binding bodies be meant that plural capsular saccharides-protein binding body contains the serotype capsular saccharides of phase equivalent number respectively, and its valence mumber is all between 2～24.

4. pneumococcal conjugated vaccine according to claim 2; It is characterized in that; Said more than two 2～24 valency serotype capsular saccharides-protein binding bodies be meant that plural capsular saccharides-protein binding body contains the serotype capsular saccharides of unequal valence mumber respectively, and its valence mumber is all between 2～24.

5. pneumococcal conjugated vaccine according to claim 2; It is characterized in that; Said more than two 2～24 valency serotype capsular saccharides-protein binding bodies be meant that plural capsular saccharides-protein binding body contains the serotype capsular saccharides that equates with unequal valence mumber, and its valence mumber is all between 2～24.

6. streptococcus pneumoniae-meningococcus-b type hemophilus influenza combined vaccine; Said combined vaccine antigen comprises a plurality of serotype capsular saccharides of streptococcus pneumoniae, meningococcus A crowd, C crowd, W135 crowd, Y crowd's capsular saccharides and b type hemophilus influenza capsular saccharides; The capsular saccharides and the protein carrier of these different bacterium serotypes or sero-group carry out chemical bond, are present in the vaccine with divalent serotype or sero-group capsular saccharides-protein binding body and/or 3～24 valency serotypes or sero-group capsular saccharides-protein binding bodily form formula; Said divalent serotype or sero-group capsular saccharides-protein binding body are meant that two different serotypes and/or sero-group capsular saccharides and protein carrier combine the conjugate that forms through chemical bond; Protein carrier is linked at these two different serotypes and/or sero-group capsular saccharides together through chemical bond, and the chemical bonded refractory of two different serotype and/or sero-group capsular saccharides foundation and protein carrier is combined in to form on the structure and links; Said 3～24 valency serotypes or sero-group capsular saccharides-protein binding body are meant that 3～24 different serotypes and/or sero-group capsular saccharides and protein carrier combine the conjugate that forms through chemical bond; Protein carrier is linked at these 3～24 different serotypes and/or sero-group capsular saccharides together through chemical bond, and the chemical bonded refractory of 3～24 different serotypes and/or sero-group capsular saccharides foundation and protein carrier is combined in to form on the structure and links.

7. streptococcus pneumoniae-meningococcus-b type hemophilus influenza combined vaccine; Said combined vaccine antigen comprises a plurality of serotype capsular saccharides of streptococcus pneumoniae, meningococcus A crowd, C crowd's capsular saccharides and b type hemophilus influenza capsular saccharides; The capsular saccharides and the protein carrier of these different bacterium serotypes or sero-group carry out chemical bond, are present in the vaccine with divalent serotype or sero-group capsular saccharides-protein binding body and/or 3～24 valency serotypes or sero-group capsular saccharides-protein binding bodily form formula; Said divalent serotype or sero-group capsular saccharides-protein binding body are meant that two different serotypes and/or sero-group capsular saccharides and protein carrier combine the conjugate that forms through chemical bond; Protein carrier is linked at these two different serotypes and/or sero-group capsular saccharides together through chemical bond, and the chemical bonded refractory of two different serotype and/or sero-group capsular saccharides foundation and protein carrier is combined in to form on the structure and links; Said 3～24 valency serotypes or sero-group capsular saccharides-protein binding body are meant that 3～24 different serotypes and/or sero-group capsular saccharides and protein carrier combine the conjugate that forms through chemical bond; Protein carrier is linked at these 3～24 different serotypes and/or sero-group capsular saccharides together through chemical bond, and the chemical bonded refractory of 3～24 different serotypes and/or sero-group capsular saccharides foundation and protein carrier is combined in to form on the structure and links.

