CN101590225B - Vaccine combining DTP (diphtheria,tetanus and pertussis) and A plus C group meningococcus-b type haemophilus influenzae - Google Patents
Vaccine combining DTP (diphtheria,tetanus and pertussis) and A plus C group meningococcus-b type haemophilus influenzae Download PDFInfo
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- CN101590225B CN101590225B CN2009100671560A CN200910067156A CN101590225B CN 101590225 B CN101590225 B CN 101590225B CN 2009100671560 A CN2009100671560 A CN 2009100671560A CN 200910067156 A CN200910067156 A CN 200910067156A CN 101590225 B CN101590225 B CN 101590225B
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Abstract
The invention provides a vaccine combining DTP and A plus C group meningococcus-b type haemophilus influenzae. Capsular polysaccharide is extracted from A and C group neisseria meningitides and a culture solution of b type haemophilus influenzae respectively; after purification and activation; the capsular polysaccharide is combined with tetanus toxoid; and original solution of A and C group meningococcal polysaccharide, original solution of the b type haemophilus influenzae combined vaccine and original solution of DTAP (diphtheria,tetanus and acellular pertussis) are mixed according to certain ratio to obtain the effect of multi-immunity by one needle.
Description
Technical field:
The present invention provides a kind of whooping cough, A+C group meningitis cocci-b type hemophilus influenza combined vaccine; With the acellular whooping cough combined vaccine of absorption, AC group meningitis cocci-b type hemophilus influenza combined vaccine; Important eqpidemic diseases such as prevention of brain perimyelitis, pneumonia, pertussis, diphtheria and tetanus belong to the production of vaccine preparing technical field.
Background technology:
Epidemic cerebrospinal meningitis (epidemic encephalitis) mainly morbidity crowd is an infant, and A, C group meningitis cocci polysaccharide conjugate vaccine are mainly used in the infant prevention and use.The 1980s, along with the immunity of succeeding in developing and bring sth. into the plan of epidemic encephalitis polysaccharide vaccine, the inoculation number rises year by year in the global range, and sickness rate descends year by year.The epidemic encephalitis morbidity of China is main with A crowd; Therefore the vaccine of exploitation is A crowd's polysaccharide or the two valency polysaccharide vaccines of A+C crowd; But 2000 in Guizhou, broke out epidemic encephalitis respectively in Hebei in 2004, cause local fear, prove through Epidemiological study; Cause the popular C of being crowd bacterial strain, this has just caused the attention of disease control department.Existing A+C group meningitis cocci polysaccharide vaccine safety, effectively, protective rate can reach 80-90%.But this vaccine is poor to the immune effect of infant below 2 years old, and the inoculation potion can not reach antibody protection level, and antibody disappears very soon.Because of this vaccine the same with other polysaccharide vaccine; Belong to T cell dependent/non-dependent antigen, the immune system of infant below 2 years old is undeveloped mature still, a little less than the antibody response reaction to most of capsular polysaccharides; Only produce low-level specific antibody; Polysaccharide conjugate can stimulate T cell dependency antibody synthetic of infant, but and booster response, compare the antibody ratio that has also improved immunoglobulin (IgG) with non-binding thing vaccine.
B type hemophilus influenza (Hib) infects through droplet transmission, causes primary diseases such as the nasopharyngitis of child below five years old, meeting pharyngitis; Antibacterial gets into blood or through blood brain barrier, causes serious affecting conditions such as pneumonia, purulent meningitis, arthritis, osteomyelitis, pericarditis and septicemia from the part.WHO points out that the whole world has the Hib of 3,000,000 examples to infect every year, and wherein dead 60,000 examples (2%) are main in developed country with meningitis, are main in developing country with pneumonia.
