Preparation method of type B haemophilus influenzae capsular polysaccharide and combined vaccine

A technology of Haemophilus influenzae and capsular polysaccharide is applied in the field of preparation of Haemophilus influenzae type B capsular polysaccharide and combined vaccine, which can solve problems such as failure to develop successfully, and achieves reducing medical risks, reducing the number of vaccinations, and improving immunity. effect of effect

Inactive Publication Date: 2016-06-08
CHENGDU KANGHUA BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The DTaP vaccine to prevent diphtheria, tetanus and pertussis has been widely used in infant immunization in China, and the Hib conjugate vaccine to prevent infant meningitis and pneumonia has been successfully developed and included in the local expanded immunization program. The immunization procedures of the two vaccines are the same. At present, DTaP-Hib combined vaccines have been commercially available abroad, but DTaP-Hib combined vaccines have not been successfully developed in China.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] A preparation method of Haemophilus influenzae type B capsular polysaccharide, comprising the following steps:

[0034] (1) Establishment of Haemophilus influenzae type B seed batch strains

[0035] Haemophilus influenzae type B CMCC58547 or CMCC58534 strains were used, which came from China Institute for the Control of Pharmaceutical and Biological Products. The number of passages after the main seed batch is opened shall not exceed 5 generations, and the number of passages from the opening of the working seed batch to the inoculation fermenter shall not exceed 5 generations.

[0036] (2) Preparation of seeds for production

[0037] Start the batch of working seed strains, inoculate them on the liquid or solid Hib comprehensive medium, and prepare a suitable number of seeds for production.

[0038] (3) Preparation of culture medium for production

[0039] Hib comprehensive culture medium is used, and it must not contain harmful or other allergen substances.

[0040...

Embodiment 2

[0050] A preparation method of Haemophilus influenzae type B capsular polysaccharide, comprising the following steps:

[0051] (1) Fermentation culture: Cultivate 100L of bacterial liquid with 300 liters of fermentation tanks, inoculate initial concentration A600=0.18, culture pH is 7.5, culture temperature is 36 ℃, cultivates 10~15 hours, harvests bacterial concentration A600≥4.0. During the cultivation process, samples were taken for pure bacteria test and smear Gram staining microscopic examination. If contamination with miscellaneous bacteria was found, it should be discarded.

[0052] (2) Sterilization precipitation: When the bacteria are in the late stage of logarithmic growth or the early stage of the stationary phase, the culture is terminated, the sample is taken for the determination of the concentration of the bacteria liquid and the inspection of the pure bacteria, and then the appropriate concentration of formaldehyde solution or sodium deoxycholate is added to the...

Embodiment 3

[0061] A combined vaccine containing capsular polysaccharide of Haemophilus influenzae type b comprises capsular polysaccharide of Haemophilus influenzae type b, acellular pertussis vaccine, diphtheria toxoid vaccine and tetanus toxoid vaccine; its preparation method is:

[0062] It includes adding acellular pertussis stock solution, refined diphtheria toxoid, and refined tetanus toxoid to aluminum hydroxide for adsorption, then adding Haemophilus influenzae type B capsular polysaccharide stock solution, then diluting with normal saline, adding thimerosal to contain thimerosal per ml One-half, adjust the pH value to 5.8, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDC to react for 1 hour; then adjust the pH to 7.0, then lower the temperature to 4°C, and finally Dialyze in 0.2mol / L NaCl solution, sterilize after concentration by ultrafiltration, and store at 4°C.

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Abstract

The invention discloses a preparation method of a type B haemophilus influenzae capsular polysaccharide. The preparation method comprises the steps of fermentation culture, sterilization and precipitation, polysaccharide extraction and purification, polysaccharide derivatization and polysaccharide protein conjugate preparation. The invention further discloses a multivalent combined vaccine which is prepared through the steps that an acellular pertussis stock solution, refined diphtheria toxoid and refined tetanus toxoid are added into aluminum hydroxide to be adsorbed and then added into a type B haemophilus influenzae capsular polysaccharide stock solution, and the mixed solution is diluted with normal saline. According to the preparation method, the prior art is optimized, and the high-purity low-impurity-content type B haemophilus influenzae capsular polysaccharide can be obtained; meanwhile, the type B haemophilus influenzae capsular polysaccharide is combined with multiple component vaccines to form the multivalent vaccine, the multivalent vaccine can be immune to multiple diseases simultaneously, the inoculating times are decreased, the medical risk is reduced, and the better immune effect can be obtained through one-time inoculating.

Description

technical field [0001] The invention relates, in particular, to a preparation method of Haemophilus influenzae type B capsular polysaccharide and a combined vaccine. Background technique [0002] Haemophilus influenzae is a common pathogenic bacterium, which is only pathogenic to humans, and can be divided into encapsulated strains and non-encapsulated strains. Capsular strains are divided into six serotypes (a-f) according to the specific antigens of capsular polysaccharides, among which type b antigens are the most virulent and are the main pathogenic bacteria of children with sepsis, meningitis and pneumonia. The Hib capsule is composed of lipopolysaccharide and capsular polysaccharide, the main component is ribose phosphate alcohol phosphate (polyriboylribitolphosphate, PRP), its structure includes a ribose, a ribitol and a phosphate base. [0003] Hib is transmitted by droplets and mainly infects children under 5 years old. Neonates transmit serum bactericidal antibod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/00C12P19/04A61K39/116A61K39/10A61K39/08A61K39/05A61K39/102A61P31/04C12R1/02
CPCA61K39/0018A61K39/102A61K2039/70C08B37/00C08B37/0003C12P19/04A61K2300/00
Inventor 侯文礼
Owner CHENGDU KANGHUA BIOLOGICAL PROD
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