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Method for preparing pneumococcal capsular polysaccharide and pneumococcal vaccine

A technology of pneumococcus and capsular polysaccharides, which is applied in the direction of antibacterial drugs and bacterial antigen components, can solve the problems of ethanol operation and storage safety hazards, extremely high requirements for production plants, and increase production costs, and achieve excellent purification effects and eliminate Potential safety hazards and the effect of automation

Pending Publication Date: 2022-08-05
LANZHOU INST OF BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, phenol is harmful to the human body and the environment, and ethanol also has major safety hazards in operation and storage. It has extremely high requirements on the production plant and increases production costs.

Method used

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  • Method for preparing pneumococcal capsular polysaccharide and pneumococcal vaccine
  • Method for preparing pneumococcal capsular polysaccharide and pneumococcal vaccine
  • Method for preparing pneumococcal capsular polysaccharide and pneumococcal vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: Purification of type 1 pneumococcal capsular polysaccharide

[0083] Type 1 pneumococcus was fermented in a bioreactor, and after growing to the logarithmic growth phase, sodium deoxycholate was added to lyse the bacteria. The supernatant was collected by centrifugation, and concentrated by ultrafiltration with an ultrafiltration membrane with a molecular weight cut-off of 300 Kd to obtain a type 1 pneumococcal lysate.

[0084] Sodium deoxycholate was added to the lysate of type 1 pneumococcus to make the final concentration 0.5%, the pH was adjusted to 4.5, left at room temperature for 10 minutes, centrifuged (centrifugal force: 6000 g, centrifugation time: 25 minutes), and the supernatant was collected , adjust the pH to 7.0, the solution is slightly cloudy at this time. Add sodium deoxycholate again to make the final concentration 0.5%, adjust the pH to 4.5, stand at room temperature for 10 minutes, centrifuge (centrifugal force: 6000g, centrifugation tim...

Embodiment 2

[0087] Example 2: Purification of type 1 pneumococcal capsular polysaccharide

[0088] Type 1 pneumococcal lysate was prepared as described in Example 1.

[0089] Calcium chloride was added to the lysate of type 1 pneumococcus to make the final concentration 0.2 mol / L, the pH was adjusted to 2.5, left standing at room temperature for 5 minutes, centrifuged (centrifugal force: 6000 g, centrifugation time: 25 minutes), and the supernatant was collected. solution, adjust the pH to 7.0. The supernatant was concentrated by ultrafiltration through a 300Kd membrane pack. Add sodium deoxycholate to the ultrafiltration concentrate to make the final concentration 0.5%, adjust the pH to 4.5, stand at room temperature for 10 minutes, centrifuge (centrifugal force: 6000g, centrifugation time: 25 minutes), collect the supernatant, The pH was adjusted to 7.0, at which point the solution was slightly hazy. Add sodium deoxycholate again to make the final concentration 0.5%, adjust the pH to...

Embodiment 3

[0092] Example 3: Purification of type 5 pneumococcal capsular polysaccharide

[0093] Type 5 pneumococcus was fermented in a bioreactor, and after growth to the logarithmic growth phase, sodium deoxycholate was added to lyse the bacteria. The supernatant was collected by centrifugation, and concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cut-off of 100 Kd to obtain a type 5 pneumococcal lysate.

[0094] Sodium deoxycholate was added to the pneumococcal type 5 lysate to make the final concentration 1%, the pH was adjusted to 4.3, left standing at room temperature for 20 minutes, centrifuged (centrifugal force: 6000 g, centrifugation time: 20 minutes), and the supernatant was collected , adjust the pH to 7.0, the solution is slightly cloudy at this time. Add sodium deoxycholate again to make the final concentration 1%, adjust pH to 4.3, stand at room temperature for 20 minutes, centrifuge (centrifugal force: 6000g, centrifugation time:...

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Abstract

The invention relates to a method for preparing pneumococcal capsular polysaccharide and a pneumococcal vaccine. A method of making a pneumococcal capsular polysaccharide comprises providing a pneumococcal lysate of a selected serotype; removing proteins and nucleic acids from the lysate, the removal of proteins and the removal of nucleic acids being performed out of order in separate steps; the method for removing the protein comprises the following steps: I) mixing a sample to be treated with sodium deoxycholate to enable the pH value of the mixed system to be 3-6, centrifuging to obtain supernate, and adjusting the pH value of the supernate to be 6-8 to obtain first supernate; iI) removing sodium deoxycholate in the first supernate; the method for removing the nucleic acid comprises the following steps: A) mixing a sample to be treated with divalent salt to enable the pH value of the mixed system to be 1-4, centrifuging to obtain supernate, and adjusting the pH value of the supernate to be 6-8 to obtain second supernate; and B) removing the divalent salt added in the step A) in the second supernatant. The method is simple and ingenious, and an excellent pneumococcus capsular polysaccharide purification effect is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing pneumococcal capsular polysaccharide and pneumococcal vaccine. Background technique [0002] Pneumococcus is the primary pathogen of respiratory tract infections, the most common pathogen causing community-acquired pneumonia in children, and the main pathogen causing fatal pneumonia in the elderly. More than 90 serotypes have been found, of which about 30 serotypes are pathogenic to humans. Capsular polysaccharide is the main virulence factor of pneumococcus. The research results show that pneumococcal capsular polysaccharide vaccine has good immunogenicity and safety. [0003] The existing pneumococcal capsular polysaccharide purification method generally adopts phenol extraction method and ethanol fractional precipitation method. However, phenol is harmful to the human body and the environment, and ethanol also has major safety hazards in terms ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/00A61K39/09A61P31/04
CPCC08B37/0003A61K39/092A61P31/04
Inventor 任克明张轶张新庄李阿妮任珍芸罗树权
Owner LANZHOU INST OF BIOLOGICAL PROD
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