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Method for purifying Hemophilus influenzae type b capsular polysaccharide

A technology of Haemophilus influenzae and capsular polysaccharide, applied in the field of vaccines

Inactive Publication Date: 2013-10-16
CHENGDU OLYMVAX BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no process using hydroxyapatite for the purification of the capsular polysaccharide of Haemophilus influenzae type b

Method used

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  • Method for purifying Hemophilus influenzae type b capsular polysaccharide
  • Method for purifying Hemophilus influenzae type b capsular polysaccharide
  • Method for purifying Hemophilus influenzae type b capsular polysaccharide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Pack a hydroxyapatite chromatography column (5cm×20cm) in advance, equilibrate the column with 20mM PB buffer (pH6.9) for 2 column volumes until OD 206nm The baseline is stable. Then, 50 mg of Hib capsular polysaccharide raw sugar was dissolved in 50 ml of 20 mM PB buffer (pH 6.9) and loaded on the chromatography column. After loading the sample, wash 2 column volumes with buffer A (20mM PB buffer, pH6.9), and collect the flow-through peak. Then, the protein bound to the chromatography column was eluted with buffer B (20mM PB buffer, 0.2M sodium chloride, pH6.9) to regenerate the chromatography column.

[0033] Pack a G-25 glucose gel column (5cm×20cm) in advance, equilibrate the column with pyrogen-free water for injection until the OD206nm baseline is stable. Then load the collected flow-through peaks on the G-25 glucose gel column. The pyrogen-free water for injection was used as the mobile phase for elution, and the elution peaks containing Hib capsular polysacchari...

Embodiment 2

[0037] Pack a hydroxyapatite chromatography column (5cm×20cm) in advance, equilibrate the column with 20mM PB buffer (pH6.9) for 2 column volumes until the OD206nm baseline is stable. Then 100 mg of Hib capsular polysaccharide raw sugar was dissolved in 50 ml of 20 mM PB buffer (pH 6.9) and loaded on the chromatography column. After loading the sample, wash 2 column volumes with buffer A (20mM PB buffer, pH6.9), and collect the flow-through peak. Then, the protein bound to the chromatography column was eluted with buffer B (20mM PB buffer, 0.2M sodium chloride, pH6.9) to regenerate the chromatography column.

[0038] Pack a G-25 glucose gel column (5cm×20cm) in advance, equilibrate the column with pyrogen-free water for injection until the OD206nm baseline is stable. Then load the collected flow-through peaks on the G-25 glucose gel column. The pyrogen-free water for injection was used as the mobile phase for elution, and the elution peaks containing Hib capsular polysaccharide...

Embodiment 3

[0043] Pack a hydroxyapatite chromatography column (5cm×20cm) in advance, equilibrate the column with 20mM PB buffer (pH6.9) for 2 column volumes until the OD206nm baseline is stable. Then 100 mg of Hib capsular polysaccharide raw sugar was dissolved in 50 ml of 20 mM PB buffer (pH 6.9) and loaded on the chromatography column. After loading the sample, wash 2 column volumes with buffer A (20mM PB buffer, pH6.9), and collect the flow-through peak. Then, the protein bound to the chromatography column was eluted with buffer B (20mM PB buffer, 0.2M sodium chloride, pH6.9) to regenerate the chromatography column.

[0044] Pack a G-25 glucose gel column (5cm×20cm) in advance, equilibrate the column with pyrogen-free water for injection until the OD206nm baseline is stable. Then load the collected flow-through peaks on the G-25 glucose gel column. The pyrogen-free water for injection was used as the mobile phase for elution, and the elution peaks containing Hib capsular polysaccharide...

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Abstract

The invention discloses a method for purifying Hemophilus influenzae type b (Hib) capsular polysaccharide. According to the method, the Hib capsular polysaccharide is purified by using hydroxyapatite. The method comprises the following steps of: dissolving crude Hib capsular polysaccharide into 20mM of phosphate buffer solution, and loading an obtained solution of the crude Hib capsular polysaccharide on a hydroxyapatite chromatographic column; washing two column volumes by using 20mM of phosphate buffer solution, and collecting flow-through peaks; and eluting combined protein by using a high-salt buffer solution, and performing buffer solution replacement on the collected flow-through peaks by using a G-25 glucose gel column to obtain a stock solution of fine Hib capsular polysaccharide finally. The method has the advantages that: the capsular polysaccharide is purified by using the hydroxyapatite, so that the harm to environments and human bodies, which is caused by using phenol, is avoided; and the purity of the obtained Hib capsular polysaccharide can reach industrial standard.

Description

Technical field [0001] The invention relates to the field of vaccines, in particular to a method for purifying type b capsular polysaccharide of Haemophilus influenzae. Background technique [0002] During the preparation of Hib vaccine, the preparation of capsular polysaccharide is a key step. In our country, the extraction of capsular polysaccharides is similar to that of meningococcus and typhoid Vi polysaccharides. This process uses cetyltrimethylammonium bromide (CTAB) to form a quaternary amine complex with acidic polysaccharides to precipitate the polysaccharides. However, CTAB also has different degrees of precipitation for nucleic acids and proteins. Therefore, to obtain higher purity capsular polysaccharides, the precipitated nucleic acids and proteins must be further removed. The conventional method of removing protein is to use phenol extraction to remove protein. However, the disadvantage of this process is that a large amount of waste phenol is produced. Phenol i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/102C08B37/00A61K39/02A61P31/04
Inventor 吴强罗力心关晓峰伍长华
Owner CHENGDU OLYMVAX BIOPHARM