Seeing that A, C group meningitis cocci and b type hemophilus influenza cause the effectiveness of severity of disease and vaccine, research and develop this vaccine and be extremely important.Using in 38 countries of Hib vaccine at present, the principle that its immune programme for children is abideed by is: 1) accomplish the fundamental immunity program between the monthly age at 2-6 after the birth, every dose of injection interval at least 1 month; 2) the birth back can be carried out booster immunization one time between 12-24 month.Abide by this principle, with the acellular whooping cough combined vaccine of absorption, AC group meningitis cocci-b type hemophilus influenza combined vaccine, 2-6 accomplishes fundamental immunity between the monthly age in the birth back, accomplishes booster immunization between 12-24 month.Can prevent the epidemic cerebrospinal meningitis that A, C crowd cause, disease, pertussis, diphtheria and the tetanus that Hib causes simultaneously by this immune programme for children, reduce the immunization programs for children number of times simultaneously.
Summary of the invention:
The present invention provides a kind of whooping cough, A+C group meningitis cocci-b type hemophilus influenza combined vaccine; Adopt A, C group meningitis Nai Seshi diplococcus, b type hemophilus influenza culture fluid; Extract capsular polysaccharide respectively, after purification, activation, combine with tetanus toxoid; Then A, C group meningitis polysaccharide conjugate vaccine stock solution, b type hemophilus influenza combined vaccinogen liquid are mixed with acellular whooping cough stock solution by a certain percentage, obtain the effect of a pin panimmunity.
Combined vaccine of the present invention contains following component and content:
A group meningitis cocci polysaccharide: 10 μ g
C group meningitis cocci polysaccharide: 10 μ g
B type hemophilus influenza polysaccharide: 10 μ g
Lactose: 2.5～3.0mg
Acellular pertussis stock solution: 18 μ gPN/ml
Diphtheria toxoid: 25Lf/ml
Tetanus toxoid: 7Lf/ml
Aluminium hydroxide content: 1.0～1.5mg/ml
The preparation technology of combined vaccine of the present invention may further comprise the steps:
Strain select for use: select A, C group meningitis Neisseria gonorrhoeae for use, b type hemophilus influenza strain is used for the preparation of A, C group meningitis cocci-b type hemophilus influenza combined vaccine.
2. produce and use seed
Breakdown work seed lot strain, through suitably going down to posterity, behind assay approval, inoculation improvement doup closes culture medium or other appropriate medias, and seed is used in the suitable production of preparation quantity.
3. produce and use culture medium
Adopt the improvement doup to close culture medium or other appropriate medias through ratifying.Culture medium should not contain with cetyl trimethyl ammonium bromide and forms sedimentary composition.
4. cultivate
Adopt the culture tank liquid culture.Pure bacterium inspection is carried out in sampling in incubation, and smear is done gram stain microscopy, as finding pollution microbes, should discard.
5. results and antibacterial
Stop cultivating in the later stage of exponential phase or the early stage of resting stage.Bacterial concentration mensuration and the inspection of pure bacterium are carried out in sampling, and qualified back adds the formalin antibacterial in the culture fluid of results, or the antibacterial of employing heating means.Do not damage its polysaccharide antigen again and be advisable to guarantee antibacterial safety.
6. enucleation acid
Supernatant is collected in the centrifugal back of germ-resistant culture fluid (single batch or many batch mixings close), add cetyl trimethyl ammonium bromide, fully mixing forms deposition; Precipitate after centrifugal adds an amount of calcium chloride solution, and its ultimate density is 1mol/L, and the polysaccharide cetyl trimethyl ammonium bromide is dissociated, and adding ethanol to ultimate density is 25%, and 2-8 ℃ left standstill 1-3 hour or spent the night, the clarifying supernatant of centrifugal collection.
7. deposition polysaccharide
In above-mentioned supernatant, adding cooling ethanol to ultimate density is 80%, shake well, and centrifugal collecting precipitation is respectively washed more than 2 times with dehydrated alcohol and acetone, and precipitate is the polysaccharide semifinished product, should be kept at below-20 ℃, and is to be purified.
