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Epidemic encephalitis polysaccharide-protein conjugated vaccine and preparation method thereof

A protein and polysaccharide technology, applied in the direction of pharmaceutical formulations, medical preparations with non-active ingredients, medical preparations containing active ingredients, etc., can solve the problems of increasing the overall production cost of products, polysaccharide loss, etc., and achieve polysaccharide-protein ratio stability, The effect of low production cost and simple process flow

Inactive Publication Date: 2013-01-09
CANSINO BIOLOGICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These linkers are generally inert, that is, they will not chemically react with human proteins or other macromolecules under physiological conditions, and will be removed as much as possible in the preparation process of conjugated vaccines, but these linkers exist in the conjugated product, Can cause the human immune system to generate an immune response against these linkers, and produce corresponding ineffective antibodies
At present, polysaccharide conjugate vaccines for meningitis not only use linker technology, but also need to depolymerize the polysaccharide to reduce its molecular weight, but in this process, the polysaccharide will be lost, thereby increasing the overall production cost of the product

Method used

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  • Epidemic encephalitis polysaccharide-protein conjugated vaccine and preparation method thereof
  • Epidemic encephalitis polysaccharide-protein conjugated vaccine and preparation method thereof
  • Epidemic encephalitis polysaccharide-protein conjugated vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Preparation of purified meningococcal polysaccharide powder from Neisseria meningitidis serotypes W-135 and Y

[0043] Neisseria meningitidis serotypes W-135 and Y were fermented separately.

[0044] After centrifuging the 30L fermented liquid obtained respectively, the supernatant was obtained and placed in a stainless steel tank, concentrated to 3L by ultrafiltration using a 100KD membrane bag, and 300ml of cetyltrimethyl bromide with a mass concentration of 10% was added to the concentrated solution ammonium chloride (CTAB) aqueous solution, stirred evenly, and then stood still at 4°C for 2 hours. The rested solution was centrifuged to obtain a precipitate, and sterile 1M sodium chloride aqueous solution was added to 1 L of the precipitate, and the precipitate was dissolved at 40°C. Add absolute ethanol to the solution to a final volume concentration of 25%, stir evenly and stand at -20°C for 2 hours, then centrifuge to obtain the supernatant; add absolute ethanol t...

Embodiment 2

[0047] Preparation of carrier protein

[0048] Lyophilized seed cultures expressing diphtheria CRM197 protein were reconstituted and incubated for 16 hours. Transfer a portion of the culture to a 0.5-liter shaker flask containing growth medium and incubate the flask on a rotary shaker at 34.5-36.5°C for 8 hours. Transfer a portion of the culture from the flask to a 4-liter shaker flask containing growth medium Liter shaker flasks, and incubate the flasks on a rotary shaker at 34.5-36.5°C for 18 hours. The culture from this 4 liter shake flask was used to inoculate a fermenter containing 30 L of growth medium. The fermenters were incubated for 28 hours at 30-36.5°C, pH 7.4. The fermenter contents are filtered through centrifuges and depth filters into collectors.

[0049] The obtained 30L fermentation broth was concentrated to 2L with a 30KD membrane bag, and 500mM phosphate buffer (sodium salt) was added to the concentrated solution to a final concentration of 10mM. After ...

Embodiment 3

[0053] Activation of Neisseria meningitidis serotype Y polysaccharide

[0054] Dissolve 5g of the purified ECM Y-type polysaccharide in 1L of sodium acetate buffer (50mM pH5.0), and stir for 20 minutes with a magnetic stirrer with a magnet at room temperature to fully dissolve the purified ECM polysaccharide , add NaIO according to the ratio in Table 1 4 , and react respectively by the temperature and time shown in each reaction in Table 1, so that the adjacent dihydroxyl groups on the meningitis polysaccharide are oxidized into aldehyde groups.

[0055] Table 1. Activated meningitis polysaccharides with different aldehyde group introduction ratios obtained under different reaction conditions

[0056]

[0057] Prepare an ultrafiltration system equipped with a 30K MWCO filter membrane, place the reaction solution in the reflux vessel of this system, perform 10 equal-volume ultrafiltration replacements with purified water, and remove the remaining NaIO in the reaction 4 and...

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Abstract

The invention discloses an epidemic encephalitis polysaccharide-protein conjugated vaccine and a preparation method thereof. The vaccine is represented by [S-C]n-[N-P]m, wherein S represents an epidemic encephalitis polysaccharide molecule; C represents an atom C into which an aldehyde group is introduced in the epidemic encephalitis polysaccharide molecule; P represents a CRM197 protein molecule; N represents an atom N of lysine side chain amine in the protein molecule; and the epidemic encephalitis polysaccharide is at least one of a W-135 type epidemic encephalitis polysaccharide and a Y type epidemic encephalitis polysaccharide. In the vaccine disclosed by the invention, the Y type and W-135 type epidemic encephalitis polysaccharides are not subjected to a depolymerization technical process, and the Y type and W-135 type epidemic encephalitis polysaccharides are connected with proteins without using any connector. The method has the advantages of simple process flow, high yield, low production cost, relatively stable polysaccharide-protein ratio, capability of performing relative manual control in a horizontal range, and great improvement on the quality stability of the vaccine.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a meningococcal polysaccharide-protein conjugated vaccine and a preparation method thereof. Background technique [0002] Neisserza meningitides is the leading cause of bacterial meningitis worldwide. During the last 30 years, the incidence of endemic meningococcal disease in developing countries ranged from 10 to 25 per 100,000 (Reido, F.X. et al., 1995). The case fatality rate ranges from 10 to 20%. [0003] Pathogenic meningococci are encapsulated by a polysaccharide capsule that adheres to the outer membrane surface of the organism. Thirteen different meningococcal serotypes have been identified based on the immunological specificity of capsular polysaccharides. Five of these 13 serotypes cause the majority of meningococcal disease; they include serotypes A, B, C, W-135 and Y. Serotype A accounts for most of the epidemic diseases. Serotypes B, C and Y cause most ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/095A61K39/385A61K47/48A61P31/04
Inventor 朱涛宇学峰刘正邵忠琦毛慧华邱东旭
Owner CANSINO BIOLOGICS INC
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