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Novel method for removing protein impurities in groups A, C meningococcal capsular polysaccharide

A technology of meningococcal and capsular polysaccharides, which is applied to protein impurities in the capsular polysaccharides of group C meningococci and removes the A field, which can solve the problems of high cost, great harm, and incapability of large-scale production, and achieve low production costs , simple operation, suitable for large-scale production

Active Publication Date: 2013-09-04
CHENGDU OLYMVAX BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of refining and purifying capsular polysaccharides, removing protein impurities is a very important step. At present, the commonly used methods include cold phenol extraction and ultrafiltration after protease hydrolysis, among which ultrafiltration after protease hydrolysis is the most effective, but due to the cost High, can not be applied to large-scale production, so cold phenol extraction has become the main method for production-scale refining and purification of capsular polysaccharide deproteinization
However, the cold phenol extraction method requires the use of a large amount of phenol, which is very harmful to the human body and the environment.

Method used

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  • Novel method for removing protein impurities in groups A, C meningococcal capsular polysaccharide
  • Novel method for removing protein impurities in groups A, C meningococcal capsular polysaccharide

Examples

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Effect test

Embodiment 1

[0013] Embodiment 1: the new method of removing protein impurity in the capsular polysaccharide of group A meningococcus, it comprises the following steps:

[0014] S1. Dissolve 500 mg of the crude Group A epidemic cerebrospinal meningitis capsular polysaccharide with water for injection, dilute it into a polysaccharide solution of 5 mg / ml, add 2%, sodium deoxycholate solution of pH8.0, and the volume ratio is 0.5: 1. Mix well and stir at 2°C for 0.5h;

[0015] S2. Adjust the pH to 2 with 1mol / L hydrochloric acid, stir at 2°C for 3min, and then centrifuge at 8000rpm for 1.5h;

[0016] S3. Take the supernatant, adjust the pH to 7 with 1mol / L sodium hydroxide, and repeat steps S1 and S2 once;

[0017] S4. Take the supernatant, adjust the pH to 7 with 1mol / L sodium hydroxide, dilute 5 times with water for injection, ultrafilter to the original volume through a 250kd ultrafiltration membrane, repeat dilution, ultrafiltration concentration twice, and the last ultrafiltration conce...

Embodiment 2

[0018] Embodiment 2: the new method of removing protein impurity in the capsular polysaccharide of group A meningococci, it comprises the following steps:

[0019] S1. Dissolve 700 mg of crude Group A epidemic cerebrospinal meningitis capsular polysaccharide with water for injection, dilute it into a polysaccharide solution of 10 mg / ml, add 2%, sodium deoxycholate solution of pH8.0, and the volume ratio is 1.5: 1. Mix well and stir at 6°C for 1.5h;

[0020] S2. Adjust the pH to 3 with 1mol / L hydrochloric acid, stir at 6°C for 7min, and then centrifuge at 8000rpm for 1.5-2.5h;

[0021] S3. Take the supernatant, adjust the pH to 7 with 1mol / L sodium hydroxide, and repeat steps S1 and S2 once;

[0022] S4. Take the supernatant, adjust the pH to 7 with 1mol / L sodium hydroxide, dilute 15 times with water for injection, ultrafilter to the original volume through a 350kd ultrafiltration membrane, repeat dilution, ultrafiltration concentration 5 times, and the last ultrafiltration co...

Embodiment 3

[0023] Embodiment 3: the new method of removing protein impurity in the capsular polysaccharide of group A meningococci, it comprises the following steps:

[0024] S1. Dissolve 1000 mg of crude Group A meningococcal meningitis capsular polysaccharide with water for injection, dilute it into a polysaccharide solution of 8 mg / ml, add 2%, sodium deoxycholate solution of pH8.0, and the volume ratio is 1: 1. Mix well and stir at 4°C for 1 hour;

[0025] S2. Adjust the pH to 2 with 1mol / L hydrochloric acid, stir at 4°C for 5min, and then centrifuge at 8000rpm for 2h;

[0026] S3. Take the supernatant, adjust the pH to 7 with 1mol / L sodium hydroxide, and repeat steps S1 and S2 once;

[0027] S4. Take the supernatant, adjust the pH to 7 with 1mol / L sodium hydroxide, dilute 10 times with water for injection, ultrafilter to the original volume through a 300kd ultrafiltration membrane, repeat dilution, ultrafiltration concentration 3 times, and the last ultrafiltration concentration to...

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Abstract

The invention discloses a novel method for removing protein impurities in groups A, C meningococcal capsular polysaccharide. According to the novel method, under the acidic pH condition, an ionic detergent sodium deoxycholate has characteristics of forming micelle and absorbing the precipitate protein and is utilized to separate capsular polysaccharide and the protein. Refined polysaccharides produced by the novel method all accord with pharmacopoeia requirements. Recovery rate of polysaccharide is similar to that of polysaccharide by a traditional cold phenol extraction method. In addition, the novel method provided by the invention has characteristics of simple operation, low production cost and no harm to production personnel and the environment, and is more suitable for large-scale production.

Description

technical field [0001] The invention relates to the technical field of methods for removing protein impurities in meningococcal capsular polysaccharides, in particular to a new method for removing protein impurities in group A and C meningococcal capsular polysaccharides. Background technique [0002] Neisseria meningitidis (Nm) is the primary pathogenic bacteria of meningitis and fulminant sepsis, and the incidence of epidemic meningitis (hereinafter referred to as meningitis) is often sporadic, and there is no obvious difference among cases. Associated outbreaks, usually in small-scale fashion, can turn into catastrophic and unpredictable endemic epidemics in some areas. There are about 500,000 meningitis cases and 50,000 deaths worldwide each year. Nm is an aerobic, Gram-negative, capsuled, usually in pairs (diplococcus). Nm can be divided into 13 serogroups according to the chemical composition of capsular polysaccharides, and can be divided into different serotypes an...

Claims

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Application Information

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IPC IPC(8): C08B37/00
Inventor 陈克平伍长华关晓峰樊钒
Owner CHENGDU OLYMVAX BIOPHARM
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