8. streptococcus pneumoniae-meningococcus-b type hemophilus influenza combined vaccine; Said combined vaccine antigen comprises a plurality of serotype capsular saccharides of streptococcus pneumoniae, meningococcus A crowd, C crowd, W135 crowd, Y crowd's capsular saccharides and b type hemophilus influenza capsular saccharides, and the capsular saccharides of these streptococcus pneumoniae different serotypes is present in the vaccine with divalent serotype capsular saccharides-protein binding body and/or 3～24 valency serotype capsular saccharides-protein binding bodily form formula; The capsular saccharides of the different sero-groups of these meningococcuss is present in the vaccine with 4 valency sero-group capsular saccharides-protein binding bodily form formula; The capsular saccharides of said b type hemophilus influenza is present in the vaccine with monovalent serum type capsular saccharides-protein binding bodily form formula; Said divalent serotype capsular saccharides-protein binding body is meant that two different serotype capsular saccharides and protein carrier combine the conjugate that forms through chemical bond; Protein carrier is linked at these two different serotypes capsular saccharides together through chemical bond, and the chemical bonded refractory of two different serotype capsular saccharides foundations and protein carrier is combined in to form on the structure and links; Said 3～24 valency serotype capsular saccharides-protein binding bodies are meant that 3～24 different serotype capsular saccharides and protein carrier combine the conjugate that forms through chemical bond; Protein carrier is linked at these 3～24 different serotype capsular saccharides together through chemical bond, and the chemical bonded refractory of 3～24 different serotypes capsular saccharides foundations and protein carrier is combined in to form on the structure and links; Said 4 valency sero-group capsular saccharides-protein binding bodies are meant that 4 different sero-group capsular saccharides and protein carrier combine the conjugate that forms through chemical bond; Protein carrier is linked at these 4 different sero-group capsular saccharides together through chemical bond, and the chemical bonded refractory of 4 different sero-group capsular saccharides foundations and protein carrier is combined in to form on the structure and links; Said monovalent serum type capsular saccharides-protein binding body is meant that a serotype capsular saccharides and protein carrier combine the conjugate that forms through chemical bond.

9. streptococcus pneumoniae-meningococcus-b type hemophilus influenza combined vaccine, said combined vaccine antigen comprises a plurality of serotype capsular saccharides of streptococcus pneumoniae, meningococcus A crowd, C crowd's capsular saccharides and b type hemophilus influenza capsular saccharides; The capsular saccharides of these streptococcus pneumoniae different serotypes is present in the vaccine with divalent serotype capsular saccharides-protein binding body and/or 3～24 valency serotype capsular saccharides-protein binding bodily form formula; The capsular saccharides of the different sero-groups of these meningococcuss is present in the vaccine with divalent sero-group capsular saccharides-protein binding bodily form formula; The capsular saccharides of said b type hemophilus influenza is present in the vaccine with monovalent serum type capsular saccharides-protein binding bodily form formula; Said divalent serotype capsular saccharides-protein binding body is meant that two different serotype capsular saccharides and protein carrier combine the conjugate that forms through chemical bond; Protein carrier is linked at these two different serotypes capsular saccharides together through chemical bond, and the chemical bonded refractory of two different serotype capsular saccharides foundations and protein carrier is combined in to form on the structure and links; Said 3～24 valency serotype capsular saccharides-protein binding bodies are meant that 3～24 different serotype capsular saccharides and protein carrier combine the conjugate that forms through chemical bond; Protein carrier is linked at these 3～24 different serotype capsular saccharides together through chemical bond, and the chemical bonded refractory of 3～24 different serotypes capsular saccharides foundations and protein carrier is combined in to form on the structure and links; Said divalent sero-group capsular saccharides-protein binding body is meant that 2 different sero-group capsular saccharides and protein carrier combine the conjugate that forms through chemical bond; Protein carrier is linked at these 2 different sero-group capsular saccharides together through chemical bond, and the chemical bonded refractory of 2 different sero-group capsular saccharides foundations and protein carrier is combined in to form on the structure and links; Said monovalent serum type capsular saccharides-protein binding body is meant that a serotype capsular saccharides and protein carrier combine the conjugate that forms through chemical bond.

10. according to each described combined vaccine of claim 1-5 or according to each described combined vaccine of claim 6-9; It is characterized in that said streptococcus pneumoniae serotype capsular saccharides is selected from the capsular saccharides of streptococcus pneumoniae 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, the preparation of 33F type bacterial strain.

11. according to each described combined vaccine of claim 1-5 or according to each described combined vaccine of claim 6-9; It is characterized in that said streptococcus pneumoniae serotype capsular saccharides is selected from the capsular saccharides of streptococcus pneumoniae 1,2,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F, the preparation of 23F type bacterial strain.