8. polysaccharide purification
Semifinished product is dissolved in the 1/10 saturated neutral SAS, makes its concentration reach 10-20mg/ml; Extract for several times with cold phenol (the 100g crystalline phenol is dissolved in the 1/10 saturated acetic acid sodium solution of 40ml) by 1: 2 capacity, centrifugal collection supernatant, and with 0.1mol/L calcium chloride solution or other suitable solution dialysis, adding ethanol to final concentration is 75%～80%; The precipitate of centrifugal collection is respectively washed more than 2 times with dehydrated alcohol and acetone, and dry back is purified polysaccharide stock solution with sterilized water for injection dissolution precipitation thing.Leaching process should carry out below 15 ℃
9. polysaccharide activation, derivation combine with tetanus toxoid
Through cyanogen bromide-activated, ADH (1, the 6-AH) reaction that adds suitable proportion is spent the night with the polysaccharide of physics and chemistry assay approval, considers or other proper method are removed the Bromine cyanide. and the ADH of derivation not, filtration sterilization ,-20 ℃ of preservations with ultra then.Sampling is examined and determine; Behind the assay approval, polysaccharide-AH derivant and tetanus toxoid mixed in equal amounts with suitable concentration add EDAC (carbodiimide) then; Mixture 2-8 ℃ keeps certain hour; With 0.2M NaCl solution dialysed overnight, reactant with Sepharose CL-4B column chromatography purification, is collected V
0The part eluent transfers pH to neutral with NaOH, and aseptic filtration both had been a conjugate stock solution, 2-8 ℃ of preservation.
10. semi-finished product preparation and packing
After combining Seedling stock solution, b type hemophilus influenza combined vaccinogen liquid to mix in proportion the A of assay approval, the C group meningitis cocci polysaccharide, carry out packing on request with acellular pertussis stock solution, branch loading amount 0.5mL/ bottle, it is to be checked to put 2-8 ℃ of storehouse.
11. finished product calibrating
Related request according to " the AC group meningitis cocci-b type hemophilus influenza combined vaccine system inspection tentative specification " worked out and existing " adsorbing manufacturing of acellular whooping cough combined vaccine and vertification regulation " is examined and determine comprehensively.
The invention has the advantages that:
1. contain aluminium hydroxide in the acellular whooping cough combined vaccine, as the adjuvant composition, following characteristics are arranged: 1) adsorption rate is high, can reach fast, high after the injection of first pin and continue immune effect of a specified duration; 2) an amount of aluminium adjuvant released antigen progressively, long period immune stimulatory cell; 3) stimulate humoral immunization, produce IgG, the IgE of higher titre, activate the Th2 cell.The ingenious adjuvant aluminium hydroxide that utilizes of the present invention is processed whooping cough+AC group meningitis cocci-b type hemophilus influenza combined vaccine, behind the inoculation human body, not only strengthens the whooping cough immune effect, can also strengthen A, C group meningitis cocci-Hib combined vaccine immune effect.
2. inoculate disease, pertussis, diphtheria and tetanus that a pin just can prevent epidemic cerebrospinal meningitis, Hib to cause;
3. improve vaccine coverage rate and rate of vaccination, reduce that child's multiple injection vaccine brought misery, reduce inoculation and managerial expense, the minimizing head of a family, child and doctor's inconvenience and workload.
The specific embodiment:
Embodiment 1:
200901 batches of whooping coughs, A+C group meningitis cocci-b type hemophilus influenza combined vaccines prepare:
1, gets A group meningitis cocci 29201 bacterial strains, C group meningitis cocci 29205 bacterial strains, b type hemophilus influenza 58534 bacterial strains; Be inoculated in suitable agar culture medium; In the environment of 35-37 ℃ of carbon dioxide, cultivated 16-20 hour, smear is done gram stain microscopy, and the inoculation fermentation jar is cultivated.
2, adopt the culture tank liquid culture.Pure bacterium inspection is carried out in sampling in incubation, and smear is done gram stain microscopy, as finding pollution microbes, should discard.Stop cultivating in the later stage of exponential phase or the early stage of resting stage.Bacterial concentration mensuration and the inspection of pure bacterium are carried out in sampling, and qualified back adds the formalin antibacterial in the culture fluid of results, or the antibacterial of employing heating means.Do not damage its polysaccharide antigen again and be advisable to guarantee antibacterial safety.
