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Method for purifying capsular polysaccharide

A technology of capsular polysaccharides and bacterial capsular polysaccharides, applied in the field of vaccines, can solve the problems of complex steps and low yield of extracted substances

Active Publication Date: 2010-06-09
SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the follow-up steps of this method are relatively complicated, requiring multi-step filtration, sieving filtration or ultrafiltration, and the yield of the extracted substance is low.

Method used

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  • Method for purifying capsular polysaccharide
  • Method for purifying capsular polysaccharide
  • Method for purifying capsular polysaccharide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0106] A preferred embodiment of the present invention comprises the following steps:

[0107] (1) Bacteria are fermented and cultivated in a bioreactor, and formaldehyde is added in the late stage of logarithmic growth to sterilize;

[0108] (2) Precipitation of soluble polysaccharides using cationic detergents, especially cetyltrimethylammonium salts (eg cetyltrimethylammonium bromide). Before the precipitation, the supernatant can be collected by centrifugation of the bacterial culture and the culture, or the collected supernatant can be concentrated;

[0109] (3) After precipitation, the precipitate was collected by centrifugation and redissolved. Preference is given to aqueous ethanol. The final ethanol concentration was between 20% and 30%. The optimum final ethanol concentration depends on the bacterial serogroup / type;

[0110] (4) Centrifuge the redissolved precipitate to collect the supernatant, and reprecipitate the polysaccharide. Preference is given to aqueous...

Embodiment 1

[0134] Example 1. Extraction of group C meningococcal meningitis polysaccharide

[0135] Open the freeze-dried group C ECM strains (bacteria number: CMCC 29025, purchased from the China Medical Bacteria Collection and Management Center), inoculate it into a common agar medium containing 10% sheep blood, and cultivate it under a carbon dioxide environment at 35-37°C for 16 Hour.

[0136] The cultured bacteria were inoculated into semi-comprehensive slant medium, and cultured for 16 hours at 35-37°C in a carbon dioxide environment. Scrape the bacterial lawn and inoculate until 50ml of improved semi-comprehensive medium is installed (prepared by yourself according to "Manufacture and Application of Microbial Culture Medium" , the medium mainly The ingredients are yogurt hydrolyzate, sodium glutamate, glycine, yeast dialyzate, ammonium chloride, magnesium sulfate, glucose, etc.) in a triangular flask, shake culture at 35-37°C for 3 hours, press 1% (v / v ) ratio to inoculate a 5L ...

Embodiment 2

[0139] Example 2. Extracting group C meningococcal meningitis polysaccharide from 5L tank culture

[0140] Freeze-dried Group C ECM strains (bacteria number: CMCC29205, purchased from the China Medical Bacteria Collection and Management Center) were opened, and the bacteria were cultured, sterilized and the culture supernatant was harvested as in Example 1. The incubation time in a 5 L fermenter was 10 hours. Add cetyltrimethylammonium bromide at 0.1% (w / v), and place at 2-8°C overnight to precipitate polysaccharides. The precipitated polysaccharide complexes were collected by centrifugation at 4000 rpm for 30 minutes. Precipitate the complex with 1M CaCl 2 Dissolve, add ethanol to 20% (v / v), most impurities such as nucleic acid and protein will not dissolve. The clarified supernatant was collected by centrifugation at 8000 rpm for 60 minutes. Add ethanol to 70% (v / v) to precipitate polysaccharides. The precipitated polysaccharide was collected by centrifugation, washed t...

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Abstract

The invention relates to a treatment method for purifying capsular polysaccharide, comprising the following steps of: (a), adding a lower alcohol with low final concentration into a cell capsular polysaccharide precipitate, and dissolving the capsular polysaccharide to form a mixed liquid; (b), carrying out solid-liquid separation on the mixed liquid and collecting a supernate; (c), adding a lower alcohol with high final concentration into the obtained supernate to form a rough precipitate containing the cell capsular polysaccharide; (d) adding the lower alcohol with the low final concentration into the rough precipitate so that the polysaccharide in the rough precipitate is in a dissolving state; (e) adding the lower alcohol with the high final concentration into the supernate so as to form the capsular polysaccharide precipitate; and (f) collecting the obtained capsular polysaccharide precipitate to obtain a purified cell capsular polysaccharide. The method conveniently, controllably and effectively improves recycling rates of the capsular polysaccharide and improves the security without using toxic reagents of phenol or chloroform.

Description

technical field [0001] The invention relates to the field of vaccines, in particular to a treatment method for capsular polysaccharides of capsulated bacteria. Background technique [0002] It is well known in the art that vaccines can be used to prevent pathogenic bacterial infections. Currently, vaccines commonly used to prevent pathogenic bacterial infections include bacterial vaccines and subunit vaccines. Bacterial vaccines can be prepared by inactivating bacteria or attenuating pathogenic bacteria, while subunit vaccines are prepared by extracting proteins and / or polysaccharides of pathogenic bacteria. [0003] Polysaccharide vaccines are a class of vaccines prepared by purifying bacterial polysaccharides, such as capsular polysaccharides. Polysaccharide vaccines already on the market include: Bacillus influenzae type b polysaccharide vaccine, meningococcal polysaccharide vaccine, pneumococcal polysaccharide vaccine and typhoid Vi polysaccharide vaccine, etc. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/00A61K39/095A61K31/715A61P31/04
CPCY02A50/30
Inventor 朱为江元翔荣家康
Owner SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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