CN104383532A - Bacterial polysaccharide protein conjugate vaccine using hepatitis B surface antigen as carrier protein and preparation method of bacterial polysaccharide protein conjugate vaccine - Google Patents

Bacterial polysaccharide protein conjugate vaccine using hepatitis B surface antigen as carrier protein and preparation method of bacterial polysaccharide protein conjugate vaccine Download PDF

Info

Publication number
CN104383532A
CN104383532A CN201410723098.3A CN201410723098A CN104383532A CN 104383532 A CN104383532 A CN 104383532A CN 201410723098 A CN201410723098 A CN 201410723098A CN 104383532 A CN104383532 A CN 104383532A
Authority
CN
China
Prior art keywords
polysaccharide
hepatitis
surface antigen
bacterial
type
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201410723098.3A
Other languages
Chinese (zh)
Inventor
马波
陈玉秋
吴凯
黄镇
何建东
范荣坤
陶佳明
白锐琼
钱雯
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
YUNNAN WOSEN BIOTECHNOLOGY CO Ltd
Original Assignee
YUNNAN WOSEN BIOTECHNOLOGY CO Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by YUNNAN WOSEN BIOTECHNOLOGY CO Ltd filed Critical YUNNAN WOSEN BIOTECHNOLOGY CO Ltd
Priority to CN201410723098.3A priority Critical patent/CN104383532A/en
Publication of CN104383532A publication Critical patent/CN104383532A/en
Pending legal-status Critical Current

Links

Classifications

    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a bacterial polysaccharide protein conjugate vaccine using a hepatitis B surface antigen as carrier protein and a preparation method of the bacterial polysaccharide protein conjugate vaccine. According to the vaccine, protein is the hepatitis B surface antigen, and a bacterial polysaccharide is selected from any one or more of a haemophilus influenza type b polysaccharide, group A, group C, group Y and group W135 meningococcal polysaccharides, a salmonella typhi type Vi polysaccharide, a group B streptococcus type Ia polysaccharide and the like, pneumococcus serotype type 1, 2 and the like, and salmonella paratyphi type A or salmonella paratyphi type B. Animal experiments show that the antibody positive conversion rates of the bacterial polysaccharide and the hepatitis B surface antigen in the vaccine are both more than 85%, so that the vaccine is relatively high in antibody positive conversion rate; carrier protein plays a role in transforming the bacterial polysaccharide from a T-cell-independent antigen into a T-cell-dependent antigen, and also can be used for preventing diseases caused by hepatitis B virus; and by adopting the bacterial polysaccharide protein conjugate vaccine disclosed by the invention, the problem of performing immunization inoculation on infants and young children under 2 years old can be solved, the function of one injection with multiple immune effects also can be achieved, and the use crowd and coverage rate of the vaccine can be expanded.