3, supernatant is collected in the centrifugal back of germ-resistant culture fluid (single batch or many batch mixings close), add cetyl trimethyl ammonium bromide, fully mixing forms deposition; Precipitate after centrifugal adds an amount of calcium chloride solution, and its ultimate density is 1mol/L, and the polysaccharide cetyl trimethyl ammonium bromide is dissociated, and adding ethanol to ultimate density is 25%, and 2-8 ℃ left standstill 1-3 hour or spent the night, the clarifying supernatant of centrifugal collection.In above-mentioned supernatant, adding cooling ethanol to ultimate density is 80%, shake well, and centrifugal collecting precipitation is respectively washed more than 2 times with dehydrated alcohol and acetone, and precipitate is the polysaccharide semifinished product, should be kept at below-20 ℃, and is to be purified.
4, semifinished product is dissolved in the 1/10 saturated neutral SAS, makes its concentration reach 10-20mg/ml; Extract for several times with cold phenol (the 100g crystalline phenol is dissolved in the 1/10 saturated acetic acid sodium solution of 40ml) by 1: 2 capacity, centrifugal collection supernatant, and with 0.1mol/L calcium chloride solution or other suitable solution dialysis, adding ethanol to final concentration is 75%～80%; The precipitate of centrifugal collection is respectively washed more than 2 times with dehydrated alcohol and acetone, and dry back is purified polysaccharide stock solution with sterilized water for injection dissolution precipitation thing.Leaching process should carry out below 15 ℃.
With the polysaccharide of physics and chemistry assay approval through cyanogen bromide-activated, ADH (1, the 6-AH) reaction that adds suitable proportion is spent the night, consider or other proper method are removed the Bromine cyanide. and the ADH of derivation not with ultra then, filtration sterilization, lyophilizing (or-20 ℃) is preserved.Sampling is examined and determine; Behind the assay approval, polysaccharide-AH derivant and tetanus toxoid mixed in equal amounts with suitable concentration add EDAC (carbodiimide) then; Mixture 2-8 ℃ keeps certain hour; With 0.2M NaCl solution dialysed overnight, reactant with Sepharose CL-4B column chromatography purification, is collected V
0The part eluent transfers pH to neutral with NaOH, and aseptic filtration both had been a conjugate stock solution, 2-8 ℃ of preservation.
6. semi-finished product preparation and packing
After combining Seedling stock solution, b type hemophilus influenza combined vaccinogen liquid to mix in proportion the AC group meningitis cocci polysaccharide of assay approval, carry out packing on request with acellular pertussis stock solution, branch loading amount 0.5mL/ bottle, it is to be checked to put 2-8 ℃ of storehouse.
7. finished product calibrating
Related request according to " the AC group meningitis cocci-b type hemophilus influenza combined vaccine system inspection tentative specification " worked out and existing " adsorbing manufacturing of acellular whooping cough combined vaccine and vertification regulation " is examined and determine comprehensively.
Embodiment 2:
200902 batches of whooping coughs, A+C group meningitis cocci-b type hemophilus influenza combined vaccines prepare:
1, gets A group meningitis cocci 29201 bacterial strains, C group meningitis cocci 29205 bacterial strains, b type hemophilus influenza 58534 bacterial strains; Be inoculated in suitable agar culture medium; In the environment of 35-37 ℃ of carbon dioxide, cultivated 16-20 hour, smear is done gram stain microscopy, and the inoculation fermentation jar is cultivated.
2, adopt the culture tank liquid culture.Pure bacterium inspection is carried out in sampling in incubation, and smear is done gram stain microscopy, as finding pollution microbes, should discard.Stop cultivating in the later stage of exponential phase or the early stage of resting stage.Bacterial concentration mensuration and the inspection of pure bacterium are carried out in sampling, and qualified back adds the formalin antibacterial in the culture fluid of results, or the antibacterial of employing heating means.Do not damage its polysaccharide antigen again and be advisable to guarantee antibacterial safety.
