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Method for hydrogen-bonding adsorption purification of oxytocin by using agarose comprising beta-cyclodextrin ligand

A technology of hydrogen bond adsorption and oxytocin, which is applied in the field of hydrogen bond adsorption chromatography separation and purification, can solve the problems of high biocompatibility and biosafety, limited variety of media, and difficult to enlarge, and achieves biocompatibility and biosafety. High safety, easy to scale up, and strong crude product tolerance

Active Publication Date: 2011-11-23
浙江华军药业有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are commercial preparative-grade reversed-phase columns, there are still some shortcomings in applying reversed-phase chromatography technology to the large-scale production of polypeptide drugs: First, the sample load of reversed-phase high-performance liquid chromatography is low, especially for For crude products with more impurities, excessive crude samples will cause irreversible collapse of the chromatographic column; second, the variety of media is limited and it is not easy to scale up; third, the number of times the media can be reused is limited, and the requirements for sample pretreatment are higher; fourth, Conventional silica gel matrix is ​​bonded with alkyl ligands. This kind of chromatographic separation medium is used for the production of clinical drugs. Its biocompatibility and biosafety are not as high as those of dextran and agarose.

Method used

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  • Method for hydrogen-bonding adsorption purification of oxytocin by using agarose comprising beta-cyclodextrin ligand
  • Method for hydrogen-bonding adsorption purification of oxytocin by using agarose comprising beta-cyclodextrin ligand
  • Method for hydrogen-bonding adsorption purification of oxytocin by using agarose comprising beta-cyclodextrin ligand

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Embodiment 1

[0030] Example 1, β-cyclodextrin ligand agarose gel hydrogen bond adsorption chromatography medium separation and purification liquid phase synthesis of crude oxytocin (initial purity about 17%)

[0031] 1) Mobile phase composition: acetonitrile-acetic acid-water system, the initial mobile phase is 30% acetonitrile, the prepared mobile phase is filtered with a 0.45 μm filter membrane, ultrasonically degassed or vacuum degassed for 20 minutes, and left at room temperature for 0.5 hours for later use;

[0032] 2) The crude product of chemically synthesized oxytocin was dissolved in the initial mobile phase, prepared into a 50 mg / mL solution, ultrasonically assisted for 5 minutes, and filtered through a 0.45 μm filter membrane;

[0033] 3) The β-cyclodextrin ligand agarose gel hydrogen bond adsorption chromatography medium is packed into a column, the diameter of the column bed is 10mm, and the height of the column bed is 30cm. 10% of volume;

[0034] 4) Elution method: After th...

Embodiment 2

[0038] Example 2, β-cyclodextrin ligand agarose gel hydrogen bond adsorption chromatography medium separation and purification solid-phase synthesis of crude oxytocin (initial purity about 58%)

[0039] 3) Mobile phase composition: acetonitrile-acetic acid-water system, the initial mobile phase is 20% acetonitrile, the prepared mobile phase is filtered with a 0.45 μm filter membrane, ultrasonically degassed or vacuum degassed for 20 minutes, and left at room temperature for 0.5 hours for later use;

[0040] 4) The crude product of chemically synthesized oxytocin was dissolved in the initial mobile phase, prepared into a 50 mg / mL solution, ultrasonically assisted for 3 minutes, and filtered through a 0.45 μm filter membrane;

[0041] 3) The β-cyclodextrin ligand agarose gel hydrogen bond adsorption chromatography medium is packed into a column with a column bed diameter of 16 mm and a column bed height of 30 cm. The initial mobile phase is used to equilibrate the volume of 2 col...

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Abstract

The invention relates to a method for chemically synthesizing oxytocin by using hydrogen-bonding adsorption chromatographic separation and purification. In the method, a high-concentration and high-crosslinking-degree agarose gel medium, on the surface of which beta-cyclodextrin ligand is bonded, is adopted; and the oxytocin is separated and purified from a rough oxytocin product which is obtained through chemical synthesis (comprising liquid-phase synthesis and solid-phase synthesis) according to a hydrogen-bonding adsorption principle. The method is used for producing oxytocin raw material medicines, has the advantages of high selectivity, high sample loading capacity, high medium recycling rate and the like; and the defects of low medium recycling rate and low sample loading capacity existing in the conventional reverse phase chromatography are overcome.

Description

technical field [0001] The present invention relates to a hydrogen bond adsorption chromatographic separation and purification method of chemically synthesized polypeptide drug, oxytocin, specifically using an agarose gel medium bonded with β-cyclodextrin ligand on the surface, and using hydrogen bond adsorption chromatographic Principle Separation and purification of chemically synthesized oxytocin. Background technique [0002] There are a large number and variety of biologically active peptides in nature, involving various toxins, hormones, antibacterial peptides, pheromones, cytokines, etc., which play an important role in regulating various life activities, and are widely involved in molecular recognition, signal transduction, etc. Guidance, enzyme activity regulation, immune regulation, cell differentiation and individual development regulation and other processes. Since the invention of peptide artificial synthesis technology at the beginning of last century, dozens ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/16C07K1/36C07K1/34C07K1/22
Inventor 高留根顾铭谷海涛周天琼周正兵俞保彬
Owner 浙江华军药业有限公司
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