Description

A kind of hepatitis B surface antigen is bacterial polysaccharide protein conjugate vaccine of carrier protein and preparation method thereof
Technical field
The present invention relates to vaccine for man technical field, being specifically related in order to hepatitis B surface antigen is the unit price or polyvalent vaccine goods and preparation method thereof that bacterial polysaccharide protein conjugate prepared by carrier protein is made.
Background technology
Bacterial polysaccharides is one of Major Virulence Factors of antibacterial, has good immunogenicity, can bring out body and produce effective protection antibody.But bacterial polysaccharides belongs to T-independent antigen, to the immune system physiogeny infant of incomplete less than 2 years old, effective protection antibody can not be induced, and can not immunological memory be produced.And by after bacterial polysaccharide antigen and carrier protein covalent bond; bacterial polysaccharides can be converted into T cell dependence antigen by T-independent antigen; make bacterial polysaccharides also can produce the protection of effective antibody to the infant of less than 2 years old, thus have also been enlarged the use crowd of vaccine.But the bacterial polysaccharide protein combined vaccine of list marketing both at home and abroad at present, the disease only for preventing bacterial polysaccharide antigen to cause, and carrier protein only plays effect bacterial polysaccharides being converted into T cell dependence antigen by T-independent antigen.
Given this, applicants have invented a kind of is the vaccine that bacterial polysaccharide protein conjugates prepared by protein carrier is made in order to hepatitis B surface antigen, and hepatitis B surface antigen wherein not only serves effect bacterial polysaccharides combined vaccine being converted into T cell dependence antigen by T-independent antigen; And serve the disease of effectively preventing carrier protein (hepatitis B surface antigen) to cause.
Summary of the invention
The technical problem to be solved in the present invention overcomes prior art to there is carrier protein in vaccine and only play the defect of effect bacterial polysaccharides being converted into T cell dependence antigen by T-independent antigen.
For solving the problems of the technologies described above, the invention provides a kind of hepatitis B surface antigen is bacterial polysaccharide protein conjugate vaccine of carrier protein and preparation method thereof, after this vaccine uses, the specific antibody of the antigen of directed toward bacteria polysaccharide can be produced, the specific antibody for hepatitis B surface antigen can be produced again, overcome carrier protein in vaccine and only play the defect of effect bacterial polysaccharides being converted into T cell dependence antigen by T-independent antigen, reach the effect that a pin is exempted from more.
Technical scheme of the present invention is as follows:
1. one kind is the bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen, it is characterized in that: the albumen contained in described vaccine is hepatitis B surface antigen, the bacterial polysaccharides contained in described vaccine is selected from b type hemophilus influenza polysaccharide, neisseria meningitis scorching pneumoniae serotype group A group, C group, Y group, W135 group's polysaccharide, salmonella typhi Vi type polysaccharide, B group streptococcus Ia, Ib, II, III, V-type, Pneumococcus serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F, bacillus paratyphosus A type or bacillus paratyphosus B-mode in any one or a few.
2. is the bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen according to technical scheme 1, it is characterized in that: each bacterial polysaccharides to be connected with hepatitis B surface antigen with covalent bond respectively and to be connected into bacterial polysaccharide protein conjugate.
3. is the bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen according to technical scheme 2, it is characterized in that: in described bacterial polysaccharide protein conjugate, the mass ratio of total bacterial polysaccharides and hepatitis B surface antigen is 1:0.1 ~ 1:5.
4. is the bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen according to technical scheme 1 or 2 or 3, it is characterized in that: described hepatitis B virus surface antigen is natural hepatitis B virus surface antigen or recombination hepatitis B surface antigen.
5. is the bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen according to technical scheme 1 or 2 or 3, it is characterized in that: the combination agent that described vaccine is liquid preparation, solid particle agent, semi-solid agent or liquid preparation and solid particle agent are combined.
6. is the bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen according to technical scheme 4, it is characterized in that: the combination agent that described vaccine is liquid preparation, solid particle agent, semi-solid agent or liquid preparation and solid particle agent are combined.
7. is the bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen according to technical scheme 1 or 2 or 3, it is characterized in that: described vaccine is injection, nasal formulations, gel or Extencap.
8. is the bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen according to technical scheme 4, it is characterized in that: described vaccine is injection, nasal formulations, gel or Extencap.
9. is the bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen according to technical scheme 1 or 2 or 3, it is characterized in that: also containing aluminium adjuvant or a kind of deoxyribonucleic acid fragment containing Cytosine-phosphate-guanine in described vaccine.
10. is the bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen according to technical scheme 4, it is characterized in that: also containing aluminium adjuvant or a kind of deoxyribonucleic acid fragment containing Cytosine-phosphate-guanine in described vaccine.
11. 1 kinds is the preparation method of the unit price bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen, comprises the following steps:
(1) bacterial polysaccharides dissolves
The NaCl of a kind of bacterial polysaccharides 0.2mol/L is dissolved as the concentration of 1 ~ 20mg/ml; Described a kind of bacterial polysaccharides is b type hemophilus influenza polysaccharide, neisseria meningitis scorching pneumoniae serotype group A group, C group, Y group, W135 group's polysaccharide, salmonella typhi Vi type polysaccharide, B group streptococcus Ia, Ib, II, III, V-type, Pneumococcus serotypes 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F, bacillus paratyphosus A type or bacillus paratyphosus B-mode in any one;
(2) bacterial polysaccharides activation
The bacterial polysaccharides that polysaccharide activator must activate is added in the bacterial polysaccharides solution that step (1) is dissolved, the quality of the polysaccharide activator added: bacterial polysaccharides mass ratio is 0.1:1 ~ 1.5:1, described polysaccharide activator is any one in Bromine cyanide., 1-cyano group-DMAP Tetrafluoroboric acid ester, carbodiimide, 3-(2-pyridyidithio) propanoic acid, N-hydroxy-succinamide ester;
(3) bacterial polysaccharides derives
By the bacterial polysaccharides of activation in the bacterial polysaccharides of the activation of step (2) gained: the mass ratio of difunctional link agent is that the ratio row of 1:2 ~ 1:5 add difunctional link agent and carry out the derivative of bacterial polysaccharides and to obtain bacterial polysaccharides derivant, described difunctional link agent is any one in adipic dihydrazide, Polyethylene Glycol, hexamethylene diamine or hexanediol;
(4) ultrafiltration of bacterial polysaccharides derivant
Bacterial polysaccharides derivant step (3) obtained obtains bacterial polysaccharides derivant through the ultrafilter membrane ultrafiltration lyophilizing of 1 ~ 30KD;
(5) bacterial polysaccharides and protein binding
After being 2 ~ 20mg/ml by the concentration that the bacterial polysaccharides derivant that step (4) obtains to be dissolved to bacterial polysaccharides derivant by the NaCl of 0.2mol/L; In bacterial polysaccharides derivant: the mass ratio of hepatitis B surface antigen is that the ratio of 1:0.4 ~ 1:2 adds in carrier protein, then adding carbodiimide to carbodiimide concentration is 0.001 ~ 0.2mol/L, maintaining pH is 5.5 ± 0.5, react 2 ~ 4 hours, obtain a kind of bacterial polysaccharides derivant and carrier protein covalent conjunct agent;
(6) a kind of for step (5) gained bacterial polysaccharides derivant and carrier protein covalent conjunct agent gel column are carried out purification, collect V 0absworption peak near (elution volume of the molecule of solvent resistant column material cannot be entered), namely aseptic filtration obtains the unit price bacterial polysaccharide protein conjugate stock solution after purification;
(7) vaccine liquid preparation is prepared
Water for injection is added in unit price bacterial polysaccharide protein conjugate stock solution after the purification of step (6) gained, sodium chloride, phosphate buffer and aluminium adjuvant, the concentration of bacterial polysaccharides is made to be 4 μ g/ml ~ 60 μ g/ml, sodium chloride concentration is 4 ~ 9mg/ml, sodium hydrogen phosphate concentration is 38 ~ 72 μ g/ml, phosphate dihydrogen sodium concentration is 24 ~ 88 μ g/ml, described aluminium adjuvant is aluminium hydroxide or aluminum phosphate, aluminum hydroxide concentration is made to be 0.5 ~ 2.5mg/ml, or aluminium phosphate concentration is 0.5 ~ 2.0mg/ml, namely making with hepatitis B surface antigen is the unit price bacterial polysaccharide protein conjugate vaccine liquid preparation of carrier protein,
Or (8) prepare vaccine solid granule
Water for injection, sodium chloride and stabilizing agent is added in unit price bacterial polysaccharide protein conjugate stock solution after the purification of step (6) gained, make that the concentration of bacterial polysaccharides is 4 μ g/ml ~ 60 μ g/ml, sodium chloride concentration is 4 ~ 9mg/ml, described stabilizing agent is lactose or sucrose or glycine, make lactose concn be 8 ~ 25mg/ml; Or sucrose concentration is 40 ~ 120mg/ml; Or glycine concentration is 20 ~ 80mg/ml, namely lyophilizing makes with hepatitis B surface antigen is the unit price bacterial polysaccharide protein conjugate vaccine solid granule of carrier protein;
Or (9) prepare the semi-solid agent of vaccine
Water for injection, sodium chloride and excipient is added in unit price bacterial polysaccharide protein conjugate stock solution after the purification of step (6) gained, make that the concentration of bacterial polysaccharides is 4 μ g/ml ~ 60 μ g/ml, sodium chloride concentration is 4 ~ 9mg/ml, described excipient is sodium carboxymethyl cellulose or carbomer, sodium carboxymethyl cellulose concentration is made to be 10 ~ 30mg/ml, or carbomer concentration is 5 ~ 20mg/ml, namely making with hepatitis B surface antigen is the semi-solid agent of unit price bacterial polysaccharide protein conjugate vaccine of carrier protein.
12. 1 kinds is the preparation method of the two valencys above bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen, comprises the following steps:
(1) a kind of bacterial polysaccharides derivant is prepared
Any one bacterial polysaccharides derivant is prepared by step in claim 11 (1) to the method for step (4);
(2) bacterial polysaccharides and protein binding
Be that after the ratio mixing of 1:1 ~ 1:4, with the NaCl of 0.2mol/L, mixed bacterial polysaccharides derivant being dissolved to mixed bacterial polysaccharides derivatives concentration is 2 ~ 20mg/ml respectively in the mass ratio between each bacterial polysaccharides derivant by two or more bacterial polysaccharides derivant; Again in total bacterial polysaccharides derivant: the mass ratio of hepatitis B surface antigen is that the ratio of 1:0.4 ~ 1:2 adds in carrier protein, then, adding carbodiimide to carbodiimide concentration is 0.001 ~ 0.2mol/L, maintaining pH is 5.5 ± 0.5, react 2 ~ 4 hours, obtain the above bacterial polysaccharides derivant of two valencys and carrier protein covalent conjunct agent;
(3) purification
The above bacterial polysaccharides derivant of step (2) gained two valency and carrier protein covalent conjunct agent gel column are carried out purification, collects V 0neighbouring absworption peak, namely aseptic filtration obtains the two valencys above bacterial polysaccharide protein conjugate stock solution after purification;
(4) vaccine liquid preparation is prepared
Water for injection is added in two valencys above bacterial polysaccharide protein conjugate stock solution after the purification of step (3) gained, sodium chloride, phosphate buffer and aluminium adjuvant, the concentration of total bacterial polysaccharides is made to be 8.8 μ g/ml ~ 300 μ g/ml, sodium chloride concentration is 4 ~ 9mg/ml, sodium hydrogen phosphate concentration is 38 ~ 72 μ g/ml, phosphate dihydrogen sodium concentration is 24 ~ 88 μ g/ml, described aluminium adjuvant is aluminium hydroxide or aluminum phosphate, aluminum hydroxide concentration is made to be 0.5 ~ 2.5mg/ml, or aluminium phosphate concentration is 0.5 ~ 2.0mg/ml, namely making with hepatitis B surface antigen is the two valencys above bacterial polysaccharide protein conjugate vaccine liquid preparation of carrier protein, the concentration of described total bacterial polysaccharides is the concentration of each bacterial polysaccharides quality sum,
Or (5) prepare vaccine solid granule
Water for injection, sodium chloride and stabilizing agent is added in two valencys above bacterial polysaccharide protein conjugate stock solution after the purification of step (3) gained, make that the concentration of total bacterial polysaccharides is 8.8 μ g/ml ~ 300 μ g/ml, sodium chloride concentration is 4 ~ 9mg/ml, described stabilizing agent is lactose or sucrose or glycine, make lactose concn be 8 ~ 25mg/ml; Or sucrose concentration is 40 ~ 120mg/ml, or glycine concentration is 20 ~ 80mg/ml, and namely lyophilizing makes with hepatitis B surface antigen is the two valencys above bacterial polysaccharide protein conjugate vaccine solid granule of carrier protein; The concentration of described total bacterial polysaccharides is the concentration of each bacterial polysaccharides quality sum;
Or (6) prepare the semi-solid agent of vaccine
Water for injection, sodium chloride and excipient is added in two valencys above bacterial polysaccharide protein conjugate stock solution after the purification of step (3) gained, make that the concentration of total bacterial polysaccharides is 8.8 μ g/ml ~ 300 μ g/ml, sodium chloride concentration is 4 ~ 9mg/ml, described excipient is sodium carboxymethyl cellulose or carbomer, sodium carboxymethyl cellulose concentration is made to be 10 ~ 30mg/ml, or carbomer concentration is 5 ~ 20mg/ml, namely making with hepatitis B surface antigen is the semi-solid agent of two valencys above bacterial polysaccharide protein conjugate vaccine of carrier protein; The concentration of described total bacterial polysaccharides is the concentration of each bacterial polysaccharides quality sum.
Vaccine provided by the invention, the method by injection, oral, collunarium, external application carries out immunity, for the disease that the microbial disease of serotype cause of disease of preventing bacterial polysaccharides corresponding and hepatitis B virus cause.
Compared with existing conjugate vaccine product, the invention has the beneficial effects as follows:
1, animal experiment shows, vaccine of the present invention can produce the Positive seroconversion rate (Conversion rate more than 85%) of higher bacterial polysaccharides, and the mice efficiency test of hepatitis B surface antigen also very well (Positive seroconversion rate more than 85%) simultaneously.Show that the carrier protein in vaccine of the present invention not only serves effect bacterial polysaccharides being converted into T cell dependence antigen by T-independent antigen, and serve prevention hepatitis B virus being caused to disease simultaneously.Vaccine of the present invention had both expanded the use crowd of vaccine, solved the immunity inoculation problem of 2 years old Infants Below, reached again the effect that a pin is exempted from more.Overcome carrier protein in existing conjugate vaccine and only play the defect of effect bacterial polysaccharides being converted into T cell dependence antigen by T-independent antigen.
2, vaccine of the present invention expands use crowd and the coverage rate of vaccine, decreases the number of times of vaccination crowd vaccinate, decreases the misery of injecting and bringing.
3, bacterial polysaccharides-hepatitis B surface antigen combined vaccine of preparing of preparation method of the present invention, well remains antigenicity and the immunogenicity of bacterial polysaccharide antigen and hepatitis B surface antigen.
Detailed description of the invention
Following embodiment is routine techniques without specified otherwise, and in each embodiment, various reagent, raw material etc. are commercially available prod, and various bacteria culture is all bought from Chinese medicine microbiology DSMZ without specified otherwise.Following embodiment for illustration of the present invention, but is not used in and limits the scope of the invention.
Embodiment 1 ~ embodiment 5 is the preparation of bacterial polysaccharides.Embodiment 6: the preparation of hepatitis B surface antigen.Embodiment 7: the preparation of bacterial polysaccharides-adipic dihydrazide derivant.Embodiment 8 (1) ~ (13): the preparation of bacterial polysaccharides-hepatitis B surface antigen conjugate stock solution; Embodiment 9 (1) ~ (13): the preparation of vaccine product.Embodiment 10: the efficiency assay of vaccine product.Embodiment 11 (1) ~ (4): the safety testing of vaccine product.Embodiment 12: result of the test statistical analysis.
The preparation of embodiment 1 B group streptococcus and streptococcus pneumoniae polysaccharides
(1) ferment: use Ia type B group streptococcus respectively, Ib type B group streptococcus, II type B group streptococcus, type III B group streptococcus, V-type B group streptococcus, 1 type streptococcus pneumoniae, 2 type streptococcus pneumoniae, 3 type streptococcus pneumoniae, 4 type streptococcus pneumoniae, 5 type streptococcus pneumoniae, 6A type streptococcus pneumoniae, 6B type streptococcus pneumoniae, 7F type streptococcus pneumoniae, 8 type streptococcus pneumoniae, 9N type streptococcus pneumoniae, 9V type streptococcus pneumoniae, 10A type streptococcus pneumoniae, 11A type streptococcus pneumoniae, 14 type streptococcus pneumoniae, 15B type streptococcus pneumoniae, 17F type streptococcus pneumoniae, 18C type streptococcus pneumoniae, 19A type streptococcus pneumoniae, 19F type streptococcus pneumoniae, 20 type streptococcus pneumoniae, 22F type streptococcus pneumoniae, 23F type streptococcus pneumoniae or the pneumococcal work seed of 33F type, be inoculated in improvement half synthetic medium respectively, cultivate 4 ~ 12 hours in 35 ~ 37 DEG C of carbon dioxide environments, as culture fluid OD 600when>=2, adding NaTDC to sodium deoxycholate concentration is that 1 ‰ (quality volume fractions) sterilize.
(2) slightly pure: collected by centrifugation supernatant, add ethanol to concentration of alcohol 40% volume fraction, then collected by centrifugation supernatant, then add ethanol to concentration of alcohol 75% volume fraction, then centrifugal collecting precipitation;
(3) consummate: the neutral sodium acetate solution of precipitation 0.3mol/L dissolves, and adds cold phenol extracted several times, removes albumen, collect supernatant, ultrafiltration, adds ethanol to concentration of alcohol 35% volume fraction, collected by centrifugation supernatant, supernatant ultrafiltration, collects backflow end, adds ethanol to concentration of alcohol 75% volume fraction, collecting precipitation, with acetone, washed with diethylether precipitates, drain, namely the Ia type B streptococcal polysaccharide of purification is obtained respectively, Ib type B streptococcal polysaccharide, II type B streptococcal polysaccharide, type III B streptococcal polysaccharide, V-type B streptococcal polysaccharide, 1 type pneumococal polysaccharide, 2 type pneumococal polysaccharides, 3 type pneumococal polysaccharides, 4 type pneumococal polysaccharides, 5 type pneumococal polysaccharides, 6A type pneumococal polysaccharide, 6B type pneumococal polysaccharide, 7F type pneumococal polysaccharide, 8 type pneumococal polysaccharides, 9N type pneumococal polysaccharide, 9V type pneumococal polysaccharide, 10A type pneumococal polysaccharide, 11A type pneumococal polysaccharide, 14 type pneumococal polysaccharides, 15B type pneumococal polysaccharide, 17F type pneumococal polysaccharide, 18C type pneumococal polysaccharide, 19A type pneumococal polysaccharide, 19F type pneumococal polysaccharide, 20 type pneumococal polysaccharides, 22F type pneumococal polysaccharide, 23F type pneumococal polysaccharide, or 33F type pneumococal polysaccharide.