3, supernatant is collected in the centrifugal back of germ-resistant culture fluid (single batch or many batch mixings close), add cetyl trimethyl ammonium bromide, fully mixing forms deposition; Precipitate after centrifugal adds an amount of calcium chloride solution, and its ultimate density is 1mol/L, and the polysaccharide cetyl trimethyl ammonium bromide is dissociated, and adding ethanol to ultimate density is 25%, and 2-8 ℃ left standstill 1-3 hour or spent the night, the clarifying supernatant of centrifugal collection.In above-mentioned supernatant, adding cooling ethanol to ultimate density is 80%, shake well, and centrifugal collecting precipitation is respectively washed more than 2 times with dehydrated alcohol and acetone, and precipitate is the polysaccharide semifinished product, should be kept at below-20 ℃, and is to be purified.
4, semifinished product is dissolved in the 1/10 saturated neutral SAS, makes its concentration reach 10-20mg/ml; Extract for several times with cold phenol (the 100g crystalline phenol is dissolved in the 1/10 saturated acetic acid sodium solution of 40ml) by 1: 2 capacity, centrifugal collection supernatant, and with 0.1mol/L calcium chloride solution or other suitable solution dialysis, adding ethanol to final concentration is 75%～80%; The precipitate of centrifugal collection is respectively washed more than 2 times with dehydrated alcohol and acetone, and dry back is purified polysaccharide stock solution with sterilized water for injection dissolution precipitation thing.Leaching process should carry out below 15 ℃.
With the polysaccharide of physics and chemistry assay approval through cyanogen bromide-activated, ADH (1, the 6-AH) reaction that adds suitable proportion is spent the night, consider or other proper method are removed the Bromine cyanide. and the ADH of derivation not with ultra then, filtration sterilization, lyophilizing (or-20 ℃) is preserved.Sampling is examined and determine; Behind the assay approval, polysaccharide-AH derivant and tetanus toxoid mixed in equal amounts with suitable concentration add EDAC (carbodiimide) then; Mixture 2-8 ℃ keeps certain hour; With 0.2M NaCl solution dialysed overnight, reactant with Sepharose CL-4B column chromatography purification, is collected V
0The part eluent transfers pH to neutral with NaOH, and aseptic filtration both had been a conjugate stock solution, 2-8 ℃ of preservation.
6. semi-finished product preparation and packing
After combining Seedling stock solution, b type hemophilus influenza combined vaccinogen liquid to mix in proportion the AC group meningitis cocci polysaccharide of assay approval, carry out packing on request with acellular pertussis stock solution, branch loading amount 0.5mL/ bottle, it is to be checked to put 2-8 ℃ of storehouse.
7. finished product calibrating
Related request according to " the AC group meningitis cocci-b type hemophilus influenza combined vaccine system inspection tentative specification " worked out and existing " adsorbing manufacturing of acellular whooping cough combined vaccine and vertification regulation " is examined and determine comprehensively.
Embodiment 3:
200903 batches of whooping coughs, A+C group meningitis cocci-b type hemophilus influenza combined vaccines prepare:
1. get A group meningitis cocci 29201 bacterial strains, C group meningitis cocci 29205 bacterial strains, b type hemophilus influenza 58534 bacterial strains; Be inoculated in suitable agar culture medium; In the environment of 35-37 ℃ of carbon dioxide, cultivated 16-20 hour, smear is done gram stain microscopy, and the inoculation fermentation jar is cultivated.
2. adopt the culture tank liquid culture.Pure bacterium inspection is carried out in sampling in incubation, and smear is done gram stain microscopy, as finding pollution microbes, should discard.Stop cultivating in the later stage of exponential phase or the early stage of resting stage.Bacterial concentration mensuration and the inspection of pure bacterium are carried out in sampling, and qualified back adds the formalin antibacterial in the culture fluid of results, or the antibacterial of employing heating means.Do not damage its polysaccharide antigen again and be advisable to guarantee antibacterial safety.
