Method for purifying pneumococcal capsular polysaccharide

A technology of Streptococcus pneumoniae and capsular polysaccharide, applied in the fields of biology and vaccines, can solve the problems of increasing operation steps and time, increasing the risk of cross-contamination, loss of immunogenicity of polysaccharides, etc. The effect of improving the quality of pneumococcal polysaccharide vaccine and improving the production process

Inactive Publication Date: 2015-04-22
SINOVAC BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Due to the different chemical properties of the capsular polysaccharides of different serotypes of Streptococcus pneumoniae, the nature and content of impurities such as proteins and nucleic acids synthesized in the fermentation process are also different. Therefore, each serotype of capsular polysaccharides needs to use organic solvents (lower alcohols, Chloroform, acetone, phenol, etc.) step-by-step precipitation, enzyme digestion, ultrafiltration and other methods to remove protein and nucleic acid contamination, but so far there is no general method that can purify the capsular polysaccharides of 23 serotypes of Streptococcus pneumoniae, in production , all adopt the process of purifying capsular polysaccharides for different serotypes respectively
[0005] For the existing purification process of

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Streptococcus pneumoniae type 1 was fermented in a bioreactor; grown to the late logarithmic growth phase, sterilized and lysed with sodium deoxycholate at a final concentration of 0.1%. Centrifuge at 9000g for 50 minutes, and carry out ultrafiltration and concentration of the feed liquid with a membrane bag with a molecular weight cut-off of 100KD. After the fermentation broth volume is concentrated by 8 times, sodium acetate is added to make the final mass percentage concentration 7%, and pre-cooled 1- Propanol to make the final concentration of the volume percentage to 20%, settling at 4°C for 8 hours, centrifuging to recover the supernatant, replacing the 1-propanol and salt ions in the alcohol supernatant by buffer ultrafiltration, and obtaining Streptococcus pneumoniae Type 1 alcohol precipitation supernatant ultrafiltrate. Adjust the pH of the supernatant ultrafiltrate of alcohol precipitation to 8.5, add MgCl 2 Make the final concentration to 2mM, add Benzonase...

Embodiment 2

[0046] Streptococcus pneumoniae type 2 is fermented in a bioreactor; grow to the late logarithmic growth phase and sterilize with a lysing agent and lyse the bacteria. After being centrifuged at 9000g for 50 minutes and concentrated by ultrafiltration of a membrane bag with a molecular weight cut-off of 100KD, the volume of the fermented liquid was concentrated 8 times, and then sodium acetate was added to make the final mass percentage concentration 5%, and 2-propanol that had been cooled in advance was added to make it The final concentration of the volume percentage is 25%, let it stand for 22 hours at 4°C, centrifuge to recover the supernatant, replace the 2-propanol and salt ions in the alcohol precipitation supernatant with buffer ultrafiltration, and obtain the alcohol precipitation supernatant ultrafiltrate . Adjust the pH to 8.0, add MgCl 2 Make the final concentration to 3mM, add Benzonase nuclease to make the final concentration to 25U / ml, stir the reaction at 37°C...

Embodiment 3

[0054]Streptococcus pneumoniae type 5 is fermented in a bioreactor; grow to the late logarithmic growth phase and use a lysing agent to sterilize and lyse the bacteria. After centrifuging at 9000g for 50 minutes and concentration by ultrafiltration, the volume of the fermented liquid was concentrated 8 times, then sodium acetate was added to make the final mass percentage concentration 4%, and pre-cooled ethanol was added to make the final volume percentage concentration 12.5%, at 4°C Set aside to settle for 20 hours, centrifuge or filter to recover the supernatant, replace ethanol and salt ions in the alcohol precipitation supernatant with buffer ultrafiltration, and obtain the alcohol precipitation supernatant ultrafiltrate. Adjust the pH to 7.0 and add MgCl 2 Make the final concentration to 3mM, add Benzonase nuclease to make the final concentration to 30U / ml, stir and react at 37°C for 6h, the reaction catalyzed by Benzonase nuclease ends; adjust the pH to 7.5, add calcium...

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Abstract

The invention relates to a method for purifying pneumococcal capsular polysaccharide and belongs to the field of biological products. The method for purifying pneumococcal capsular polysaccharide comprises the following steps: cracking bacteria of a streptococcus pneumoniae inactivated and fermented liquid, carrying out clarification and ultra-filtration to obtain ultra-filtration concentration liquid, precipitating by using low alcohols and carrying out ultra-filtration, degrading protein and nucleic acid into micromolecule fragments through enzymic catalytic reaction under the specific reaction condition by using protease and nuclease, further finely purifying after purifications processes of ultra-filtration and chromatography in subsequent processes to obtain purified pneumococcal capsular polysaccharide. The method is mild in reaction condition, simple and convenient to operate; the process is liable to control; the defects of the prior art can be overcome; poisonous and harmful chemical reagents such as phenols, chloroform and acetone are not used; the method is environmentally friendly and friendly to operator; the prepared capsular polysaccharide is high in recovery rate while the content of protein and nucleic acid is reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the field of vaccines; the invention relates to a method for purifying capsular polysaccharide, in particular to a method for extracting and purifying capsular polysaccharide of Streptococcus pneumoniae without using phenol. Background technique [0002] Streptococcus pneumoniae is the main pathogenic bacterium that causes pneumonia-like diseases and upper respiratory tract infections such as otitis media, sinusitis, and bronchitis. In severe cases, it can cause central nervous system, heart valves, and bone and joint infections through the blood circulation system. At present, the injection of polyvalent pneumonia capsular polysaccharide vaccine is an effective means to reduce the infection of Streptococcus pneumoniae in children over 2 years old and high-infected people. [0003] In the fermentation process of Streptococcus pneumoniae, in addition to synthesizing capsular polysacch...

Claims

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Application Information

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IPC IPC(8): C08B37/00
Inventor 王见冬赵浩宋大伟梁海兰谭剑韩星高强尹卫东
Owner SINOVAC BIOTECH
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