Method for purifying bacterial capsular polysaccharide

A technology of capsular polysaccharide and purification method is applied in the field of purification of bacterial capsular polysaccharide crude product, can solve the problems of polysaccharide reduction, unfavorable production, solution volume limitation, etc., achieves reduction of protein and nucleic acid impurities, easy industrial production, and process Simple and fast effects

Active Publication Date: 2017-08-15
SHANGHAI RUIZHOU BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Ethanol precipitation is usually effective in removing protein contaminants from polysaccharides, but it is difficult to meet the purity requirements of vaccines for injection
In addition, the use of ethanol can also bring the following problems: 1) the use of ethanol requires fire prevention facilities, and the cost of designing such facilities is very high; Waste liquid treatment is very difficult; 3) It is difficult to redissolve the polysaccharide after treatment, and steps such as freeze-drying are required
Since some serotype capsular polysaccharides have phosphate ions (such as 6A, 6B, 19F, etc.), the filler will bind a large number of such polysaccharide molecules during chromatography, resulting in a significant reduction in the p

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Preparation of Streptococcus pneumoniae polysaccharide serotypes 1, 3, 4, 7F, 9v, 15B, 19A, 19F, 23F, 33F capsular crude polysaccharide

[0034] Streptococcus pneumoniae fermentation broth was dissolved with sodium deoxycholate (DOC) at 4°C for 12-24h. Then add acid directly to the bacterial lysate, adjust the pH value of the lysate containing capsular polysaccharide to 3-6.5, let it stand at room temperature for more than 1 hour, and centrifuge to remove impurities such as lysate, some proteins and nucleic acids, and a large number of cell debris. Centrifuge the acidified solution for one hour at 20° C. with a relative centrifugal force greater than 10,000 g, collect the supernatant and discard the precipitate. The obtained supernatant is ultrafiltered with a molecular cut-off of 50-100kD membrane, diafiltered with pure water to further remove small molecular impurities, and finally concentrated to obtain a crude capsular polysaccharide solution.

[0035] T...

Embodiment 2

[0037]Example 2. Analysis of the treatment effect of 19F serotype capsular polysaccharide solution by different chromatography packing materials

[0038] Experimental operation

[0039] Ion-exchange chromatography (Capto DEAE, GE): The crude polysaccharide solution obtained after acidification treatment uses a membrane with a molecular weight cut-off of 10-100kD, and uses 5-50mM phosphate buffer solution at a pH of 6 to 8 to further ultrafilter and change the solution. until a constant conductance is reached. The Capto DEAE chromatographic column was equilibrated with the same buffer, and then the capsular polysaccharide solution after the exchange was applied to the column, and the flow-through was collected. After loading the samples, wash with 1-2 column volumes of 25 mM phosphate buffer, and the polysaccharides are recovered in the flow-through and washing solutions. Proteins, nucleic acids, and polysaccharides bound to the Capto DEAE column were eluted with 1M NaCl. ...

Embodiment 3

[0048] Example 3. Preparation of Streptococcus pneumoniae polysaccharide serotypes 1, 3, 4, 7F, 9v, 15B, 19A, 19F, 23F, 33F refined sugar

[0049] The crude polysaccharide solution obtained after the acidification treatment uses a membrane with a molecular weight cut-off of 10-100kD, uses 5-50mM phosphate buffer solution, and the salt ion concentration (NaCl) is preferably at 100-300mM, and is further ultrafiltered until a constant conductivity is achieved. The Hydroxyapatite chromatography column is preferably connected in series with the Capto adhere chromatography column and then equilibrated with the same buffer solution, and then the capsular polysaccharide solution after the liquid exchange is applied to the column to collect the flow-through. After the sample loading is completed, further wash with 1-2 column volumes of the same salt ion buffer, and the polysaccharides are recovered in the flow-through and wash solutions.

[0050] Table 3. Protein and nucleic acid conte...

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Abstract

The invention discloses a method for purifying bacterial capsular polysaccharide. The method comprises the following steps: treating a streptococcus pneumonia fermentation culture solution with an inactivator, adding acid for regulating the pH value to be less than or equal to 6.5, precipitating impurities, centrifuging, and carrying out microfiltration for removing precipitates, so that polysaccharide clarified liquor is obtained; carrying out ultrafiltration liquid change on the polysaccharide clarified liquor by adopting a membrane with the molecular weight cut-off of 10-100KDa and concentrating, and carrying out chromatography on the obtained capsular polysaccharide solution by combining compound type ion exchange chromatography and hydroxyl phosphate grey salt chromatography, so that the purified capsular polysaccharide solution is obtained; and concentrating and carrying out ultrafiltration on the capsular polysaccharide solution, and carrying out sterile filtration and storing. By combining the compound type ion exchange chromatography and the hydroxyl phosphate grey salt chromatography, the contents of protein and nucleic acid impurities can be effectively reduced to be lower than 1%, the quality of a purified polysaccharide product is higher than European Union pharmacopoeia standard requirements, and the recovery rate is more than 60%. The method disclosed by the invention has the advantages that the technology is simple and rapid, the enlargement is easy, and industrial production can be realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a method for purifying crude bacterial capsular polysaccharides. Background technique [0002] Streptococcus pneumoniae is a Gram-positive bacterium with more than 90 serotypes, most of which are coated with a layer of capsular polysaccharide. Capsular polysaccharides can evade recognition by the immune system and prevent phagocytosis by immune cells, thus allowing bacteria to multiply in the body and cause disease. Years of research and clinical studies have proved that the capsular polysaccharide of Streptococcus pneumoniae as a vaccine can induce specific antibodies and has a good immune protection against the corresponding Streptococcus pneumoniae. The composition and structure of the capsular polysaccharides of different serotypes of Streptococcus pneumoniae are different, so it is usually necessary to use a combination of polysaccharides of different serotypes ...

Claims

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Application Information

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IPC IPC(8): C08B37/00
CPCC08B37/0003
Inventor 祝先潮陈春光
Owner SHANGHAI RUIZHOU BIOTECH CO LTD
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