Described improvement half synthetic medium is:
Embodiment 2: the preparation of meningococcal polysacharide
Use the work seed of the scorching coccus of A group, C group, Y group or W135 group's neisseria meningitis respectively, prepare corresponding meningococcal polysacharide respectively as follows:
The work seed of unlatching A group or C group or Y group or the scorching coccus of W135 group's neisseria meningitis is inoculated in the agar culture medium containing 10% (volume fraction) Sanguis caprae seu ovis, cultivate 16 ~ 20 hours in 35 ~ 37 DEG C of carbon dioxide environments, it is qualified that Gram’s staining mirror is picked up, be inoculated in fermentation tank, adopt the comprehensive culture medium culturing of meningococcus improvement half to sterilize by formalin to during the exponential phase later stage.Collected by centrifugation supernatant adds cetyl trimethyl ammonium bromide, fully mixes precipitation; Centrifugal collecting precipitation, add calcium chloride to dissociate, collected by centrifugation supernatant, adding ethanol to concentration of alcohol is 25% volume fraction, hold over night, collected by centrifugation supernatant, adding ethanol to concentration of alcohol is 80% volume fraction, centrifugal collecting precipitation, uses ethanol and washing with acetone respectively, drains and obtain rough polysaccharide.Rough polysaccharide is dissolved in 1/10 (volume fraction) saturated neutral sodium acetate aqueous solution, and with cold phenol extracted several times, collected by centrifugation supernatant, supernatant ultrafiltration, collect backflow end, adding ethanol to concentration of alcohol is 25% volume fraction, hold over night.Collected by centrifugation supernatant, supernatant ultrafiltration, collect backflow end, adding ethanol to concentration of alcohol is 80% volume fraction, centrifugal collecting precipitation, wash respectively with ethanol and acetone, drain, namely obtain the A meningococcal polysaccharide of purification, C meningococcal polysaccharide, Y meningococcal polysaccharide or W135 meningococcal polysaccharide respectively.
Described meningococcus improves half synthetic medium:
The preparation (according to " Chinese Pharmacopoeia " 2010 editions method preparations) of embodiment 3:b type hemophilus influenza polysaccharide
Open b type hemophilus influenza work seed, be inoculated in Hib synthetic medium, holding temperature 37 ± 0.5 DEG C, 10%CO 2concentration cultivates 20 hours, and it is qualified that Gram’s staining mirror is picked up, and is inoculated in fermentation tank, adopts Hib synthetic medium liquid culture to sterilize to during the exponential phase later stage by formalin.The bacterium liquid collected by centrifugation supernatant of formaldehyde sterilization adds cetyl trimethyl ammonium bromide and fully mixes precipitate polysaccharides; Centrifugal collecting precipitation, adds calcium chloride and dissociates, collected by centrifugation supernatant, and adding ethanol to concentration of alcohol is 25% volume fraction, hold over night, collected by centrifugation supernatant, adding ethanol to concentration is 80%, centrifugal collecting precipitation, uses ethanol, washing with acetone precipitate successively, drains and be crude polysaccharides.Crude polysaccharides is dissolved in 1/10 (volume fraction) saturated neutral sodium acetate aqueous solution, and with cold phenol extracting 3-7 time, collected by centrifugation supernatant, supernatant ultrafiltration, collect backflow end, adding ethanol to concentration of alcohol is 25% volume fraction, hold over night.Collected by centrifugation supernatant, supernatant ultrafiltration, collect backflow end, adding ethanol to concentration of alcohol is 75% volume fraction, and centrifugal collecting precipitation, washs respectively with ethanol and acetone, drain, and obtains the b type hemophilus influenza polysaccharide of purification.
Described Hib Synthetical cultivation based formulas is:
Embodiment 4: the preparation (according to " Chinese Pharmacopoeia " 2010 editions method preparations) of Salmonella typhi Vi polysaccharide
Open Salmonella typhi Vi work seed, being inoculated in Salmonella typhi improves in half synthetic medium, holding temperature 35 ~ 37 DEG C, it is qualified that Gram’s staining mirror is picked up, be inoculated in fermentation tank, when 35 ~ 37 DEG C of stir culture are to the exponential phase later stage, adding formalin to formalin concentration is 1.5-2% carry out sterilization 30min.Bacterium liquid collected by centrifugation supernatant after sterilization, adds cetyl trimethyl ammonium bromide and fully mixes precipitate polysaccharides; Centrifugal collecting precipitation, adding calcium chloride to calcium chloride concentration is that 0.5 ~ 1.5mol/L dissociates, collected by centrifugation supernatant, adding ethanol to concentration of alcohol is 25% volume fraction, hold over night, collected by centrifugation supernatant, adding ethanol to concentration of alcohol is 80% volume fraction, centrifugal collecting precipitation, uses ethanol, washing with acetone precipitate successively, drains and be crude polysaccharides.Crude polysaccharides is dissolved in 1/10 (volume fraction) saturated neutral sodium acetate aqueous solution, with cold phenol extracting 3-7 time, collected by centrifugation supernatant, supernatant ultrafiltration, collect backflow end, adding ethanol to concentration of alcohol is 75% volume fraction, centrifugal collecting precipitation, wash respectively with ethanol and acetone, drain, obtain the Salmonella typhi Vi refined polysaccharide of purification.
Described Salmonella typhi improves half synthetic medium:
Embodiment 5: the preparation of A type and Salmonella paratyphi B O-SP (O specific polysaccharide)
Open paratyphosus A bacillus work seed or Salmonella paratyphi B work seed, adopt the comprehensive culture medium culturing of Salmonella typhi improvement half, 35 ~ 37 DEG C are shaken to educate and spend the night, pure bacterium passed examination, turn and be inoculated in fermentation tank, 37 DEG C of deep ventilation stir culture 6-8 are little when the exponential phase later stage, and adding formalin to concentration of formaldehyde is that 2% (volume fraction) carries out sterilization stirring 30min hold over night, collected by centrifugation thalline.Get 200g wet thallus to be fully suspended in 1800ml water for injection, be heated to 68 DEG C, add the phenol 2000ml of 90% of equal-volume 68 DEG C, 68 DEG C are stirred 1 hour, 7300 × g, 10 DEG C, centrifugal 1 hour, phase liquid in collection; In, mend apyrogeneity water for injection (PFW) in lower phase liquid to 2000ml, 68 DEG C stir 1 hour; 7300g, draws upper phase liquid in centrifugal 1 hour, merges phase liquid on twice for 10 DEG C, adds ethanol to concentration of alcohol and is 25% volume fraction, adds sodium acetate (NaAc) to NaAc concentration and be 10mM, add calcium chloride (CaCl 2) to CaCl 2concentration is 2mM, and 2 ~ 8 DEG C are spent the night; 7300 × g, 10 DEG C centrifugal 1 hour, and collect supernatant, adding ethanol to concentration of alcohol is 75% volume fraction, hold over night, 7300 × g, 10 DEG C, centrifugal 1 hour.Collecting precipitation.Precipitate after dissolving with PFW, again carry out hot phenol and carry, phase liquid in collection, ultrafiltration, lyophilizing, be crude product lipopolysaccharide (LPS).Dissolve LPS with 1% glacial acetic acid and become 10mg/ml, water-bath boils 90 minutes, 7300 × g, and 10 DEG C centrifugal 1 hour, collects supernatant, adjusts pH to 6.5 ~ 7.0.Supernatant ultrafiltration, collect backflow end, purify by Sephadex G-75 chromatography, collect the 1st and the 2nd peak, after merging, lyophilizing is paratyphosus A bacillus O-SP or Salmonella paratyphi B O-SP.
Described Salmonella typhi improves half synthetic medium:
Embodiment 6: the preparation of hepatitis B surface antigen solution
Fermentation: get restructuring (Hansenula yeast) hepatitis B strain (purchased from microbe research center, Beijing) 1 (1ml/ props up) and be inoculated in the YNB culture medium (commercialization culture medium) of 500ml, 30 DEG C, 200rpm cultivates 24 hours; At A 600nmmeasure light absorption value under condition, when absorbance reaches 12, by culture transferred species in the YNB culture medium of 5L, in 30 DEG C, 200rpm cultivates 24 hours; A 600nmmeasure light absorption value, when absorbance reaches 12, by culture transferred species to (fermentation tank producer model: Bei Lang D50) in fermentation tank.Adopt two carbon source two-stage fermentation technology to complete restructuring Hansenula yeast high density fermentation and expression, control dissolved oxygen in sweat and be not less than 20%; After cell density reaches 0.50g/ml, enter the abduction delivering stage, add methanol and induce, methanol concentration is not higher than 5g/L.During fermentation ends, cell density is about to 0.60g/ml, and whole sweat carries out 100 hours, results fermentation culture medium.
Slightly pure: the culture of results is in 4 DEG C, and 5000rpm, centrifugal 20 minutes, collects centrifuged deposit and add buffer solution, wash 2 times.By cell suspension in cell breakage liquid, and add polysorbate 20, ball mill smudge cells, cell crashing ratio reaches more than 95%, add concentration 50% (volume fraction) polyethylene glycol 6000, in 2-8 DEG C of effect 10 hours, 4 DEG C, 7000rpm, centrifugal 20 minutes, collects supernatant.Add silica gel solution 4 DEG C of stirring and adsorbing in supernatant after 16 hours, 4 DEG C, 4000rpm, centrifugal 20 minutes, remove supernatant.In precipitation, add silica gel eluent, 56 DEG C of intensification De contamination, 4 DEG C, 4000rpm, centrifugal 15 points, remove precipitation, collect supernatant.
Consummate: the first step, adopt chromatographic media to be Capto adhere column chromatography purification method, ultraviolet monitoring wavelength is 280nm, collects absorbance A 280nmbe greater than the eluting peak of 0.5, eluent is Tris-HCl and sodium chloride buffer system.Second step, by the eluting peak collected, adopt Q Sepharose HP column purification, eluent is 50mmol/L Tris-HCl, 50mmol/L Tris-HCl and 1mol/L sodium chloride mixed liquor (1:1), and monitoring wavelength 280nm, collects absorbance A 280nmbe greater than the eluting peak of 0.5.3rd step, adopts Potassium bromide density zone ultracentrifugation by the refined solution collected, and under room temperature, 20000rpm, centrifugal 24 hours, collects object peak.Finally, adopt column chromatography desalination, chromatographic media is Sepharose 4FF, and monitoring wavelength 280nm, collects absorbance A 280nmbe greater than the eluting peak of 0.2, be concentrated into concentration and be greater than 10mg/ml, through the filter membrane aseptic filtration in 0.22um aperture, be restructuring (Hansenula yeast) hepatitis B surface antigen solution (hereinafter referred to as hepatitis B surface antigen).
Embodiment 7: the preparation of unit price bacterial polysaccharides-adipic dihydrazide derivant
B type hemophilus influenza polysaccharide; Neisseria meningitis scorching pneumoniae serotype group A group, C group, Y group or W135 group's polysaccharide; Salmonella typhi Vi type polysaccharide; B group streptococcus Ia, Ib, II, III or V-type; Pneumococcus serotypes 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F or 33F type polysaccharide; The preparation of each unit price bacterial polysaccharides-adipic dihydrazide derivant of the B-mode polysaccharide of bacillus paratyphosus A type, bacillus paratyphosus is prepared respectively as follows:
(1) bacterial polysaccharides dissolves: take bacterial polysaccharides 200mg, be dissolved as 5mg/ml by the NaCl solution of 0.2mol/L,
(2) activation of bacterial polysaccharides: the Bromine cyanide. adding 100mg in the bacterial polysaccharides solution that step (1) is dissolved, control pH10.5 ± 0.5 reaction 30min, obtains the bacterial polysaccharides of activation;
(3) bacterial polysaccharides derives: 1, the 6-adipic dihydrazide adding 800mg in the bacterial polysaccharides of the activation of step (2) gained, maintains pH8.5 ± 0.5 reaction 60min and obtains bacterial polysaccharides-adipic dihydrazide derivant;
(4) ultrafiltration of bacterial polysaccharides derivant: the ultrafilter membrane ultrafiltration of bacterial polysaccharides-adipic dihydrazide derivant 30KD that step (3) is obtained, namely lyophilizing obtains following unit price bacterial polysaccharides-adipic dihydrazide derivant respectively:
Ia type B streptococcal polysaccharide-adipic dihydrazide derivant.Ib type B streptococcal polysaccharide-adipic dihydrazide derivant.II type B streptococcal polysaccharide-adipic dihydrazide derivant.Type III B streptococcal polysaccharide-adipic dihydrazide derivant.V-type B streptococcal polysaccharide-adipic dihydrazide derivant.1 type pneumococal polysaccharide-adipic dihydrazide derivant.2 type pneumococal polysaccharide-adipic dihydrazide derivants.3 type pneumococal polysaccharide-adipic dihydrazide derivants.4 type pneumococal polysaccharide-adipic dihydrazide derivants.5 type pneumococal polysaccharide-adipic dihydrazide derivants.6A type pneumococal polysaccharide-adipic dihydrazide derivant.6B type pneumococal polysaccharide-adipic dihydrazide derivant.7F type pneumococal polysaccharide-adipic dihydrazide derivant.8 type pneumococal polysaccharide-adipic dihydrazide derivants.9N type pneumococal polysaccharide-adipic dihydrazide derivant.9V type pneumococal polysaccharide-adipic dihydrazide derivant.10A type pneumococal polysaccharide-adipic dihydrazide derivant.11A type pneumococal polysaccharide-adipic dihydrazide derivant.14 type pneumococal polysaccharide-adipic dihydrazide derivants.15B type pneumococal polysaccharide-adipic dihydrazide derivant.17F type pneumococal polysaccharide-adipic dihydrazide derivant.18C type pneumococal polysaccharide-adipic dihydrazide derivant.19A type pneumococal polysaccharide-adipic dihydrazide derivant.19F type pneumococal polysaccharide-adipic dihydrazide derivant.20 type pneumococal polysaccharide-adipic dihydrazide derivants.22F type pneumococal polysaccharide-adipic dihydrazide derivant.23F type pneumococal polysaccharide-adipic dihydrazide derivant.33F type pneumococal polysaccharide-adipic dihydrazide derivant.B type hemophilus influenza polysaccharide-adipic dihydrazide derivant.A meningococcus group polysaccharide-adipic dihydrazide derivant.C group meningitis cocci group polysaccharide-adipic dihydrazide derivant.Y group meningitis cocci group polysaccharide-adipic dihydrazide derivant.W135 group meningitis cocci group polysaccharide-adipic dihydrazide derivant.Salmonella typhi Vi type polysaccharide-adipic dihydrazide derivant.Paratyphosus A bacillus O-SP-adipic dihydrazide derivant.Or Salmonella paratyphi B O-SP-adipic dihydrazide derivant.
The preparation of embodiment 8 bacterial polysaccharideses-hepatitis B surface antigen conjugate stock solution
(1) preparation of unit price bacterial polysaccharides-hepatitis B surface antigen conjugate stock solution
Each unit price bacterial polysaccharides-adipic dihydrazide derivant that embodiment 7 is prepared respectively prepares unit price bacterial polysaccharides-hepatitis B surface antigen conjugate stock solution respectively as follows:
Take unit price bacterial polysaccharides-adipic dihydrazide derivant 150mg, be dissolved in the NaCl of 15ml 0.2mol/L, add hepatitis B surface antigen solution 15ml (hepatitis B surface antigen concentration is 10mg/ml) prepared by embodiment 6, then adding carbodiimide (EDAC) to EDAC concentration is 0.1mol/L, maintain pH5.5 ± 0.5 (2 ~ 8 DEG C) reaction 4 hours, obtain unit price bacterial polysaccharides derivant and carrier protein covalent conjunct agent, then carry out purification with gel column, collect V 0neighbouring absworption peak, namely aseptic filtration obtains following unit price bacterial polysaccharides-hepatitis B surface antigen conjugate stock solution respectively:
B type hemophilus influenza polysaccharide-hepatitis B surface antigen conjugate stock solution, A meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, C meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, Y meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, W135 meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, salmonella typhi Vi polysaccharide-hepatitis B surface antigen conjugate stock solution, Ia type B streptococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, Ib type B streptococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, II type B streptococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, type III B streptococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, V-type B group streptococcus type polysaccharide-hepatitis B surface antigen conjugate stock solution, 1 type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 2 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 3 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 4 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 5 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 6A type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 6B type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 7F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 8 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 9N type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 9V type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 10A type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 11A type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 14 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution.15B type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 17F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 18C type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 19A type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 19F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 20 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 22F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 23F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 33F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, paratyphosus A bacillus O-SP-hepatitis B surface antigen conjugate stock solution or Salmonella paratyphi B O-SP-hepatitis B surface antigen conjugate stock solution.
The preparation of (2) 13 valency pneumococal polysaccharides-hepatitis B surface antigen covalent conjunct agent stock solution
Take the 1 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg prepared by embodiment 7 respectively, 3 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg, 4 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg, 5 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg, 6A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg, 6B type pneumococal polysaccharide-adipic dihydrazide derivant 20mg, 7F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg, 9V type pneumococal polysaccharide-adipic dihydrazide derivant 10mg, 14 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg, 18C type pneumococal polysaccharide-adipic dihydrazide derivant 10mg, 19A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg, 19F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg, with 23F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg, by various polysaccharide derivates mixed dissolution in the NaCl of 14ml 0.2mol/L, add hepatitis B surface antigen 15.4ml (hepatitis B surface antigen concentration is 10mg/ml) prepared by embodiment 6, then carbodiimide (EDAC) 0.5368g is added, maintain pH5.5 ± 0.5 maintenance reaction and obtain 13 valency pneumococal polysaccharide-adipic dihydrazide derivant and carrier protein covalent conjunct agents in 4 hours, then purification is carried out with gel column, collect V 0neighbouring absworption peak, namely aseptic filtration obtains 13 valency pneumococal polysaccharides-hepatitis B surface antigen covalent conjunct agent stock solution.
The preparation of (3) 15 valency pneumococal polysaccharides-hepatitis B surface antigen covalent conjunct agent stock solution