3. supernatant is collected in the centrifugal back of germ-resistant culture fluid (single batch or many batch mixings close), add cetyl trimethyl ammonium bromide, fully mixing forms deposition; Precipitate after centrifugal adds an amount of calcium chloride solution, and its ultimate density is 1mol/L, and the polysaccharide cetyl trimethyl ammonium bromide is dissociated, and adding ethanol to ultimate density is 25%, and 2-8 ℃ left standstill 1-3 hour or spent the night, the clarifying supernatant of centrifugal collection.In above-mentioned supernatant, adding cooling ethanol to ultimate density is 80%, shake well, and centrifugal collecting precipitation is respectively washed more than 2 times with dehydrated alcohol and acetone, and precipitate is the polysaccharide semifinished product, should be kept at below-20 ℃, and is to be purified.
4. semifinished product is dissolved in the 1/10 saturated neutral SAS, makes its concentration reach 10-20mg/ml; Extract for several times with cold phenol (the 100g crystalline phenol is dissolved in the 1/10 saturated acetic acid sodium solution of 40ml) by 1: 2 capacity, centrifugal collection supernatant, and with 0.1mol/L calcium chloride solution or other suitable solution dialysis, adding ethanol to final concentration is 75%～80%; The precipitate of centrifugal collection is respectively washed more than 2 times with dehydrated alcohol and acetone, and dry back is purified polysaccharide stock solution with sterilized water for injection dissolution precipitation thing.Leaching process should carry out below 15 ℃.
With the polysaccharide of physics and chemistry assay approval through cyanogen bromide-activated, ADH (1, the 6-AH) reaction that adds suitable proportion is spent the night, consider or other proper method are removed the Bromine cyanide. and the ADH of derivation not with ultra then, filtration sterilization, lyophilizing (or-20 ℃) is preserved.Sampling is examined and determine; Behind the assay approval, polysaccharide-AH derivant and tetanus toxoid mixed in equal amounts with suitable concentration add EDAC (carbodiimide) then; Mixture 2-8 ℃ keeps certain hour; With 0.2M NaCl solution dialysed overnight, reactant with Sepharose CL-4B column chromatography purification, is collected V
0The part eluent transfers pH to neutral with NaOH, and aseptic filtration both had been a conjugate stock solution, 2-8 ℃ of preservation.
6. semi-finished product preparation and packing
After combining Seedling stock solution, b type hemophilus influenza combined vaccinogen liquid to mix in proportion the A of assay approval, the C group meningitis cocci polysaccharide, carry out packing on request with acellular pertussis stock solution, branch loading amount 0.5mL/ bottle, it is to be checked to put 2-8 ℃ of storehouse.
7. finished product calibrating
Related request according to " the AC group meningitis cocci-b type hemophilus influenza combined vaccine system inspection tentative specification " worked out and existing " adsorbing manufacturing of acellular whooping cough combined vaccine and vertification regulation " is examined and determine comprehensively.
Test Example 1:
Requirement according to " the AC group meningitis cocci-b type hemophilus influenza combined vaccine system inspection tentative specification " worked out and existing " adsorbing manufacturing of acellular whooping cough combined vaccine and vertification regulation " is examined and determine the result to whooping cough, A+C group meningitis cocci-b type hemophilus influenza combined vaccine comprehensively
Table one whooping cough, A+C group meningitis cocci-b type hemophilus influenza combined vaccine potency test
Table two whooping cough+AC group meningitis cocci-b type hemophilus influenza combined vaccine potency test
Result of the test shows; Whooping cough, A+C group meningitis cocci-b type hemophilus influenza combined vaccine are safe, effectively, meet the requirement of " the AC group meningitis cocci-b type hemophilus influenza combined vaccine system inspection tentative specification " worked out and existing " adsorbing manufacturing of acellular whooping cough combined vaccine and vertification regulation " fully.
