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Refining method of streptococcus pneumoniae capsular polysaccharide

A Streptococcus pneumoniae and refining method technology is applied in the field of refining Streptococcus pneumoniae capsular polysaccharides, which can solve the problems that the polysaccharide solution does not meet the European Pharmacopoeia standards, increase the cost of large-scale production, and have many steps to achieve large-scale production. Industrialized production, short purification time and simple preparation process

Active Publication Date: 2021-11-05
沈阳礼慧医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional polysaccharide refining process has many and complicated steps. Most of the polysaccharide solutions refined by simple processes do not meet the European Pharmacopoeia standards. To obtain refined polysaccharide solutions that meet the European Pharmacopoeia standards, further single or multiple chromatographic steps are required.
However, the use of chromatography to purify capsular polysaccharides, on the one hand, affects the yield due to the nature of the filler, the volume of the processed solution is small, and the scale-up production is limited; on the other hand, it also increases the cost of large-scale production

Method used

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  • Refining method of streptococcus pneumoniae capsular polysaccharide
  • Refining method of streptococcus pneumoniae capsular polysaccharide
  • Refining method of streptococcus pneumoniae capsular polysaccharide

Examples

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Embodiment 1

[0047] Example 1: Refining of Type 3, Type 12F and Type 19F Streptococcus pneumoniae capsular polysaccharide

[0048] In this embodiment, the purification of capsular polysaccharide of Streptococcus pneumoniae type 3 is taken as an example for illustration.

[0049] Add sodium deoxycholate to 50L of type 3 Streptococcus pneumoniae fermentation broth to make the final concentration (m / v) 0.1%, and lyse for 60 minutes. Slowly add aluminum sulfate to the lysate to a final concentration (m / v) of 0.5%, and stir for 30 minutes. Add acetic acid to adjust the pH to 4.0, heat up to 45°C, stir for 80 minutes, centrifuge at 16000rpm for 60 minutes, collect the supernatant, filter through 0.45 μm micropores, and collect the filtrate. Concentrate the filtrate 5 times, pass through the ultrafiltration membrane ultrafiltration with a molecular weight cut-off of 30kD, wash and filter with citrate-phosphate buffer solution with 6 times the volume of the concentrated solution, and then wash an...

Embodiment 2

[0055] Example 2: Refining of Type 1, Type 4, Type 6A, Type 6B, Type 7F Streptococcus pneumoniae capsular polysaccharide

[0056] In this example, the purification of the capsular polysaccharide of Streptococcus pneumoniae type 1 is taken as an example for illustration.

[0057] Add sodium deoxycholate to 50L of Streptococcus pneumoniae capsular polysaccharide fermentation liquid type 1 to make the final concentration (m / v) 0.1%, and lyse for 45 minutes. Slowly add aluminum sulfate to the lysate to a final concentration (m / v) of 1%, and stir for 20 min. Add phosphoric acid to adjust the pH to 3.75, heat up to 50°C, stir for 45 minutes, cool down to room temperature, centrifuge at 16,000 rpm for 60 minutes, collect the supernatant, filter through 0.45 μm micropores, and collect the filtrate. Concentrate the filtrate 6 times, ultrafilter through an ultrafiltration membrane with a molecular weight cut-off of 100kD, wash and filter with citrate-phosphate buffer solution with 7 ti...

Embodiment 3

[0063] Embodiment 3: refining of capsular polysaccharide of Streptococcus pneumoniae of type 5, type 9V, type 10A, and type 22F

[0064] In this embodiment, the purification of capsular polysaccharide of Streptococcus pneumoniae type 5 is taken as an example for illustration.

[0065] Add sodium deoxycholate to 50L of Streptococcus pneumoniae capsular polysaccharide fermentation broth type 5 to make the final concentration (m / v) 0.1%, and lyse for 50 minutes. Slowly add aluminum sulfate to the lysate to a final concentration (m / v) of 1.5%, and stir for 40 min. Add nitric acid to adjust the pH to 3.75, raise the temperature to 55°C, stir for 70 minutes, cool down to room temperature, centrifuge at 16000rpm for 60 minutes, collect the supernatant, filter at 4.0-9.0μm depth, and collect the filtrate. Concentrate the filtrate 8 times, ultrafilter through an ultrafiltration membrane with a molecular weight cut-off of 100KD, wash and filter with 9 times the volume of the concentrat...

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Abstract

The invention belongs to the technical field of biology, and particularly discloses a refining method of streptococcus pneumoniae capsular polysaccharide, the refining method comprises the following steps: preparing a sample solution containing streptococcus pneumoniae capsular polysaccharide, flocculating, heating and carrying out acid precipitation, centrifuging, filtering, and ultrafiltration and the like. The refining method at least has one of the following beneficial effects: according to the refining method of the streptococcus pneumoniae capsular polysaccharide, a refined streptococcus pneumoniae capsular polysaccharide solution which is far higher than the European pharmacopoeia standard can be prepared through conventional methods such as flocculation, heating acid precipitation, centrifugation, filtration and ultrafiltration; moreover, the refining method does not need purification steps such as chromatography and carbon filtration, so that the recovery rate is increased, the process steps are shortened, and large-scale industrial production is facilitated.

Description

technical field [0001] The application belongs to the field of biotechnology, more specifically, it relates to a method for refining capsular polysaccharide of Streptococcus pneumoniae. Background technique [0002] Pneumococcal disease remains one of the most important public health problems worldwide. According to the different sites of Streptococcus pneumoniae infection, pneumococcal disease can be divided into invasive pneumococcal disease (IPD) and non-invasive pneumococcal disease (NIPD). IPD refers to the infection caused by Streptococcus pneumoniae invading the original sterile parts and tissues, mainly including meningitis, sepsis and bacterial pneumonia, etc., and the incidence rate is high in children under 5 years old, especially infants and young children under 2 years old. The maternally-inherited antibodies of infants gradually disappear from the age of 6 months. Therefore, the period from 6 months to 2 years old is the period with the highest incidence of IP...

Claims

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Application Information

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IPC IPC(8): C08B37/00A61K39/09A61P31/04
CPCC08B37/0003A61K39/092A61P31/04
Inventor 吴慧张晶玉
Owner 沈阳礼慧医药科技有限公司