Take the 1 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg prepared by embodiment 7 respectively; 3 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 4 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 5 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 6A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 6B type pneumococal polysaccharide-adipic dihydrazide derivant 20mg; 7F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 9V type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 14 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 18C type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 19A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 19F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 22F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 23F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 33F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; Be in the NaCl of 0.2mol/L in 16ml concentration by various polysaccharide derivates mixed dissolution, add hepatitis B surface antigen 17.6ml (hepatitis B surface antigen concentration is 10mg/ml) prepared by embodiment 6, add carbodiimide (EDAC) 0.6134g, maintain pH5.5 ± 0.5 maintenance reaction and obtain 15 valency pneumococal polysaccharide-adipic dihydrazide derivant and carrier protein covalent conjunct agents in 4 hours, then carry out purification with gel column, collect V 0neighbouring absworption peak, namely aseptic filtration obtains 15 valency pneumococal polysaccharides-hepatitis B surface antigen covalent conjunct agent stock solution.
The preparation of (4) 23 valency pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution
Take the 1 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg prepared by embodiment 7 respectively; 2 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 3 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 4 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 5 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 6A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 6B type pneumococal polysaccharide-adipic dihydrazide derivant 20mg; 7F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 8 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 9N type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 9V type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 10A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 11A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 14 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 15B type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 17F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 18C type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 19A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 19F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 20 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 22F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 23F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 33F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; ; To be dissolved in the NaCl of 24ml 0.2mol/L after various polysaccharide derivates mixing, add hepatitis B surface antigen 26.4ml (hepatitis B surface antigen concentration is 10mg/ml) prepared by embodiment 6, add carbodiimide (EDAC) 0.9202g, maintain pH5.5 ± 0.5 maintenance reaction and obtain 23 valency pneumococal polysaccharide-adipic dihydrazide derivant and carrier protein covalent conjunct agents in 4 hours, then carry out purification with gel column, collect V 0neighbouring absworption peak, namely aseptic filtration obtains 23 valency pneumococal polysaccharides-hepatitis B surface antigen covalent conjunct agent stock solution.
(5) preparation of ACYW135 meningococcal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution
Take the A meningococcal polysaccharide-adipic dihydrazide derivant 30mg prepared by embodiment 7 respectively, C meningococcal polysaccharide-adipic dihydrazide derivant 30mg, Y meningococcal polysaccharide-adipic dihydrazide derivant 30mg, W135 meningococcal polysaccharide-adipic dihydrazide derivant 30mg will be dissolved in the NaCl of 12ml 0.2mol/L after each group's polysaccharide derivates mixing, add hepatitis B surface antigen 12.0ml (hepatitis B surface antigen concentration is 10mg/ml) prepared by embodiment 6, add carbodiimide (EDAC) 0.4601g, maintain pH5.5 ± 0.5 maintenance reaction and obtain 4 valency ACYW135 meningococcal polysaccharide-adipic dihydrazide derivant and carrier protein covalent conjunct agents in 4 hours, then purification is carried out with gel column, collect V 0neighbouring absworption peak, namely aseptic filtration obtains ACYW135 meningococcal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution.
(6) preparation of b type hemophilus influenza polysaccharide 13 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution
Take the b type hemophilus influenza polysaccharide-adipic dihydrazide derivant 40mg, the 1 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg that prepare by embodiment 7 respectively; 3 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 4 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 5 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 6A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 6B type pneumococal polysaccharide-adipic dihydrazide derivant 20mg; 7F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 9V type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 14 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 18C type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 19A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 19F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 23F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; To be dissolved in the NaCl of 18ml 0.2mol/L after various polysaccharide derivates mixing, add hepatitis B surface antigen 18.0ml (hepatitis B surface antigen concentration is 10mg/ml) prepared by embodiment 6, add carbodiimide (EDAC) 0.4601g, maintain pH5.5 ± 0.5 and maintain reaction 4 hours, then carry out purification with gel column, collect V 0neighbouring absworption peak, namely aseptic filtration obtains b type hemophilus influenza 13 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution.
(7) preparation of b type hemophilus influenza polysaccharide 15 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution
Take respectively and prepare b type hemophilus influenza polysaccharide-adipic dihydrazide derivant 40mg by embodiment 7,1 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 3 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 4 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 5 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 6A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 6B type pneumococal polysaccharide-adipic dihydrazide derivant 20mg; 7F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 9V type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 14 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 18C type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 19A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 19F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 22F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 23F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 33F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; To be dissolved in the NaCl of 20ml 0.2mol/L after various polysaccharide derivates mixing, add hepatitis B surface antigen 20.0ml (hepatitis B surface antigen concentration is 10mg/ml) prepared by embodiment 6, add carbodiimide (EDAC) 0.8134g, maintain pH5.5 ± 0.5 and maintain reaction 4 hours, then carry out purification with gel column, collect V 0neighbouring absworption peak, namely aseptic filtration obtains b type hemophilus influenza 15 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution.
(8) preparation of b type hemophilus influenza polysaccharide 23 valency pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution
Take respectively and prepare b type hemophilus influenza polysaccharide-adipic dihydrazide derivant 40mg by embodiment 7,1 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 2 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 3 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 4 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 5 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 6A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 6B type pneumococal polysaccharide-adipic dihydrazide derivant 20mg; 7F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 8 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 9N type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 9V type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 10A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 11A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 14 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 15B type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 17F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 18C type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 19A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 19F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 20 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 22F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 23F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 33F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; To be dissolved in the NaCl of 28ml 0.2mol/L after various polysaccharide derivates mixing, add hepatitis B surface antigen 28.0ml (hepatitis B surface antigen concentration is 10mg/ml) prepared by embodiment 6, add carbodiimide (EDAC) 1.6202g, maintain pH5.5 ± 0.5 and maintain reaction 4 hours, then carry out purification with gel column, collect V 0neighbouring absworption peak, namely aseptic filtration obtains b type hemophilus influenza pneumonia coccus 23 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution.
(9) preparation of b type hemophilus influenza Polysaccharide A CYW135 meningococcal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution
Take respectively and prepare b type hemophilus influenza polysaccharide-adipic dihydrazide derivant 40mg by embodiment 7, A meningococcal polysaccharide-adipic dihydrazide derivant 20mg, C meningococcal polysaccharide-adipic dihydrazide derivant 20mg, Y meningococcal polysaccharide-adipic dihydrazide derivant 20mg, W135 meningococcal polysaccharide-adipic dihydrazide derivant 20mg, to be dissolved in the NaCl of 12ml 0.2mol/L after each polysaccharide derivates mixing, add hepatitis B surface antigen 12.0ml (hepatitis B surface antigen concentration is 10mg/ml) prepared by embodiment 6, add carbodiimide (EDAC) 0.4601g, maintain pH5.5 ± 0.5 and maintain reaction 4 hours, then purification is carried out with gel column, collect V 0neighbouring absworption peak, namely aseptic filtration obtains b type hemophilus influenza Polysaccharide A CYW135 meningococcal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution.
(10) preparation of ACYW135 group meningitis cocci 13 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution
Take the A meningococcal polysaccharide-adipic dihydrazide derivant 20mg prepared by embodiment 7 respectively, C meningococcal polysaccharide-adipic dihydrazide derivant 20mg, Y meningococcal polysaccharide-adipic dihydrazide derivant 20mg, W135 meningococcal polysaccharide-adipic dihydrazide derivant 20mg, 1 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 3 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 4 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 5 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 6A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 6B type pneumococal polysaccharide-adipic dihydrazide derivant 20mg; 7F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 9V type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 14 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 18C type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 19A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 19F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 23F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; To be dissolved in the NaCl of 22ml 0.2mol/L after each polysaccharide derivates mixing, add hepatitis B surface antigen 22.0ml (hepatitis B surface antigen concentration is 10mg/ml) prepared by embodiment 6, add carbodiimide (EDAC) 0.9601g, maintain pH5.5 ± 0.5 and maintain reaction 4 hours, then carry out purification with gel column, collect V 0neighbouring absworption peak, namely aseptic filtration obtains ACYW135 group meningitis cocci 13 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution.
(11) preparation of ACYW135 group meningitis cocci 15 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution
Take the A meningococcal polysaccharide-adipic dihydrazide derivant 20mg prepared by embodiment 7 respectively, C meningococcal polysaccharide-adipic dihydrazide derivant 20mg, Y meningococcal polysaccharide-adipic dihydrazide derivant 20mg, W135 meningococcal polysaccharide-adipic dihydrazide derivant 20mg, 1 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 3 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 4 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 5 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 6A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 6B type pneumococal polysaccharide-adipic dihydrazide derivant 20mg; 7F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 9V type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 14 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 18C type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 19A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 19F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 22F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 23F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 33F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; To be dissolved in the NaCl of 24ml 0.2mol/L after each polysaccharide derivates mixing, add hepatitis B surface antigen 24.0ml (hepatitis B surface antigen concentration is 10mg/ml) prepared by embodiment 6, add carbodiimide (EDAC) 0.8601g, maintain pH5.5 ± 0.5 and maintain reaction 4 hours, then carry out purification with gel column, collect V 0neighbouring absworption peak, namely aseptic filtration obtains ACYW135 group meningitis cocci 15 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution.
(12) preparation of ACYW135 group meningitis cocci 23 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution
Take the A meningococcal polysaccharide-adipic dihydrazide derivant 20mg prepared by embodiment 7 respectively, C meningococcal polysaccharide-adipic dihydrazide derivant 20mg, Y meningococcal polysaccharide-adipic dihydrazide derivant 20mg, W135 meningococcal polysaccharide-adipic dihydrazide derivant 20mg, 1 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 2 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 3 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 4 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 5 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 6A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 6B type pneumococal polysaccharide-adipic dihydrazide derivant 20mg; 7F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 8 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 9N type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 9V type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 10A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 11A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 14 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 15B type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 17F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 18C type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 19A type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 19F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 20 type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 22F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 23F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; 33F type pneumococal polysaccharide-adipic dihydrazide derivant 10mg; By each polysaccharide derivates mixed dissolution in the NaCl of 32ml0.2mol/L, add hepatitis B surface antigen 32.0ml (hepatitis B surface antigen concentration is 10mg/ml) prepared by embodiment 6, add carbodiimide (EDAC) 1.1601g, maintain pH5.5 ± 0.5 and maintain reaction 4 hours, then carry out purification with gel column, collect V 0neighbouring absworption peak, namely aseptic filtration obtains ACYW135 group meningitis cocci 23 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution.
(13) preparation of Salmonella typhoid Vi, paratyphoid A, paratyphoid B polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution
Take the typhoid Vi polysaccharide-adipic dihydrazide derivant 50mg prepared by embodiment 7 respectively, paratyphosus A bacillus O-SP-adipic dihydrazide derivant 50mg, Salmonella paratyphi B O-SP-adipic dihydrazide derivant 50mg, be dissolved in the NaCl of 15ml 0.2mol/L after mixing, add hepatitis B surface antigen 16.5ml (hepatitis B surface antigen concentration is 10mg/ml) prepared by embodiment 6, add carbodiimide (EDAC) 0.6601g, maintain pH5.5 ± 0.5 and maintain reaction 4 hours, then carry out purification with gel column, collect V 0neighbouring absworption peak, namely aseptic filtration obtains Salmonella typhoid Vi, paratyphoid A, paratyphoid B polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution.
Embodiment 9: the preparation of vaccine product
(1) unit price bacterial polysaccharides-hepatitis B surface antigen combined vaccine goods (liquid preparation) preparation
C meningococcal polysaccharide-hepatitis B surface antigen knot conjugate the stock solution of gained is prepared respectively in embodiment 8 (1), Y meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, W135 meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, 1 type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 2 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 3 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 4 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 5 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 6A type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 6B type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 7F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 8 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 9N type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 9V type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 10A type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 11A type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 14 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 15B type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 17F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 18C type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 19A type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 19F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 20 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 22F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 23F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 33F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, or add water for injection in salmonella typhi Vi polysaccharide-hepatitis B surface antigen conjugate stock solution, sodium chloride, phosphate buffer and aluminum phosphate, make the concentration of bacterial polysaccharides be 25 μ g/ml, sodium chloride concentration is 8.5mg/ml, sodium hydrogen phosphate concentration is 70 μ g/ml, phosphate dihydrogen sodium concentration is 22 μ g/ml, and aluminium phosphate concentration is 0.6mg/ml, namely makes the unit price bacterial polysaccharide protein combined vaccine liquid preparation that following hepatitis B surface antigen is carrier protein respectively:
