A kind of purification process of haemophilus b-type polysaccharide

A Haemophilus, b-type technology, applied in the field of polysaccharide vaccines, can solve problems such as environmental hazards, user health hazards, corrosion, etc., to prevent environmental and personnel hazards, reduce flammable and explosive hazards, and avoid phenol The effect of using

Active Publication Date: 2021-05-14
BEIJING ZHIFEI LVZHU BIOPHARM +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, as a corrosive and highly toxic chemical reagent, phenol has serious harm to the environment and pollutes the air and water; the storage of large amounts of ethanol has high safety requirements for production plants
On the other hand, phenol has a strong corrosive effect on human skin and mucous membranes, inhibits the central nervous system, damages liver and kidney functions, and endangers the health of production operators.
At the same time, the phenol residues in the finished vaccine products also pose potential hazards to the health of users

Method used

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  • A kind of purification process of haemophilus b-type polysaccharide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1) Dissolve the crude polysaccharide in PB buffer, and perform ultrafiltration using a tangential flow ultrafiltration membrane bag to obtain an ultrafiltration solution:

[0029] Use a concentration of 5mmol / L, pH6.5 phosphate buffer to dissolve the crude polysaccharide to a concentration of 5mg / mL, the ultrafiltration volume is 10 times the volume of the crude polysaccharide solution, the temperature is controlled at 8°C, and the tangential flow ultrafiltration membrane The package model is 30KD.

[0030] 2) The ultrafiltration solution is chromatographed through a ceramic hydroxyapatite column to obtain a chromatographic solution:

[0031] Load the ultrafiltration solution onto the ceramic hydroxyapatite chromatography column, collect the flow-through peak displayed at 206nm to obtain the chromatographic fluid, and use high-concentration PB buffer to elute protein and nucleic acid; the height range of the hydroxyapatite column is 16cm , the loading volume is 0.5CV (...

Embodiment 2

[0037] 1) Dissolve the crude polysaccharide in PB buffer, and perform ultrafiltration using a tangential flow ultrafiltration membrane bag to obtain an ultrafiltration solution:

[0038] Use a concentration of 15mmol / L, pH 8 phosphate buffer to dissolve the crude polysaccharide to a concentration of 10mg / mL, the ultrafiltration volume is 50 times the volume of the crude polysaccharide solution, the temperature is controlled at 15°C, and the tangential flow ultrafiltration membrane The model is 300KD.

[0039] 2) The ultrafiltration solution is chromatographed through a ceramic hydroxyapatite column to obtain a chromatographic solution:

[0040] Load the ultrafiltration solution onto the ceramic hydroxyapatite chromatography column, collect the flow-through peak displayed at 206nm to obtain the chromatographic fluid, and use high-concentration PB buffer to elute protein and nucleic acid; the height range of the hydroxyapatite column is 24cm , the loading volume is 1.5CV (colum...

Embodiment 3

[0046] The various indicators of Haemophilus type b polysaccharide obtained by the process of the present invention are compared with the national pharmacopoeia and existing patents (patent number: CN201210037245.2) and the results are as follows:

[0047] It can be found that all indicators of the Haemophilus type b polysaccharide obtained by this process meet the requirements of the National Pharmacopoeia, and the content of protein and nucleic acid is lower than that of the polysaccharide obtained by the existing patented method.

[0048]

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Abstract

The invention discloses a type b haemophilus polysaccharide purification process, which comprises the steps of tangential flow ultrafiltration membrane bag ultrafiltration, ceramic hydroxyapatite chromatography, tangential flow ultrafiltration membrane bag ultrafiltration desalination concentration, and freeze-drying steps. The invention has the advantages of avoiding the use of phenol, preventing the harm of phenol to the environment and personnel, reducing the use of ethanol, and reducing the risk of inflammability and explosion during the use of ethanol; The combined use of limestone makes the content of protein and nucleic acid lower, and it is more meaningful for production scale-up; the combination of direct and freeze-drying process obtains polysaccharide products with low water content, and the stability of polysaccharides is enhanced.

Description

Technical field: [0001] The invention relates to polysaccharide purification in the field of polysaccharide vaccines, in particular to the purification of Haemophilus influenzae type b (Hib) polysaccharides. Background technique: [0002] In the preparation process including Hib polysaccharide vaccines, conjugate vaccines and related multivalent vaccines, the preparation of polysaccharides is a key step. At present, the extraction and purification of Hib polysaccharide in the world adopts a similar process. Cetyltrimethylammonium bromide (CTAB) is used to precipitate the supernatant of the fermented broth after centrifugation to obtain complex sugar mixed with nucleic acid and protein. After dissolving complex sugars, different concentrations of ethanol were used to precipitate crude sugars step by step. The raw sugar is extracted with phenol to remove protein, and then ethanol is used for precipitation to finally obtain Hib refined sugar with low protein content and low n...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08B37/00
CPCC08B37/0003
Inventor 高磊苏晓叶石献华程亚君李倩
Owner BEIJING ZHIFEI LVZHU BIOPHARM
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