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Method for purifying capsular polysaccharide from capsular bacterium fermentation liquid

A technology of bacterial fermentation and capsular polysaccharide, applied in antibacterial drugs, bacterial antigen components, pharmaceutical formulations, etc., can solve problems such as increasing operation steps and operation time, substances that cannot be directly degraded, human and environmental hazards, etc. The effect of reducing protein and nucleic acid content, saving time and production costs, and improving recovery

Inactive Publication Date: 2012-09-05
CANSINO BIOLOGICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Now the main capsular polysaccharide purification process often adds organic reagents such as phenol, chloroform and acetone to remove protein. These substances cannot be directly degraded and are very harmful to the human body and the environment.
Once these substances cannot be completely removed in the polysaccharide preparation, it is easy to cause harm to the human body; at the same time, these organic reagents need to be used multiple times in the purification process, which not only increases the operation steps and operation time, but also increases the cost in the production process

Method used

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  • Method for purifying capsular polysaccharide from capsular bacterium fermentation liquid
  • Method for purifying capsular polysaccharide from capsular bacterium fermentation liquid
  • Method for purifying capsular polysaccharide from capsular bacterium fermentation liquid

Examples

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Effect test

Embodiment 1

[0027] Example 1: Purification of capsular polysaccharide from Haemophilus influenzae type B fermentation broth

[0028] 1. Strain: Hib strain was purchased from ATCC, the strain number is 51654;

[0029] 2. Medium: MWSM I medium, MWSM II medium, MCDM I medium;

[0030] 3. Bacterial culture:

[0031] After melting the dry powder of the bacteria with the corresponding medium, spread it on the chocolate blood plate, and cultivate it overnight in the carbon dioxide incubator, scrape off the bacteria, put it into the first-level seed shaker flask and cultivate it for 4 hours, and then put the bacteria solution into the Put it into a 50L fermenter and cultivate it for 16 hours, and finally use formaldehyde to inactivate it.

[0032] 4. Purification of capsular polysaccharide:

[0033] Concentrate by centrifugation and ultrafiltration after fermented liquid volume is concentrated 10 times, add sodium acetate to make its mass percentage concentration be 1%, adjust pH value to be 4...

Embodiment 2

[0048] Example 2: Purification of capsular polysaccharide from N. meningitidis type A fermentation broth

[0049] 1. Strain: Neisseria meningitidis type A strain was purchased from China National Institutes for Food and Drug Control, strain number CMCC 29201;

[0050] 2. Medium: MWSM I medium, MWSM II medium, MCDM I medium;

[0051] 3. Bacterial culture:

[0052] After melting the dry powder of the bacteria with the corresponding medium, spread it on the chocolate blood plate, and cultivate it overnight in the carbon dioxide incubator, scrape off the bacteria, put it into the first-level seed shaker flask and cultivate it for 4 hours, and then put the bacteria solution into the Put it into a 50L fermenter and cultivate it for 16 hours, and finally use formaldehyde to inactivate it.

[0053] 4. Purification of capsular polysaccharide:

[0054] Concentrate the fermented liquid volume by centrifugation and ultrafiltration to 30 times, add sodium acetate to make the mass percent ...

Embodiment 3

[0069] Example 3: Purification of capsular polysaccharide from pneumococcal fermentation broth

[0070] 1. Strains: Pneumococcus strains were purchased from China National Institutes for Food and Drug Control, strain number CMCC31218;

[0071] 2. Medium: MWSM I medium, MWSM II medium, MCDM I medium;

[0072] 3. Bacterial culture:

[0073] After melting the dry powder of the bacteria with the corresponding medium, spread it on the chocolate blood plate, and cultivate it overnight in the carbon dioxide incubator, scrape off the bacteria, put it into the first-level seed shaker flask and cultivate it for 4 hours, and then put the bacteria solution into the Put it into a 50L fermenter and cultivate it for 16 hours, and finally use formaldehyde to inactivate it.

[0074] 4. Purification of capsular polysaccharide:

[0075] Concentrate by centrifugation and ultrafiltration after fermented liquid volume is concentrated 20 times, add sodium acetate to make its mass percentage conce...

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Abstract

The invention discloses a method for purifying capsular polysaccharide, which comprises the following steps of: centrifuging the capsular bacterium fermentation liquid, collecting the supernatant liquid, carrying out ultrafiltration concentration and ethanol precipitation, and collecting the supernatant liquid; carrying out ethanol precipitation, collecting precipitates, and redissolving injection water; adding protease for enzymolysis, and carrying out ultrafiltration on protein hydrolysates; adding hexadecyl trimethyl ammonium bromide and NaCl of which the mass percent final concentration is 0.2-0.3%, centrifuging, and collecting the supernatant liquid; adding hexadecyl trimethyl ammonium bromide and NaCl of which the mass percent final concentration is 0.1-0.15%, centrifuging, collecting precipitates, and dissolving the precipitates; carrying out ethanol precipitation, collecting precipitates, and redissolving injection water; and carrying out ultrafiltration and membrane filtration. Through the purification method disclosed by the invention, the recovery rate of the capsular polysaccharide is high, the contents of protein and nucleic acid are low, the step of utilizing toxic organic reagents in the traditional purification method is omitted, the purification steps are simplified, and the time and cost are saved.

Description

technical field [0001] The invention belongs to the field of vaccine production and preparation, and relates to a method for purifying capsular polysaccharides, in particular to a method for purifying capsular polysaccharides from capsular bacteria fermentation liquid. Background technique [0002] Studies in recent years have shown that invasive Haemophilus influenzae type b (Hib), Neisseria meningitidis and pneumococcus have become the main pathogenic bacteria in the respiratory tract of children in my country, causing lower respiratory tract infections, pneumonia, otitis media, meningitis, etc. A variety of diseases, children under the age of 2 have a higher infection rate. In the 1970s, WHO formulated the production and inspection procedures for Hib, Neisseria meningitidis and pneumococcus and other related bacterial capsular polysaccharide vaccines, which were revised in 1980, making the production of these polysaccharide vaccines standardized. . [0003] Currently, th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/00A61K39/09A61K39/095A61K39/102A61P31/04
Inventor 朱涛钟玥如郝擘
Owner CANSINO BIOLOGICS INC
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