C meningococcal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, Y meningococcal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, W135 meningococcal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, 1 type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, 2 type pneumococal polysaccharides-hepatitis B surface antigen combined vaccine liquid preparation, 3 type pneumococal polysaccharides-hepatitis B surface antigen combined vaccine liquid preparation, 4 type pneumococal polysaccharides-hepatitis B surface antigen combined vaccine liquid preparation, 5 type pneumococal polysaccharides-hepatitis B surface antigen combined vaccine liquid preparation, 6A type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, 6B type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, 7F type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, 8 type pneumococal polysaccharides-hepatitis B surface antigen combined vaccine liquid preparation, 9N type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, 9V type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, 10A type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, 11A type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, 14 type pneumococal polysaccharides-hepatitis B surface antigen combined vaccine liquid preparation, 15B type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, 17F type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, 18C type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, 19A type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, 19F type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, 20 type pneumococal polysaccharides-hepatitis B surface antigen combined vaccine liquid preparation, 22F type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, 23F type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, 33F type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation, or salmonella typhi Vi polysaccharide-hepatitis B surface antigen combined vaccine liquid preparation.
(2) unit price bacterial polysaccharides-hepatitis B surface antigen combined vaccine goods (solid particle agent) preparation
The b type hemophilus influenza polysaccharide-hepatitis B surface antigen conjugate stock solution of gained is prepared respectively in embodiment 8 (1), A meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, Ib type B streptococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, II type B streptococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, type III B streptococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, V-type B group streptococcus type polysaccharide-hepatitis B surface antigen conjugate stock solution, paratyphosus A bacillus O-SP-hepatitis B surface antigen conjugate stock solution, or add water for injection in Salmonella paratyphi B O-SP-hepatitis B surface antigen conjugate stock solution, sodium chloride, lactose and glycine, the concentration of bacterial polysaccharides is made to be 25 μ g/ml, sodium chloride concentration is 8.5mg/ml, lactose concn is 20mg/ml, glycine concentration is 25mg/ml, namely lyophilizing makes the unit price bacterial polysaccharide protein combined vaccine solid particle agent that following hepatitis B surface antigen is carrier protein respectively:
B type hemophilus influenza polysaccharide-hepatitis B surface antigen combined vaccine solid particle preparation, A meningococcal polysaccharide-hepatitis B surface antigen combined vaccine solid particle preparation, Ib type B streptococcal polysaccharide-hepatitis B surface antigen combined vaccine solid particle preparation, II type B streptococcal polysaccharide-hepatitis B surface antigen combined vaccine solid particle preparation, type III B streptococcal polysaccharide-hepatitis B surface antigen combined vaccine solid particle preparation, V-type B group streptococcus type polysaccharide-hepatitis B surface antigen combined vaccine solid particle preparation, paratyphosus A bacillus O-SP-hepatitis B surface antigen combined vaccine solid particle preparation, or Salmonella paratyphi B O-SP-hepatitis B surface antigen combined vaccine solid particle preparation.
(3) unit price bacterial polysaccharides-hepatitis B surface antigen combined vaccine goods (semi-solid agent) preparation
Gained b type hemophilus influenza polysaccharide-hepatitis B surface antigen conjugate stock solution is prepared respectively in embodiment 8 (1), 1 type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 2 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 3 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 4 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 5 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 6A type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 6B type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 7F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 8 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 9N type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 9V type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, water for injection is added in 10A type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, sodium chloride and carbomer, make the concentration of bacterial polysaccharides be 40 μ g/ml, sodium chloride concentration is 8.5mg/ml, carbomer concentration is 15mg/ml, namely makes the unit price bacterial polysaccharide protein combined vaccine semi-solid preparation that following hepatitis B surface antigen is carrier protein respectively:
B type hemophilus influenza polysaccharide-hepatitis B surface antigen combined vaccine semi-solid preparation.1 type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine semi-solid preparation.2 type pneumococal polysaccharides-hepatitis B surface antigen combined vaccine semi-solid preparation.3 type pneumococal polysaccharides-hepatitis B surface antigen combined vaccine semi-solid preparation.4 type pneumococal polysaccharides-hepatitis B surface antigen combined vaccine semi-solid preparation.5 type pneumococal polysaccharides-hepatitis B surface antigen combined vaccine semi-solid preparation.6A type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine semi-solid preparation.6B type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine semi-solid preparation.7F type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine semi-solid preparation.8 type pneumococal polysaccharides-hepatitis B surface antigen combined vaccine semi-solid preparation.9N type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine semi-solid preparation.9V type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine semi-solid preparation.10A type pneumococal polysaccharide-hepatitis B surface antigen combined vaccine semi-solid preparation.
(4) 13 valency pneumococal polysaccharides-hepatitis B surface antigen combined vaccine goods (liquid preparation) preparation
Get the 1 type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution executed example 8 (1) and prepare gained respectively, 3 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 4 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 5 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 6A type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 6B type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 7F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 9V type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 14 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 18C type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 19A type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 19F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, after each appropriate mixing of 23F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, add Aluminium phosphate adjuvant, sodium chloride, sodium hydrogen phosphate, sodium dihydrogen phosphate, water for injection, makes into every milliliter containing 1 type, 3 types, 4 types, 5 types, 6A type, 7F type, 9V type, 14 types, 18C type, 19A type, 19F type, each 4.4 micrograms of 23F type pneumococal polysaccharide, 6B type pneumococal polysaccharide 8.8 microgram, sodium chloride concentration is 8.5 milligrams, aluminum phosphate 1 milligram, sodium hydrogen phosphate 38 microgram, namely the even suspension of sodium dihydrogen phosphate 88 microgram obtains 13 valency pneumococal polysaccharide-hepatitis B surface antigen combined vaccines.
(5) 15 valency pneumococal polysaccharides-hepatitis B surface antigen combined vaccine goods (liquid preparation) preparation
Get the 1 type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution executed example 8 (1) and prepare gained respectively, 3 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 4 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 5 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 6A type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 6B type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 7F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 9V type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 14 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 18C type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 19A type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 19F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 22F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution 23F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, after each appropriate mixing of 33F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, add Aluminium phosphate adjuvant, sodium chloride, sodium hydrogen phosphate, sodium dihydrogen phosphate, water for injection, makes into every milliliter containing 6B type pneumococal polysaccharide 8.8 microgram, each 4.4 micrograms of all the other 14 type pneumococal polysaccharides, sodium chloride concentration is 8.5 milligrams, aluminum phosphate 1 milligram, sodium hydrogen phosphate 38 microgram, the even suspension of sodium dihydrogen phosphate 88 microgram, obtains 15 valency pneumococal polysaccharide-hepatitis B surface antigen combined vaccines.
(6) 23 valency pneumococal polysaccharides-hepatitis B surface antigen combined vaccine goods (liquid preparation) preparation
Get the 1 type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution executed example 8 (1) and prepare gained respectively, 2 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 3 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 4 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 5 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 6A type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 6B type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 7F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 8 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 9N type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 9V type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 10A type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 11A type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 14 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 15B type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 17F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 18C type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 19A type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 19F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 20 type pneumococal polysaccharides-hepatitis B surface antigen conjugate stock solution, 22F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, 23F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution, after 33F type pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution mixes in right amount, add Aluminium phosphate adjuvant, sodium chloride, sodium hydrogen phosphate, sodium dihydrogen phosphate, water for injection, make into every milliliter containing 6B type pneumococal polysaccharide 8.8 microgram, each 4.4 micrograms of all the other 22 type pneumococal polysaccharides, sodium chloride concentration is 8.5 milligrams, aluminum phosphate 1 milligram, sodium hydrogen phosphate 38 microgram, the even suspension of sodium dihydrogen phosphate 88 microgram, obtain 23 valency pneumococal polysaccharide-hepatitis B surface antigen combined vaccines.
(7) ACYW135 meningococcal polysaccharide-hepatitis B surface antigen combined vaccine goods (solid particle agent) preparation
Get respectively and execute after example 8 (1) prepares the A meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution of gained, C meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, Y meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, each appropriate mixing of W135 meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, add sodium chloride, lactose, water for injection, makes into every milliliter of homogeneous solution containing each 10 micrograms of A, C, Y, W135 meningococcal polysaccharide, 8.5 milligrams, sodium chloride, lactose 25 milligrams; Every bottle of subpackage 0.6 milliliter; Vacuum lyophilization, obtains the lyophilized products that moisture is no more than 3%, obtains ACYW135 meningococcal polysaccharide-hepatitis B surface antigen combined vaccine.
(8) b type hemophilus influenza 13 valency pneumococal polysaccharide-hepatitis B surface antigen covalent bond vaccine product (liquid preparation) preparation
B type hemophilus influenza 13 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution that Example 8 (6) prepares gained is appropriate, add Aluminium phosphate adjuvant, sodium chloride, sodium hydrogen phosphate, sodium dihydrogen phosphate, water for injection, make into every milliliter containing b type hemophilus influenza polysaccharide 20 microgram, 6B type pneumococal polysaccharide 8.8 microgram, each 4.4 micrograms of all the other 12 type pneumococal polysaccharides, 8.5 milligrams, sodium chloride, aluminum phosphate 1 milligram, sodium hydrogen phosphate 38 microgram, the even suspension of sodium dihydrogen phosphate 88 microgram, obtain b type hemophilus influenza 13 valency pneumococal polysaccharide-hepatitis B surface antigen covalent bond vaccine.
(9) b type hemophilus influenza 15 valency pneumococal polysaccharide-hepatitis B surface antigen covalent bond vaccine product (liquid preparation) preparation
B type hemophilus influenza 15 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution that Example 8 (7) prepares gained is in right amount each, add sodium chloride, sodium hydrogen phosphate, sodium dihydrogen phosphate, water for injection, make into every milliliter of homogeneous solution containing b type hemophilus influenza polysaccharide 20 microgram, 6B type pneumococal polysaccharide 10 microgram, each 5 micrograms of all the other 14 type pneumococal polysaccharides, 8.5 milligrams, sodium chloride, sodium hydrogen phosphate 38 microgram, sodium dihydrogen phosphate 88 microgram; Obtain b type hemophilus influenza 15 valency pneumococal polysaccharide-hepatitis B surface antigen covalent bond vaccine.
(10) b type hemophilus influenza 23 valency pneumococal polysaccharide-hepatitis B surface antigen covalent bond vaccine product (semi-solid agent) preparation
B type hemophilus influenza 23 valency pneumococal polysaccharide-hepatitis B surface antigen conjugate stock solution that Example 8 (8) prepares gained is in right amount each, add carbomer 910, sodium chloride, water for injection, make into every milliliter containing b type hemophilus influenza polysaccharide 20 microgram, 6B type pneumococal polysaccharide 10 microgram, each 5 micrograms of all the other 22 type pneumococal polysaccharides, carbomer 91010 milligrams, 8.5 milligrams, sodium chloride, sodium hydroxide adjust ph to 6.5 is added in stirring, obtain homogeneous transparent gel, obtain b type hemophilus influenza 23 valency pneumococal polysaccharide-hepatitis B surface antigen covalent bond vaccine.
(11) b type hemophilus influenza, ACYW135 meningococcal polysaccharide-hepatitis B surface antigen combined vaccine goods (solid particle agent) preparation
Example 8 (1) prepares the A meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution of gained respectively, C meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, Y meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, W135 meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, b type hemophilus influenza polysaccharide-hepatitis B surface antigen conjugate stock solution is in right amount each, add sodium chloride, lactose, water for injection, make into every milliliter containing A, C, Y, each 10 micrograms of W135 meningococcal polysaccharide, b type hemophilus influenza polysaccharide 20 microgram, 8.5 milligrams, sodium chloride, the homogeneous solution that lactose is 25 milligrams, vacuum lyophilization, obtains the lyophilized products that moisture is no more than 3%, obtain b type hemophilus influenza, ACYW135 meningococcal polysaccharide-hepatitis B surface antigen combined vaccine.
(12) ACYW135 group meningitis cocci 13 valency pneumococal polysaccharide-hepatitis B surface antigen combined vaccine goods (liquid preparation) preparation
Example 8 (1) prepares the A meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution of gained respectively, C meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, Y meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, W135 meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, 13 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solutions prepared by embodiment 8 (2) are in right amount each, add sodium chloride, sodium hydrogen phosphate, sodium dihydrogen phosphate, water for injection, make into every milliliter containing A, C, Y, each 20 micrograms of W135 meningococcal polysaccharide, 6B type pneumococal polysaccharide 10 microgram, each 5 micrograms of all the other 12 type pneumococal polysaccharides, 8.5 milligrams, sodium chloride, sodium hydrogen phosphate 38 microgram, the homogeneous solution of sodium dihydrogen phosphate 88 microgram, obtain ACYW135 group meningitis cocci 13 valency pneumococal polysaccharide-hepatitis B surface antigen combined vaccine.
(13) ACYW135 group meningitis cocci 15 valency pneumococal polysaccharide-hepatitis B surface antigen combined vaccine goods (semi-solid agent) preparation
Example 8 (1) prepares the A meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution of gained respectively, C meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, Y meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, W135 meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, 15 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solutions prepared by embodiment 8 (3) are in right amount each, add carbomer 910, sodium chloride, water for injection, make into every milliliter containing A, C, Y, each 20 micrograms of W135 meningococcal polysaccharide, 6B type pneumococal polysaccharide 10 microgram, each 5 micrograms of all the other 14 type pneumococal polysaccharides, carbomer 91010 milligrams, 8.5 milligrams, sodium chloride, sodium hydroxide adjust ph to 6.5 is added in stirring, obtain homogeneous transparent gel, obtain ACYW135 group meningitis cocci 15 valency pneumococal polysaccharide-hepatitis B surface antigen combined vaccine.
(14) ACYW135 group meningitis cocci 23 valency pneumococal polysaccharide-hepatitis B surface antigen combined vaccine goods (solid particle agent) preparation
Example 8 (1) prepares the A meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution of gained respectively, C meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, Y meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, W135 meningococcal polysaccharide-hepatitis B surface antigen conjugate stock solution, 23 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solutions prepared by embodiment 8 (4) are in right amount each, add sodium chloride, lactose, water for injection, make into every milliliter containing A, C, Y, each 10 micrograms of W135 meningococcal polysaccharide, each 10 micrograms of pneumococal polysaccharide of 23 types, 8.5 milligrams, sodium chloride, the homogeneous solution that lactose is 25 milligrams, vacuum lyophilization, obtains the lyophilized products that moisture is no more than 3%, obtain ACYW135 group meningitis cocci 23 valency pneumococal polysaccharide-hepatitis B surface antigen combined vaccine.
(15) Salmonella typhoid Vi, paratyphoid A, paratyphoid B polysaccharide-hepatitis B surface antigen combined vaccine goods (solid particle agent) preparation
Unit price typhoid Vi polysaccharide-hepatitis B surface antigen conjugate stock solution prepared by difference Example 8 (1), unit price paratyphosus A bacillus O-SP-hepatitis B surface antigen conjugate stock solution, after each appropriate mixing of paratyphoid B O-SP-hepatitis B surface antigen conjugate stock solution, add glycine, water for injection, make into every milliliter containing typhoid Vi polysaccharide 40 microgram, first paratyphoid fever polysaccharide, each 50 micrograms of paratyphoid B polysaccharide, the homogeneous solution that glycine is 20 milligrams, lyophilisation is no more than 3% to moisture, obtain typhoid Vi polysaccharide-hepatitis B surface antigen conjugate, paratyphoid A polysaccharide-hepatitis B surface antigen conjugate, paratyphoid B polysaccharide-hepatitis B surface antigen conjugate lyophilized preparation.
The efficiency assay of embodiment 10 vaccine product
(1) polysaccharide efficacy test in vaccine product
The vaccine preparation of preparation in embodiment 9 (1) ~ (13), carries out polysaccharide efficacy test in vaccine product by following method respectively.
