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Purifying method for bacterial capsular polysaccharide

A technology of bacterial capsular polysaccharide and purification method, which is applied in the directions of pharmaceutical formulations, medical preparations containing active ingredients, and antibody medical ingredients, etc. The effect of reducing toxin content, expanding production scale, and easy production scale

Active Publication Date: 2016-11-23
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are three existing methods for removing impurity proteins in bacterial capsular polysaccharides: first, use phenol extraction to remove impurity proteins, and use ultracentrifugation to remove endotoxins, but phenol has strong toxicity and strong corrosiveness, which is harmful to the work. Hazard to human health; phenol is also a serious hazard to the environment
Secondly, column chromatography is used to remove impurity proteins and purify capsular polysaccharides, but this method has a small amount of processing and high cost, and the purification methods required for different bacterial capsular polysaccharides are different, and no enterprise has yet used it for vaccine production
Finally, relevant literature and patent reports use ethanol precipitation to remove protein, but this method requires a large amount of ethanol, which is a flammable reagent and requires relatively high requirements for plant and equipment. There is a big difference in the effect

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] A method for purifying bacterial capsular polysaccharide, said purification method comprising the following steps:

[0032] (a) Dissolving polysaccharide: use pre-cooled NaAC and NaCl mixed solution to dissolve the rough sugar containing bacterial capsular polysaccharide to obtain mixed solution A;

[0033] (b) Extraction and purification: Add non-ionic surfactant to the mixed solution obtained in step (a), put it in ice bath for 5-15 minutes, then place it at room temperature for 40-60 minutes, then separate the solid and liquid, and collect the Serum;

[0034] (c) collecting the supernatant obtained in step (b), repeating the treatment of step (b) 3 to 5 times for the supernatant, and collecting the final supernatant to obtain the mixed solution B;

[0035](d) Add absolute ethanol to the mixture B to a final concentration of 70-90%, mix well, let stand at 4°C for 20-40 minutes, then separate the solid and liquid, and collect the precipitate A;

[0036] (e) with CaCl...

Embodiment 2

[0045] In this example, TritonX114 was selected as a non-ionic surfactant to extract and purify bacterial capsular polysaccharide from meningococcus group A rough sugar. The specific preparation method is as follows:

[0046] (a) Dissolving polysaccharide: use 400ml pre-cooled 0.4MNaAC / 6%NaCl mixed solution to dissolve 2.6g group A meningococcal crude sugar to obtain mixed solution A;

[0047] (b) Extraction and purification: Add 104ml TritonX114 to the mixture obtained in step (a), bathe in ice for 10min, then place it at room temperature for 30min, centrifuge at 15000g, 20°C for 30min, and collect the supernatant;

[0048] (c) collecting the supernatant obtained in step (b), repeating the treatment of step (b) 3 times for the supernatant, and collecting the final supernatant to obtain the mixed solution B;

[0049] (d) Add absolute ethanol to the mixture B to a final concentration of 80%, mix well, let stand at 4°C for 20-40min, centrifuge at 6500g, 4°C for 30min, and coll...

Embodiment 3

[0061] In this example, TritonX114 was selected as a non-ionic surfactant to extract and purify bacterial capsular polysaccharide from meningococcus group C crude sugar. The specific preparation method is as follows:

[0062] (a) Dissolving polysaccharide: use 400ml pre-cooled 0.4MNaAC / 6%NaCl mixed solution to dissolve 2.6g meningococcal group C crude sugar to obtain mixed solution A;

[0063] (b) Extraction and purification: Add 104ml TritonX114 to the mixture obtained in step (a), bathe in ice for 10min, then place it at room temperature for 30min, centrifuge at 15000g, 20°C for 30min, and collect the supernatant;

[0064] (c) collecting the supernatant obtained in step (b), repeating the treatment of step (b) 3 to 5 times for the supernatant, and collecting the final supernatant to obtain the mixed solution B;

[0065] (d) Add absolute ethanol to the mixture B to a final concentration of 80%, mix well, let stand at 4°C for 20-40min, centrifuge at 6500g, 4°C for 30min, and...

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Abstract

The invention discloses a purifying method for bacterial capsular polysaccharide. According to the purifying method, the bacterial capsular polysaccharide is purified with a phase separation method through a nonionic surfactant. Impurity protein and endotoxin are required to be removed as many as possible in a preparation process of a bacterial capsular polysaccharide vaccine, so that side reactions of the vaccine can be reduced. Conventionally used phenol extraction method, column chromatography and ethyl alcohol precipitation method all have many defects. Compared with phenol, the nonionic surfactant has characteristics of being free of corrosion or carcinogenicity and the like and cannot harm the environment. Compared with column chromatography, the purifying method has the advantages that the handling capacity is high, time saving and economical performance are realized, production scale is easy to expand, and the purifying method for the bacterial capsular polysaccharide is safe, environment-friendly and efficient.

Description

technical field [0001] The invention belongs to the field of polysaccharide purification, in particular to a method for purifying bacterial capsular polysaccharide. Background technique [0002] Bacterial infectious disease is a disease that is harmful to the population. At present, the main drugs used to treat bacterial infectious diseases are antibiotics. However, the abuse of antibiotics and other antibacterial drugs has led to the rapid increase of drug-resistant bacteria, making it unable to effectively control infection. Bacterial vaccines can improve the resistance of susceptible populations to bacteria and reduce the incidence of pathogenic bacterial infections. Therefore, the role of bacterial vaccines in the control and prevention of bacterial infectious diseases cannot be underestimated. A variety of bacterial vaccines are currently on the market, mainly divided into inactivated vaccines, live attenuated vaccines, and component vaccines. Among them, the bacterial...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/00
CPCC08B37/00C08B37/0003A61K39/095
Inventor 寸韡肖红剑毕研伟李育中李智华丁晨高丹丹闫玲梅
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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