12. according to each described combined vaccine of claim 1-5 or according to each described combined vaccine of claim 6-9; It is characterized in that said protein carrier is selected from tetanus toxoid, diphtheria toxoid, the nontoxic variant of diphtheria toxin, diphtherotoxin, B group meningitis cocci outer membrane protein, streptococcus pneumoniae surface protein A, PsaA pneumococcal surface adhesion A, pneumolysin, hemophilus influenza D albumen, pertussis toxin, PT, cholera toxin, purified protein derivative (PPD) of tuberculin, Pseudomonas aeruginosa exotoxin A, keyhole limpet hemocyanin.

13. according to each described combined vaccine of claim 1-5 or according to each described combined vaccine of claim 6-9; It is characterized in that said protein carrier is to derive from pneumococcal albumen PspA, PsaA, Ply to merge the pneumoprotein complex that forms.

14., it is characterized in that each serotype of finished product vaccine or sero-group capsular saccharides dosage are 0.1～50 μ g according to each described combined vaccine of claim 1-5 or according to each described combined vaccine of claim 6-9.

15., it is characterized in that the finished product vaccine comprises adjuvant according to each described combined vaccine of claim 1-5 or according to each described combined vaccine of claim 6-9.

Method according to claim 15, wherein the combination vaccine, wherein said adjuvant is selected from aluminum hydroxide, calcium phosphate adjuvant, cholera toxin B subunit, saponin Q21, lipids, lipid monophosphate A, MF59, Escherichia coli heat-labile toxin.

17. combined vaccine according to claim 16 is characterized in that, described aluminium adjuvant is selected from aluminium hydroxide, aluminum phosphate or aluminum sulfate.

18., it is characterized in that the finished product vaccine does not contain any antiseptic according to each described combined vaccine of claim 1-5 or according to each described combined vaccine of claim 6-9.

19., it is characterized in that the finished product vaccine comprises the antiseptic that is selected from thimerosal, 2-phenoxyethanol, benzyl alcohol according to each described combined vaccine of claim 1-5 or according to each described combined vaccine of claim 6-9.

20. according to each described combined vaccine of claim 1-5 or according to each described combined vaccine of claim 6-9; It is characterized in that the capsular saccharides in described divalent serotype or the sero-group capsular saccharides-protein binding body is capsular polysaccharide and/or pod membrane oligosaccharide; Capsular saccharides in described 3～24 valency serotypes or the sero-group capsular saccharides-protein binding body is capsular polysaccharide and/or pod membrane oligosaccharide; Said divalent serotype or sero-group capsular saccharides-protein binding body are meant that two different serotypes and/or sero-group capsular saccharides and protein carrier combine the conjugate that forms through chemical bond; Protein carrier is linked at these two different serotypes and/or sero-group capsular saccharides together through chemical bond, and the chemical bonded refractory of two different serotype and/or sero-group capsular saccharides foundation and protein carrier is combined in to form on the structure and links; Said 3～24 valency serotypes or sero-group capsular saccharides-protein binding body are meant that 3～24 different serotypes and/or sero-group capsular saccharides and protein carrier combine the conjugate that forms through chemical bond; Protein carrier is linked at these 3～24 different serotypes and/or sero-group capsular saccharides together through chemical bond, and the chemical bonded refractory of 3～24 different serotypes and/or sero-group capsular saccharides foundation and protein carrier is combined in to form on the structure and links.

21. a method for preparing the bacterial capsule sugar combined vaccine that contains divalent serotype or sero-group capsular saccharides-protein binding body and/or 3～24 valency serotypes or sero-group capsular saccharides-protein binding body said method comprising the steps of:

A) from different serotypes or sero-group bacterial cultures, extract respectively, the purification capsular saccharides, process single serotype or sero-group capsular saccharides, i.e. monovalent serum type or sero-group capsular saccharides;

B) the monovalent serum type of step a) gained or the sero-group capsular saccharides capsular saccharides in two above serotypes or sero-group is merged according to ratio, become divalent or 3～24 valency serotypes or sero-group capsular saccharides;