Female BAl BIc/c mice 120; Divide 5 groups, wherein sample sets often organizes 20, and negative control (normal saline) organizes 40.Every mice femoribus internus subcutaneous injection sample solution 0.2ml (containing polysaccharide antigen 2.5ug).Once, within the 21st day, 35 days, each treated animal eye socket adopts whole blood in each immunity in 0th day, 14 days, 28 days, 37 degree of water-baths 30 minutes, and 4 degree of placements 30 minutes, the centrifugal 10min of 5000rpm, collects serum.Conversion rate and the antibody level of serum of each serum sample is detected by ELISA method
The efficacy test of hepatitis B surface antigen in vaccine product
The vaccine preparation of preparation in embodiment 9 (1) ~ (13), carries out the efficacy test of hepatitis B surface antigen in vaccine product respectively by following method
Female BAl BIc/c mice, 140, divide 6 groups, wherein sample sets often organizes 20, and negative control (normal saline) organizes 40; Every mouse peritoneal injected sample solution 1.0ml.Mouse feeder was observed after 4-6 week, and eyeball is taken a blood sample about 1ml.Mouse Blood is put room temperature and is placed the centrifugal 10min of more than 1hr, 5000rpm, carries out antibody level of serum detection by Hepatitis B surface antibody diagnostic kit (Shanghai Kehua Bio-engineering Co., Ltd) description.If blank 1 part, negative control 5 parts, positive control 2 parts, draw immune serum to be checked and the every hole of negative and positive control serum adds 50 μ l; Except blank control wells, every hole adds enzyme conjugates 50 μ l, carefully mixes, shrouding, hatches 30min for 37 DEG C.Discard liquid in hole after washing plate, cleaning mixture dosage 350 μ l, washes plate 5 times.Carefully patting dry rear every hole adds developer A liquid and each 50 μ l of B liquid successively for the last time, carefully mixes, shrouding, hatches colour developing 15min for 37 DEG C.Every hole adds stop buffer 50 μ l, mixing.Detect with microplate reader 450nm (reference wavelength 630nm), with blank control wells school zero, read the absorbance (after cessation reaction in 10min reading) in each hole.Calculating antibody Conversion rate and ED 50value (be not less than 1.0 μ g be judged to qualified).
The safety testing of embodiment 11 vaccine product
The vaccine preparation of preparation in embodiment 9 (1) ~ (13), carries out the safety testing of vaccine product respectively by following method
(1) acute toxicity test
According to " GLP ", (office of State Food and Drug Administration makes No. 2, on 09 01st, 2003), " prevention biological product preclinical safety evaluations drug evaluation rule " (State Food and Drug Administration, in December, 2005), " chemicals acute toxicity test technological guidance principle " (State Food and Drug Administration, 2005 03 month) carries out acute toxicity test in mice.
2 groups are established in test, are respectively matched group and combined vaccine group, often organize 10 mices, male and female half and half.Combined vaccine group and matched group all with mouse muscle injection is maximum can the corresponding test sample of administration volume 0.25mL/ single intramuscular injection (bilateral hindlimb muscle) or reference substance (0.9% sodium chloride injection).Administration was defined as test the 1st day the same day.Within after administration continuous 14 days, observe each group of mice general status; In test the 1st (before administration), within 3,7,11,14 days, measure body weight; Test and measure food ration on the 1st, 8,13 day; 14 days observe terminate rear to all survival mice carry out hematology, blood biochemistry detect and gross anatomy observe.
(2) muscular irritation test
According to " GLP ", (office of State Food and Drug Administration makes No. 2, on 09 01st, 2003), " prevention biological product preclinical safety evaluations drug evaluation rule " (State Food and Drug Administration, in December, 2005), " Chemical induced irritation, anaphylaxis and hemolytic investigative technique guideline " (State Food and Drug Administration, 2005 03 month) carries out Japan large ear rabbit and tests through the muscular irritation of quadriceps femoris single injection.
Establish 3 groups altogether, be respectively matched group and combined vaccine is low, high dose group, often organize 4 rabbits, male and female half and half.Combined vaccine is low, high dose is respectively 1 dose of (0.5mL)/side and 3 doses of (1.5mL)/sides, and matched group administration volume is 1.5mL/ side.Each group gives corresponding test sample or reference substance (0.9% sodium chloride injection) by set dosage or volume through rabbit bilateral quadriceps femoris single injection, and within about 48 hours and 16 days, respectively get 2 rabbits and implement peaceful and comfortable post mortem after injection, carry out histopathological examination after grading is observed to injection site quadriceps femoris.
(3) hypersensitive test
According to " GLP ", (office of State Food and Drug Administration makes No. 2, on 09 01st, 2003), " prevention biological product preclinical safety evaluations drug evaluation rule " (State Food and Drug Administration, in December, 2005), " Chemical induced irritation, anaphylaxis and hemolytic investigative technique guideline " (State Food and Drug Administration, 2005 03 month) carries out the whole body initiatively irritated and passive hypersensitive test of Cavia porcellus.
A: whole body is initiatively irritated
Test establishes 4 groups altogether, be respectively that negative control group, positive controls and combined vaccine are low, high dose group (priming dose is respectively 0.5,1mL/ only, be equivalent to 1,2 times of clinical plan dosage), often organize 6 Cavia porcelluss, male and female half and half.During sensitization, combined vaccine low dose group press 0.5mL/ only, high dose group and negative control group by 1mL/ the corresponding test sample of intramuscular injection or 0.9% sodium chloride injection, positive controls presses the oralbumin solution of 0.5mL/ lumbar injection 8mg/mL.The next day sensitization 1 time, continuous sensitization 5 times.After last sensitization the 10th day, each group Cavia porcellus excited through single intravenous injection, and booster dose is priming dose 2 times.The general reaction that after observing booster injection, Cavia porcellus occurs and death condition.
B. passive allergy
Test point two stages: (1) antiserum prepare stage: 16 Cavia porcelluss (male and female half and half) are divided into 4 groups at random.Be respectively that negative control group, positive controls and combined vaccine are low, high dose group (priming dose is 0.5,1mL/ only, be equivalent to 1,2 times of clinical plan dosage respectively).During sensitization, combined vaccine low dose group press 0.5mL/ only, high dose group and negative control group by 1mL/ the corresponding test sample of intramuscular injection or 0.9% sodium chloride injection, positive controls presses the oralbumin solution of 0.5mL/ lumbar injection 8mg/mL.The next day sensitization 1 time, continuous sensitization 5 times.Within after last sensitization 10 days, prepare antiserum from abdominal aortic blood.(2) locus coeruleus experimental stage: 24 Cavia porcelluss (male and female half and half) are divided at random 4 groups of setting identical with the antiserum prepare stage.Antiserum prepared by each group carries out skin passive sensitization in the corresponding injection point intradermal injection of guinea pig back skin, each dilution factor 0.1mL/ point after diluting by 1 ﹕ 2,1 ﹕ 8,1 ﹕ 32.1% Evans blue that intradermal injection added 0.5mL through the antigen of intravenous injection and the 1st stage same dose after 4 hours excites.Inject after 30 minutes, measure each dilution factor injection point skin locus coeruleus diameter after each group Cavia porcellus implements euthanasia and take pictures.
(4) hemolysis in vitro test
According to " GLP ", (office of State Food and Drug Administration makes No. 2, on 09 01st, 2003), " prevention biological product preclinical safety evaluations drug evaluation rule " (State Food and Drug Administration, in December, 2005), " Chemical induced irritation, anaphylaxis and hemolytic investigative technique guideline " (State Food and Drug Administration, 2005 03 month) carries out hemolysis in vitro test.
By 2% rabbit erythrocyte suspension, combined vaccine (5 dosage groups, be 0.1,0.2,0.3,0.4,0.5mL/ pipe), 0.9% sodium chloride injection (negative control group), sterilized water for injection (positive controls) be in after the mixing of setting ratio, be placed in 37 DEG C ± 0.5 DEG C calorstat, respectively observed once respectively at 0,15,30,45,60,120,180 minute.Often group makees 3 parallel pipes.
Embodiment 12 result of the test statistical analysis
(1) conjugate stock solution testing result
Form 1 unit price Seedling conjugate stock solution testing result
Form 2 13 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution testing result
Form 3 b type hemophilus influenza 13 valency pneumococal polysaccharide-hepatitis B surface antigen covalent bond vaccine
Form 4 ACYW135 meningococcal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution testing result
Form 5 ACYW135 group meningitis cocci 13 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution testing result
Form 6 Salmonella typhoid Vi, paratyphoid A, paratyphoid B polysaccharide-hepatitis B surface antigen covalent conjunct agent stock solution testing result
(2) animal is for polysaccharide immunogenic result of the test in vaccine product
In form 7 univalent vaccine, animal is for polysaccharide immunogenic result of the test in vaccine product
In form 8 13 valency pneumococal polysaccharide-hepatitis B surface antigen covalent bond vaccine, animal is for polysaccharide immunogenic result of the test in vaccine product
In form 9 ACYW135 meningococcal polysaccharide-hepatitis B surface antigen covalent bond vaccine, animal is for polysaccharide immunogenic result of the test in vaccine product
In form 10 b type hemophilus influenza 13 valency pneumococal polysaccharide-hepatitis B surface antigen covalent conjunct agent vaccine, animal is for polysaccharide immunogenic result of the test in vaccine product
Form 11 Salmonella typhoid Vi, paratyphoid A, paratyphoid B polysaccharide-hepatitis B surface antigen covalent bond
In vaccine, animal is for polysaccharide immunogenic result of the test in vaccine product
In form 12 ACYW135 group meningitis cocci 13 valency pneumococal polysaccharide-hepatitis B surface antigen covalent bond vaccine, animal is for polysaccharide immunogenic result of the test in vaccine product
(3) animal is for the Study On Immunogenicity result of hepatitis B surface antigen in vaccine product
In form 13 vaccine, animal is for the Study On Immunogenicity result of hepatitis B surface antigen in vaccine product
(4) the safety testing result of vaccine product
A acute toxicity test
2 groups are established in test altogether, are respectively matched group and vaccine group, often organize 10 mices, male and female half and half.Vaccine group and matched group all with mouse muscle injection is maximum can the test sample of administration volume 0.25mL/ single intramuscular injection (bilateral hindlimb muscle) corresponding lot number or reference substance (0.9% sodium chloride injection).Administration was defined as test the 1st day the same day.
Within after administration continuous 14 days, observe each group of mice general status; In test the 1st (before administration), within 3,7,11,14 days, measure body weight; Test and measure food ration on the 1st, 8,13 day; 14 days observe terminate rear to all survival mice carry out hematology, blood biochemistry detect and gross anatomy observe.
Main result is as follows:
1. general status
After administration in 14 day observation period, each group mice has no dead and dying, and general status is good, and autonomic activities is normal, and skin is cleaned by hair, and fecaluria is normal, has no signs of toxicity and occurs.
2. body weight and food ration
After administration in 14 day observation period, female, the male Mus body weight of each group all normally increases, and food ration also no abnormality seen changes.
3. hematological examination
At the end of within after administration 14 days, observing, each group murine interleukin counting (WBC) and classification, red blood cell count(RBC) (RBC), hemoglobin (HGB), red cell volume (HCT), mean corpuscular volume (MCV) (MCV), mean corpusular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) (MCHC), reticulocyte count and the hematological indices such as percentage ratio (RET, RET%), platelet count (PLT) are showed no abnormal change.
4. blood bio-chemistry checking
At the end of within after administration 14 days, observing, the visible slight rising of vaccine group mice total bilirubin (TBIL).In addition, mouse alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate amino transferase (AST), total protein (TP), albumin (ALB), blood glucose (GLU), T-CHOL (CHOL), triglyceride (TG), creatine phosphokinase (CK), carbamide (Urea), creatinine (Crea) and electrolyte (K is respectively organized +, Na +, Cl -) blood biochemistry index such as concentration is showed no the abnormal change relevant to test sample.
5. gross anatomy is observed
At the end of within after administration 14 days, observing, the gross anatomy of each group mice is observed, and the size of the main organs such as brain, heart, liver, spleen, lungs, kidney, reproductive system, gastrointestinal tract or tissue, form, color or quality are showed no the visible abnormal change of naked eyes.
In sum, under this experimental condition, NIH mice single intramuscular injection 0.25mL/ vaccine only, the slight total bilirubin of mice may be caused to raise, in addition, after administration, in 14 days, mice general status is good, and body weight, food ration, peripheral blood cell counts, blood biochemistry detect and gross anatomy observation is showed no obvious abnormalities change.
B. muscular irritation test
Test establishes 3 groups altogether, is respectively matched group and vaccine is low, high dose group, often organizes 4 rabbits, male and female half and half.Vaccine is low, high dose is respectively 1 dose of (0.5mL)/side and 3 doses of (1.5mL)/sides, and matched group administration volume is 1.5mL/ side.Each group gives corresponding test sample or reference substance (0.9% sodium chloride injection) by set dosage or volume through rabbit bilateral quadriceps femoris single injection, and within about 48 hours and 16 days, respectively get 2 rabbits and implement peaceful and comfortable post mortem after injection, carry out histopathological examination after grading is observed to injection site quadriceps femoris.
Main result is as follows:
1. general status
Duration of test respectively organizes that rabbit is showed no injection site redness, congestion, hardens, the local response such as to fester, and each group rabbit general status is good, autonomic activities normal, has no other abnormal symptom and occurs.
Gross examination of skeletal muscle
After injection about 48 hours, gross anatomy visible vaccine high dose group rabbit had 1 (1/4 ratio) individual injection site muscle mild hyperaemia, and all the other rabbit injection site muscle have no macroscopic abnormal change.
Inject latter 16 days, each group rabbit injection site muscle gross anatomy is observed and is showed no macroscopic abnormal change.
2. histopathological examination
After injection about 48 hours, there is slight myocyte degeneration/necrosis and cell infiltration/hemorrhage in matched group 2 (2/4 ratio) injection site muscle, slight ~ slight myocyte's degeneration/necrosis that vaccine low dose group has 2 (2/4 ratio) individual injection site muscle to occur and cell infiltration/hemorrhage, vaccine high dose group has 2 (2/4 ratio) individual injection site muscle to occur slightly ~ moderate myocyte degeneration/necrosis and cell infiltration/hemorrhage.Vaccine low dose group lesion degree and/or proportion and matched group roughly the same.Vaccine high dose group lesion degree and/or proportion are a little more than matched group, and prompting may to the certain zest of injection site muscle tool.
Inject latter 16 days, vaccine is low, high dose group respectively has 1 (1/4 ratio) injection site muscle to occur slight muscle fiber regeneration.
In sum, under this experimental condition, Japan large ear rabbit gives 0.5 through quadriceps femoris single injection, the vaccine of 1.5mL/ side, and the vaccine of 1.5mL/ side has certain zest to rabbit quadriceps femoris, and drug withdrawal 16 days is basic afterwards recovers normal.The vaccine of 0.5mL/ side to rabbit quadriceps femoris without obvious zest.
C. hypersensitive test
Test establishes 4 groups altogether, be respectively that negative control group, positive controls and vaccine are low, high dose group (priming dose is respectively 0.5,1mL/ only, be equivalent to 1,2 times of clinical plan dosage), often organize 6 Cavia porcelluss, male and female half and half.During sensitization, vaccine low dose group press 0.5mL/ only, high dose group and negative control group by 1mL/ the corresponding test sample of intramuscular injection or 0.9% sodium chloride injection, positive controls presses the oralbumin solution of 0.5mL/ lumbar injection 8mg/mL.The next day sensitization 1 time, continuous sensitization 5 times.After last sensitization the 10th day, each group Cavia porcellus excited through single intravenous injection, and booster dose is priming dose 2 times.The general reaction that after observing booster injection, Cavia porcellus occurs and death condition.Main result is as follows:
Each group of Cavia porcellus general status during sensitization is good, autonomic activities normal, skin is increased by hair cleaning, secretions without exception, Normal-weight, has no other abnormal responses.
Excite after administration in 30 minutes, negative control group Cavia porcellus has no allergic symptom; There is obvious allergic symptom in positive controls Cavia porcellus, and all dead in 8 minutes after administration; Vaccine is low, high dose group is showed no allergic symptom.
To sum up, under this experimental condition, Cavia porcellus intramuscular injection 0.5mL, 1mL/ vaccine is only planted by Britain, and whole body initiatively Hypersensitive tests result is negative.
D. hemolysis in vitro test
Test establish negative control group (0.9% sodium chloride injection), positive controls (sterilized water for injection) and vaccine 5 dosage groups (be respectively 0.1,0.2,0.3,0.4,0.5mL/ pipe).By 2% rabbit erythrocyte suspension, vaccine, 0.9% sodium chloride injection, sterilized water for injection in after the mixing of setting ratio, be placed in 37 DEG C ± 0.5 DEG C calorstat, respectively observed once respectively at 0,15,30,45,60,120,180 minute.Often group makees 3 parallel pipes.Result is as follows:
37 ± 0.5 DEG C leave standstill 3 hours, and the visible haemolysis of positive control pipe, negative control pipe and the equal visible red cell of vaccine pipe sink naturally, and supernatant water white transparency, the erythrocyte sunk after suitable jolting disperses again, does not occur haemolysis and aggregation.
In sum, under this experimental condition, vaccine is to rabbit erythrocyte without haemolysis and cohesion, and hemolysis in vitro result of the test is negative.
Embodiment 8 to the embodiment 11 (preparation of embodiment 8 bacterial polysaccharideses-hepatitis B surface antigen conjugate stock solution; The preparation of embodiment 9 vaccine product.The efficiency assay of embodiment 10 vaccine product.The safety testing of embodiment 11 vaccine product) result show, the conjugate of gained after the scorching ball polysaccharide of b type hemophilus influenza polysaccharide, pneumococal polysaccharide, neisseria meningitis, Salmonella typhi Vi polysaccharide, paratyphosus A bacillus O-SP polysaccharide, Salmonella paratyphi B O-SP polysaccharide combine with hepatitis B virus surface antigen (rHBsAg) respectively, after carrying out animal experiment, the Positive seroconversion rate (Conversion rate more than 85%) of higher bacterial polysaccharides can be produced; The mice efficiency test of hepatitis B surface antigen also very well (Positive seroconversion rate more than 85%) simultaneously.Result describe of the present invention a kind of be that bacterial polysaccharide protein conjugate prepared by protein carrier makes vaccine product in order to hepatitis B surface antigen; the protection antibody of directed toward bacteria polysaccharide can be produced, also can produce the protection antibody for hepatitis B surface antigen simultaneously.