Claims (2)
1. a whooping cough, A+C group meningitis cocci-b type hemophilus influenza combined vaccine, every volume contains following component and content for the 0.5ml finished product:
A group meningitis cocci polysaccharide: 10 μ g
C group meningitis cocci polysaccharide: 10 μ g
B type hemophilus influenza polysaccharide: 10 μ g
Lactose: 2.5～3.0mg
Acellular pertussis stock solution: 18 μ gPN/ml
Diphtheria toxoid: 25Lf/ml
Tetanus toxoid: 7Lf/ml
Aluminium hydroxide content: 1.0～1.5mg/ml.
2. the preparation technology of the said combined vaccine of claim 1 may further comprise the steps:
1) strain select for use: select A, C group meningitis Neisseria gonorrhoeae for use, b type hemophilus influenza strain is used for the preparation of A, C group meningitis cocci-b type hemophilus influenza combined vaccine;
2) seed is used in production
Breakdown work seed lot strain, through suitably going down to posterity, behind assay approval, inoculation improvement doup closes culture medium or other appropriate medias, and seed is used in the suitable production of preparation quantity;
3) culture medium is used in production
Adopt the improvement doup to close culture medium or through other appropriate medias of approval, culture medium should not contain with cetyl trimethyl ammonium bromide and forms sedimentary composition;
4) cultivate
Adopt the culture tank liquid culture, pure bacterium inspection is carried out in sampling in incubation, and smear is done gram stain microscopy, as finding pollution microbes, should discard;
5) results and antibacterial
Stop cultivating in the later stage of exponential phase or the early stage of resting stage; Bacterial concentration mensuration and the inspection of pure bacterium are carried out in sampling; Qualified back adds the formalin antibacterial in the culture fluid of results, or adopts the heating means antibacterial, does not damage its polysaccharide antigen again and is advisable to guarantee antibacterial safety;
6) enucleation acid
Germ-resistant list is criticized or many batches of blended culture fluid, and supernatant is collected in centrifugal back, adds cetyl trimethyl ammonium bromide, and fully mixing forms deposition; Precipitate after centrifugal adds an amount of calcium chloride solution, and its ultimate density is 1mol/L, and the polysaccharide cetyl trimethyl ammonium bromide is dissociated, and adding ethanol to ultimate density is 25%, and 2-8 ℃ left standstill 1-3 hour or spent the night, the clarifying supernatant of centrifugal collection;
7) deposition polysaccharide
In above-mentioned supernatant, adding cooling ethanol to ultimate density is 80%, shake well, and centrifugal collecting precipitation is respectively washed more than 2 times with dehydrated alcohol and acetone, and precipitate is the polysaccharide semifinished product, should be kept at below-20 ℃, and is to be purified;
8) polysaccharide purification
Semifinished product is dissolved in the 1/10 saturated neutral SAS, makes its concentration reach 10-20mg/ml; Use cold phenol by 1: 2 capacity, the 100g crystalline phenol is dissolved in the 1/10 saturated acetic acid sodium solution of 40ml, extract for several times, and centrifugal collection supernatant, and with 0.1mol/L calcium chloride solution or other suitable solution dialysis, adding ethanol to final concentration is 75%～80%; The precipitate of centrifugal collection is respectively washed more than 2 times with dehydrated alcohol and acetone, and dry back is purified polysaccharide stock solution with sterilized water for injection dissolution precipitation thing; Leaching process should carry out below 15 ℃;
9) polysaccharide activation, derivation combine with tetanus toxoid
Through cyanogen bromide-activated, ADH (1, the 6-AH) reaction that adds suitable proportion is spent the night with the polysaccharide of physics and chemistry assay approval, removes the Bromine cyanide. and the ADH of derivation not, filtration sterilization ,-20 ℃ of preservations with ultrafiltration or other proper method then; Sampling is examined and determine, behind the assay approval, with the polysaccharide-AH derivant and the tetanus toxoid mixed in equal amounts of suitable concentration; Add carbodiimide then, mixture 2-8 ℃ keeps certain hour, with 0.2M NaCl solution dialysed overnight; Reactant with Sepharose CL-4B column chromatography purification, is collected V0 part eluent, transfer pH to neutral with NaOH; Aseptic filtration is conjugate stock solution, 2-8 ℃ of preservation;
10) semi-finished product preparation and packing
After combining Seedling stock solution, b type hemophilus influenza combined vaccinogen liquid and acellular pertussis stock solution, diphtheria toxoid, tetanus toxoid to mix in proportion the A of assay approval, the C group meningitis cocci polysaccharide; Carry out packing on request; Divide loading amount 0.5mL/ bottle, it is to be checked to put 2-8 ℃ of storehouse.