C) divalent of step b) or 3～24 valency serotypes or sero-group capsular saccharides and protein carrier are carried out chemical bonded refractory and merge purification, process divalent serotype or sero-group capsular saccharides-protein binding isoantigen or 3～24 valency serotypes or sero-group capsular saccharides-protein binding isoantigen;

D) the divalent serotype of step c) or sero-group capsular saccharides-protein binding isoantigen and/or 3～24 valency serotypes or sero-group capsular saccharides-protein binding isoantigen are mixed with vaccine;

Said divalent serotype or sero-group capsular saccharides-protein binding body are meant that two different serotypes and/or sero-group capsular saccharides and protein carrier combine the conjugate that forms through chemical bond; Protein carrier is linked at these two different serotypes and/or sero-group capsular saccharides together through chemical bond, and the chemical bonded refractory of two different serotype and/or sero-group capsular saccharides foundation and protein carrier is combined in to form on the structure and links; Said 3～24 valency serotypes or sero-group capsular saccharides-protein binding body are meant that 3～24 different serotypes and/or sero-group capsular saccharides and protein carrier combine the conjugate that forms through chemical bond; Protein carrier is linked at these 3～24 different serotypes and/or sero-group capsular saccharides together through chemical bond, and the chemical bonded refractory of 3～24 different serotypes and/or sero-group capsular saccharides foundation and protein carrier is combined in to form on the structure and links.

22. a method for preparing the bacterial capsule sugar combined vaccine that contains divalent serotype or sero-group capsular saccharides-protein binding body and/or 3～24 valency serotypes or sero-group capsular saccharides-protein binding body said method comprising the steps of:

B) the monovalent serum type of step a) gained or the sero-group capsular saccharides capsular saccharides in two above serotypes or sero-group is merged according to ratio; Become divalent or 3～24 valency serotypes or sero-group capsular saccharides; Wherein, said capsular saccharides is further handled through homogenize after merging;

C) divalent of step b) or 3～24 valency serotypes or sero-group capsular saccharides are carried out chemical bond with protein carrier and combine, process divalent serotype or sero-group capsular saccharides-protein binding isoantigen or 3～24 valency serotypes or sero-group capsular saccharides-protein binding isoantigen;

D) the product detoxification treatment and the purification of step c) are processed divalent serotype or sero-group capsular saccharides-protein binding isoantigen or 3～24 valency serotypes or sero-group capsular saccharides-protein binding isoantigen;

E) the divalent serotype of step d) or sero-group capsular saccharides-protein binding isoantigen and/or 3～24 valency serotypes or sero-group capsular saccharides-protein binding isoantigen are mixed with vaccine;

23. according to claim 21 or 22 described methods, wherein, said antibacterial is selected from streptococcus pneumoniae, meningococcus, b type hemophilus influenza, salmonella typhi, staphylococcus, B group streptococcus.

24. according to claim 21 or 22 said methods, the mutual combined wt ratio of different serotypes or sero-group capsular saccharides is 1: 0.05～20 (w/w).

25. according to claim 21 or 22 described methods, the capsular saccharides in wherein said divalent serotype or the sero-group capsular saccharides-protein binding body is capsular polysaccharide and/or pod membrane oligosaccharide; Capsular saccharides in described 3～24 valency serotypes or the sero-group capsular saccharides-protein binding body is capsular polysaccharide and/or pod membrane oligosaccharide; Said divalent serotype or sero-group capsular saccharides-protein binding body are meant that two different serotypes and/or sero-group capsular saccharides and protein carrier combine the conjugate that forms through chemical bond; Protein carrier is linked at these two different serotypes and/or sero-group capsular saccharides together through chemical bond, and the chemical bonded refractory of two different serotype and/or sero-group capsular saccharides foundation and protein carrier is combined in to form on the structure and links; Said 3～24 valency serotypes or sero-group capsular saccharides-protein binding body are meant that 3～24 different serotypes and/or sero-group capsular saccharides and protein carrier combine the conjugate that forms through chemical bond; Protein carrier is linked at these 3～24 different serotypes and/or sero-group capsular saccharides together through chemical bond, and the chemical bonded refractory of 3～24 different serotypes and/or sero-group capsular saccharides foundation and protein carrier is combined in to form on the structure and links.