Claims (12)

1. one kind is the bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen, it is characterized in that: the albumen contained in described vaccine is hepatitis B surface antigen, the bacterial polysaccharides contained in described vaccine is selected from b type hemophilus influenza polysaccharide, neisseria meningitis scorching pneumoniae serotype group A group, C group, Y group, W135 group's polysaccharide, salmonella typhi Vi type polysaccharide, B group streptococcus Ia, Ib, II, III, V-type, Pneumococcus serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F, bacillus paratyphosus A type or bacillus paratyphosus B-mode in any one or a few.
2. hepatitis B surface antigen according to claim 1 is the bacterial polysaccharide protein conjugate vaccine of carrier protein, it is characterized in that: each bacterial polysaccharides to be connected with hepatitis B surface antigen with covalent bond respectively and to be connected into bacterial polysaccharide protein conjugate.
3. hepatitis B surface antigen according to claim 2 is the bacterial polysaccharide protein conjugate vaccine of carrier protein, it is characterized in that: in described bacterial polysaccharide protein conjugate, the mass ratio of total bacterial polysaccharides and hepatitis B surface antigen is 1:0.1 ~ 1:5.
4. is the bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen according to claim 1 or 2 or 3, it is characterized in that: described hepatitis B virus surface antigen is natural hepatitis B virus surface antigen or recombination hepatitis B surface antigen.
5. is the bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen according to claim 1 or 2 or 3, it is characterized in that: the combination agent that described vaccine is liquid preparation, solid particle agent, semi-solid agent or liquid preparation and solid particle agent are combined.
6. hepatitis B surface antigen according to claim 4 is the bacterial polysaccharide protein conjugate vaccine of carrier protein, it is characterized in that: the combination agent that described vaccine is liquid preparation, solid particle agent, semi-solid agent or liquid preparation and solid particle agent are combined.
7. is the bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen according to claim 1 or 2 or 3, it is characterized in that: described vaccine is injection, nasal formulations, gel or Extencap.
8. hepatitis B surface antigen according to claim 4 is the bacterial polysaccharide protein conjugate vaccine of carrier protein, it is characterized in that: described vaccine is injection, nasal formulations, gel or Extencap.
9. is the bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen according to claim 1 or 2 or 3, it is characterized in that: also containing aluminium adjuvant or a kind of deoxyribonucleic acid fragment containing Cytosine-phosphate-guanine in described vaccine.
10. hepatitis B surface antigen according to claim 4 is the bacterial polysaccharide protein conjugate vaccine of carrier protein, it is characterized in that: also containing aluminium adjuvant or a kind of deoxyribonucleic acid fragment containing Cytosine-phosphate-guanine in described vaccine.
11. 1 kinds is the preparation method of the unit price bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen, comprises the following steps:
(1) bacterial polysaccharides dissolves
The NaCl of a kind of bacterial polysaccharides 0.2mol/L is dissolved as the concentration of 1 ~ 20mg/ml; Described a kind of bacterial polysaccharides is b type hemophilus influenza polysaccharide, neisseria meningitis scorching pneumoniae serotype group A group, C group, Y group, W135 group's polysaccharide, salmonella typhi Vi type polysaccharide, B group streptococcus Ia, Ib, II, III, V-type, Pneumococcus serotypes 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F, bacillus paratyphosus A type or bacillus paratyphosus B-mode in any one;
(2) bacterial polysaccharides activation
The bacterial polysaccharides that polysaccharide activator must activate is added in the bacterial polysaccharides solution that step (1) is dissolved, the quality of the polysaccharide activator added: bacterial polysaccharides mass ratio is 0.1:1 ~ 1.5:1, described polysaccharide activator is any one in Bromine cyanide., 1-cyano group-DMAP Tetrafluoroboric acid ester, carbodiimide, 3-(2-pyridyidithio) propanoic acid, N-hydroxy-succinamide ester;
(3) bacterial polysaccharides derives
By the bacterial polysaccharides of activation in the bacterial polysaccharides of the activation of step (2) gained: the mass ratio of difunctional link agent is that the ratio row of 1:2 ~ 1:5 add difunctional link agent and carry out the derivative of bacterial polysaccharides and to obtain bacterial polysaccharides derivant, described difunctional link agent is any one in adipic dihydrazide, Polyethylene Glycol, hexamethylene diamine or hexanediol;
(4) ultrafiltration of bacterial polysaccharides derivant
Bacterial polysaccharides derivant step (3) obtained obtains bacterial polysaccharides derivant through the ultrafilter membrane ultrafiltration lyophilizing of 1 ~ 30KD;
(5) bacterial polysaccharides and protein binding
After being 2 ~ 20mg/ml by the concentration that the bacterial polysaccharides derivant that step (4) obtains to be dissolved to bacterial polysaccharides derivant by the NaCl of 0.2mol/L; In bacterial polysaccharides derivant: the mass ratio of hepatitis B surface antigen is that the ratio of 1:0.4 ~ 1:2 adds in carrier protein, then adding carbodiimide to carbodiimide concentration is 0.001 ~ 0.2mol/L, maintaining pH is 5.5 ± 0.5, react 2 ~ 4 hours, obtain a kind of bacterial polysaccharides derivant and carrier protein covalent conjunct agent;
(6) a kind of for step (5) gained bacterial polysaccharides derivant and carrier protein covalent conjunct agent gel column are carried out purification, collect V 0absworption peak near (elution volume of the molecule of solvent resistant column material cannot be entered), namely aseptic filtration obtains the unit price bacterial polysaccharide protein conjugate stock solution after purification;
(7) vaccine liquid preparation is prepared
Water for injection is added in unit price bacterial polysaccharide protein conjugate stock solution after the purification of step (6) gained, sodium chloride, phosphate buffer and aluminium adjuvant, the concentration of bacterial polysaccharides is made to be 4 μ g/ml ~ 60 μ g/ml, sodium chloride concentration is 4 ~ 9mg/ml, sodium hydrogen phosphate concentration is 38 ~ 72 μ g/ml, phosphate dihydrogen sodium concentration is 24 ~ 88 μ g/ml, described aluminium adjuvant is aluminium hydroxide or aluminum phosphate, aluminum hydroxide concentration is made to be 0.5 ~ 2.5mg/ml, or aluminium phosphate concentration is 0.5 ~ 2.0mg/ml, namely making with hepatitis B surface antigen is the unit price bacterial polysaccharide protein conjugate vaccine liquid preparation of carrier protein,
Or (8) prepare vaccine solid granule
Water for injection, sodium chloride and stabilizing agent is added in unit price bacterial polysaccharide protein conjugate stock solution after the purification of step (6) gained, make that the concentration of bacterial polysaccharides is 4 μ g/ml ~ 60 μ g/ml, sodium chloride concentration is 4 ~ 9mg/ml, described stabilizing agent is lactose or sucrose or glycine, make lactose concn be 8 ~ 25mg/ml; Or sucrose concentration is 40 ~ 120mg/ml; Or glycine concentration is 20 ~ 80mg/ml, namely lyophilizing makes with hepatitis B surface antigen is the unit price bacterial polysaccharide protein conjugate vaccine solid granule of carrier protein;
Or (9) prepare the semi-solid agent of vaccine
Water for injection, sodium chloride and excipient is added in unit price bacterial polysaccharide protein conjugate stock solution after the purification of step (6) gained, make that the concentration of bacterial polysaccharides is 4 μ g/ml ~ 60 μ g/ml, sodium chloride concentration is 4 ~ 9mg/ml, described excipient is sodium carboxymethyl cellulose or carbomer, sodium carboxymethyl cellulose concentration is made to be 10 ~ 30mg/ml, or carbomer concentration is 5 ~ 20mg/ml, namely making with hepatitis B surface antigen is the semi-solid agent of unit price bacterial polysaccharide protein conjugate vaccine of carrier protein.
12. 1 kinds is the preparation method of the two valencys above bacterial polysaccharide protein conjugate vaccine of carrier protein with hepatitis B surface antigen, comprises the following steps:
(1) a kind of bacterial polysaccharides derivant is prepared
Any one bacterial polysaccharides derivant is prepared by step in claim 11 (1) to the method for step (4);
(2) bacterial polysaccharides and protein binding
Be that after the ratio mixing of 1:1 ~ 1:4, with the NaCl of 0.2mol/L, mixed bacterial polysaccharides derivant being dissolved to mixed bacterial polysaccharides derivatives concentration is 2 ~ 20mg/ml respectively in the mass ratio between each bacterial polysaccharides derivant by two or more bacterial polysaccharides derivant; Again in total bacterial polysaccharides derivant: the mass ratio of hepatitis B surface antigen is that the ratio of 1:0.4 ~ 1:2 adds in carrier protein, then, adding carbodiimide to carbodiimide concentration is 0.001 ~ 0.2mol/L, maintaining pH is 5.5 ± 0.5, react 2 ~ 4 hours, obtain the above bacterial polysaccharides derivant of two valencys and carrier protein covalent conjunct agent;
(3) purification
The above bacterial polysaccharides derivant of step (2) gained two valency and carrier protein covalent conjunct agent gel column are carried out purification, collects V 0neighbouring absworption peak, namely aseptic filtration obtains the two valencys above bacterial polysaccharide protein conjugate stock solution after purification;
(4) vaccine liquid preparation is prepared
Water for injection is added in two valencys above bacterial polysaccharide protein conjugate stock solution after the purification of step (3) gained, sodium chloride, phosphate buffer and aluminium adjuvant, the concentration of total bacterial polysaccharides is made to be 8.8 μ g/ml ~ 300 μ g/ml, sodium chloride concentration is 4 ~ 9mg/ml, sodium hydrogen phosphate concentration is 38 ~ 72 μ g/ml, phosphate dihydrogen sodium concentration is 24 ~ 88 μ g/ml, described aluminium adjuvant is aluminium hydroxide or aluminum phosphate, aluminum hydroxide concentration is made to be 0.5 ~ 2.5mg/ml, or aluminium phosphate concentration is 0.5 ~ 2.0mg/ml, namely making with hepatitis B surface antigen is the two valencys above bacterial polysaccharide protein conjugate vaccine liquid preparation of carrier protein, the concentration of described total bacterial polysaccharides is the concentration of each bacterial polysaccharides quality sum,
Or (5) prepare vaccine solid granule
Water for injection, sodium chloride and stabilizing agent is added in two valencys above bacterial polysaccharide protein conjugate stock solution after the purification of step (3) gained, make that the concentration of total bacterial polysaccharides is 8.8 μ g/ml ~ 300 μ g/ml, sodium chloride concentration is 4 ~ 9mg/ml, described stabilizing agent is lactose or sucrose or glycine, make lactose concn be 8 ~ 25mg/ml; Or sucrose concentration is 40 ~ 120mg/ml, or glycine concentration is 20 ~ 80mg/ml, and namely lyophilizing makes with hepatitis B surface antigen is the two valencys above bacterial polysaccharide protein conjugate vaccine solid granule of carrier protein; The concentration of described total bacterial polysaccharides is the concentration of each bacterial polysaccharides quality sum;
Or (6) prepare the semi-solid agent of vaccine
Water for injection, sodium chloride and excipient is added in two valencys above bacterial polysaccharide protein conjugate stock solution after the purification of step (3) gained, make that the concentration of total bacterial polysaccharides is 8.8 μ g/ml ~ 300 μ g/ml, sodium chloride concentration is 4 ~ 9mg/ml, described excipient is sodium carboxymethyl cellulose or carbomer, sodium carboxymethyl cellulose concentration is made to be 10 ~ 30mg/ml, or carbomer concentration is 5 ~ 20mg/ml, namely making with hepatitis B surface antigen is the semi-solid agent of two valencys above bacterial polysaccharide protein conjugate vaccine of carrier protein; The concentration of described total bacterial polysaccharides is the concentration of each bacterial polysaccharides quality sum.
CN201410723098.3A 2014-12-02 2014-12-02 Bacterial polysaccharide protein conjugate vaccine using hepatitis B surface antigen as carrier protein and preparation method of bacterial polysaccharide protein conjugate vaccine Pending CN104383532A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201410723098.3A CN104383532A (en) 2014-12-02 2014-12-02 Bacterial polysaccharide protein conjugate vaccine using hepatitis B surface antigen as carrier protein and preparation method of bacterial polysaccharide protein conjugate vaccine