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| CN2009100671560A CN101590225B (en) | 2009-06-19 | 2009-06-19 | Vaccine combining DTP (diphtheria,tetanus and pertussis) and A plus C group meningococcus-b type haemophilus influenzae |
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| CN102633896A (en) * | 2012-04-23 | 2012-08-15 | 成都欧林生物科技股份有限公司 | Hib polysaccharide purifying technology |
| CN102813917B (en) * | 2012-08-17 | 2014-10-22 | 武汉生物制品研究所有限责任公司 | Acellular pertussis/Haemophilus influenzae type b - group A and C meningococcus combined vaccine |
| CN104689309A (en) * | 2015-03-27 | 2015-06-10 | 成都欧林生物科技股份有限公司 | Separated and purified acellular pertussis-diphtheria-tetanus, b-type haemophilus influenzae and A-group and C-group meningococcus combined vaccine and preparation method thereof |
| CN105646726A (en) * | 2016-02-03 | 2016-06-08 | 成都康华生物制品有限公司 | Preparation method of type B haemophilus influenzae capsular polysaccharide and combined vaccine |
| CN106075421A (en) * | 2016-08-02 | 2016-11-09 | 成都欧林生物科技股份有限公司 | A kind of preparation method of A group's C group meningitis cocci b type hemophilus influenza combined vaccine |
| CN111135294A (en) * | 2020-01-19 | 2020-05-12 | 北京智飞绿竹生物制药有限公司 | Combined vaccine |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0101562A2 (en) * | 1982-08-20 | 1984-02-29 | American Cyanamid Company | Combined haemophilus influenzae and diphtheria, pertussis, tetanus vaccine |
| CN1433322A (en) * | 1999-11-29 | 2003-07-30 | 启龙股份公司 | Compositions comprising nei sseria meningitidis antigens from serogroups B and C |
| CN1971266A (en) * | 2006-12-06 | 2007-05-30 | 云南沃森生物技术有限公司 | Method for detecting content of free polysaccharides in A, C group meningitis cocci polysaccharides combo |
| CN1970779A (en) * | 2006-12-06 | 2007-05-30 | 云南沃森生物技术有限公司 | Process for purifying polysaccharide of bacteria by using Sepharose 4FF gel |
| WO2008149238A2 (en) * | 2007-06-04 | 2008-12-11 | Novartis Ag | Formulation of meningitis vaccines |
-
2009
- 2009-06-19 CN CN2009100671560A patent/CN101590225B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0101562A2 (en) * | 1982-08-20 | 1984-02-29 | American Cyanamid Company | Combined haemophilus influenzae and diphtheria, pertussis, tetanus vaccine |
| CN1433322A (en) * | 1999-11-29 | 2003-07-30 | 启龙股份公司 | Compositions comprising nei sseria meningitidis antigens from serogroups B and C |
| CN1971266A (en) * | 2006-12-06 | 2007-05-30 | 云南沃森生物技术有限公司 | Method for detecting content of free polysaccharides in A, C group meningitis cocci polysaccharides combo |
| CN1970779A (en) * | 2006-12-06 | 2007-05-30 | 云南沃森生物技术有限公司 | Process for purifying polysaccharide of bacteria by using Sepharose 4FF gel |
| WO2008149238A2 (en) * | 2007-06-04 | 2008-12-11 | Novartis Ag | Formulation of meningitis vaccines |
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