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201410723098.3A CN104383532A (en) 2014-12-02 2014-12-02 Bacterial polysaccharide protein conjugate vaccine using hepatitis B surface antigen as carrier protein and preparation method of bacterial polysaccharide protein conjugate vaccine

Publications (1)

Publication Number Publication Date
CN104383532A true CN104383532A (en) 2015-03-04

Family

ID=52601555

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201410723098.3A Pending CN104383532A (en) 2014-12-02 2014-12-02 Bacterial polysaccharide protein conjugate vaccine using hepatitis B surface antigen as carrier protein and preparation method of bacterial polysaccharide protein conjugate vaccine

Country Status (1)

Country Link
CN (1) CN104383532A (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107261130A (en) * 2017-06-29 2017-10-20 云南沃森生物技术股份有限公司 It is a kind of with preparation methods of the DSC as the bacterial polysaccharide protein combined vaccine of activator
CN108079286A (en) * 2018-01-19 2018-05-29 云南沃森生物技术股份有限公司 A kind of 13 valency pneumococcal polysaccharide-protein combination compositions and its preparation method and application
CN108743936A (en) * 2018-06-14 2018-11-06 中国科学院过程工程研究所 A kind of bacterial polysaccharides combined vaccine and the preparation method and application thereof
CN110804097A (en) * 2019-10-30 2020-02-18 云南沃森生物技术股份有限公司 Method for simultaneously preparing two monoclonal antibodies

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007102797A2 (en) * 2005-12-22 2007-09-13 Sanofi Pasteur, Inc. Multivalent meningococcal derivatized polysaccharide-protein conjugates and vaccine
CN101861165A (en) * 2007-10-12 2010-10-13 麻省理工学院 Vaccine nanotechnology

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007102797A2 (en) * 2005-12-22 2007-09-13 Sanofi Pasteur, Inc. Multivalent meningococcal derivatized polysaccharide-protein conjugates and vaccine
CN101861165A (en) * 2007-10-12 2010-10-13 麻省理工学院 Vaccine nanotechnology

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
卢佳丽: "重组乙肝病毒表面抗原为载体的流脑多糖蛋白结合疫苗特性研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 *
赵海平等: "细菌荚膜多糖的分离纯化研究进展", 《微生物学免疫学进展》 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107261130A (en) * 2017-06-29 2017-10-20 云南沃森生物技术股份有限公司 It is a kind of with preparation methods of the DSC as the bacterial polysaccharide protein combined vaccine of activator
CN108079286A (en) * 2018-01-19 2018-05-29 云南沃森生物技术股份有限公司 A kind of 13 valency pneumococcal polysaccharide-protein combination compositions and its preparation method and application
CN108079286B (en) * 2018-01-19 2020-07-31 云南沃森生物技术股份有限公司 13-valent pneumococcal polysaccharide-protein conjugate composition and preparation method and application thereof
CN108743936A (en) * 2018-06-14 2018-11-06 中国科学院过程工程研究所 A kind of bacterial polysaccharides combined vaccine and the preparation method and application thereof
CN108743936B (en) * 2018-06-14 2022-03-15 中国科学院过程工程研究所 Bacterial polysaccharide conjugate vaccine and preparation method and application thereof
CN110804097A (en) * 2019-10-30 2020-02-18 云南沃森生物技术股份有限公司 Method for simultaneously preparing two monoclonal antibodies
CN110804097B (en) * 2019-10-30 2021-02-26 云南沃森生物技术股份有限公司 Method for simultaneously preparing two monoclonal antibodies

Similar Documents

Publication Publication Date Title
CN101951949B (en) Meningococcal vaccine formulations
CN101678094B (en) Formulation of meningitis vaccines
CN103656631B (en) multivalent pneumococcal capsular polysaccharide-protein conjugate composition and preparation method thereof
KR101588939B1 (en) Multivalent pneumococcal polysaccharide-protein conjugate composition
CN104225587B (en) Multivalent meningococcal derivatized proteinpolysaccharide conjugate and vaccine
CN1709505B (en) Polyvalent bacteria capsule polysaccharide-protein conjugate combined vaccine
CN100462102C (en) Mucosal meningococcal multivalence combined vaccines
CN104689309A (en) Separated and purified acellular pertussis-diphtheria-tetanus, b-type haemophilus influenzae and A-group and C-group meningococcus combined vaccine and preparation method thereof
CN102813917B (en) Acellular pertussis/Haemophilus influenzae type b - group A and C meningococcus combined vaccine
CN104383532A (en) Bacterial polysaccharide protein conjugate vaccine using hepatitis B surface antigen as carrier protein and preparation method of bacterial polysaccharide protein conjugate vaccine
ES2628511T3 (en) Mixture of lyophilized meningococcal vaccines with D-T-Pa vaccines
CN104998255B (en) New A CYW135 group meningitis cocci combined vaccines and preparation method thereof
CN102089009B (en) Conjugated Vi saccharides
CN102935226B (en) Typhoid fever and paratyphoid fever combined vaccine and preparation method thereof
CN101024079B (en) Pneumo-streptococcal-polysaccharide adventitia jointed vaccine and preparing method
CN101934077A (en) Multivalent meningococcal derivatized polysaccharide-proteinconjugates and vaccine
CN104069488A (en) Multivalent pneumococcus capsular polysaccharide-protein conjugated composition and preparation method thereof
CN108295253A (en) A kind of A, C group meningitis cocci-b types haemophilus influenzae/encephalitis B combined vaccine
CN104127869B (en) Multivalence B group meningitis cocci protein vaccine and preparation method thereof
CN108619508A (en) A kind of season influenza-RSV- epidemic meningitis-pneumococcus combined vaccine based on recombinant vector albumen
CN102264444B (en) Purification process
CN110917344B (en) Liquid vaccine composition and application thereof
CN109091668A (en) 16 valence streptococcus pneumonia combined vaccine compositions
CN1209163C (en) Meningococcal vaccine and its preparing method
CN110251667A (en) A kind of immune combination preparation and its preparation method and application

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20150304

RJ01 Rejection of invention patent application after publication