Method for purifying solid-phase synthetic coarse liraglutide

A crude liraglutide peptide, solid-phase synthesis technology, applied in the field of biomedicine, can solve the problems of long peptide chain, difficult purification, strong hydrophobicity, etc.

Active Publication Date: 2012-07-18
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Liraglutide is difficult to purify due to its long peptide chain and strong hydrophobicity due to the presence of palmitoyl groups

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Liraglutide was synthesized in solid phase, and the purity of the crude peptide was 50%.

[0030] Sample handling:

[0031] 2.2 g of solid crude peptide was dissolved in 10% acetonitrile / 90% water (V / V) by ultrasound, and the sample was completely dissolved, then filtered with a filter membrane, and the filtrate was collected for future use.

[0032] The first step of HPLC purification:

[0033] Purification conditions: chromatographic column: a chromatographic column with octaalkylsilane bonded silica gel as the stationary phase, and the diameter and length of the column are: 50mm×250mm. Mobile phase: A phase: 0.1% trifluoroacetic acid 85% water / 15% isopropanol aqueous solution; B phase: 0.1% trifluoroacetic acid in acetonitrile, flow rate: 55ml / min, gradient: 40% B-60% B, Detection wavelength: 275nm. The injection volume was 2.2 g.

[0034] Purification process: wash the chromatographic column with more than 50% acetonitrile, and then equilibrate the sample, and t...

Embodiment 2

[0042]Solid-phase synthesis of liraglutide: In the presence of an activator system, Fmoc-Gly-resin was obtained by coupling the resin solid-phase support and N-terminal Fmoc-protected glycine; by solid-phase synthesis, according to the main chain of liraglutide The peptide sequence is sequentially coupled with amino acids with N-terminal Fmoc protection and side chain protection, wherein the lysine side chain is protected by Alloc; the lysine side chain protection group Alloc is removed; by solid-phase synthesis, the lysine side chain Coupling of Palmitoyl-Gllu-OtBu; cleavage, removal of protecting groups and resin to obtain liraglutide crude peptide. The purity of the crude peptide was 60%.

[0043] Sample handling:

[0044] 2.5 g of solid crude peptide was dissolved in 20% acetonitrile / 80% water (V / V), sonicated to completely dissolve the sample, and then filtered with a filter membrane, and the filtrate was collected for future use.

[0045] The first step of HPLC purific...

Embodiment 3

[0054] Liraglutide was synthesized in solid phase, and the purity of the crude peptide was 58%.

[0055] Sample handling:

[0056] 3.0 g of solid crude peptide was dissolved in 30% acetonitrile / 70% water (V / V), ultrasonicated to completely dissolve the sample, and then filtered with a filter membrane, and the filtrate was collected for future use.

[0057] The first step of HPLC purification:

[0058] Purification conditions: chromatographic column: a chromatographic column with octaalkylsilane bonded silica gel as the stationary phase, and the diameter and length of the column are: 50mm×250mm. Mobile phase: A phase: 0.2% trifluoroacetic acid 65% water / 35% isopropanol solution in water; B phase: 0.2% trifluoroacetic acid in acetonitrile, flow rate: 70ml / min, gradient: 30% B-50% B, Detection wavelength: 275nm. The injection volume was 3.0 g.

[0059] Purification process: wash the chromatographic column with more than 50% acetonitrile, and then equilibrate the sample, and t...

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Abstract

The invention relates to the field of biomedicine, in particular to a method for purifying solid-phase synthetic coarse liraglutide. The method comprises the following steps of: dissolving solid-phase synthetic coarse liraglutide into acetonitrile aqueous solution to obtain coarse peptide solution; and purifying through four-step HPLC (High Performance Liquid Chromatography) to obtain the liraglutide. The method has the advantages of high purity and high yield.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for purifying solid-phase synthetic liraglutide crude peptide. Background technique [0002] Diabetes Mellitus (DM) is a high incidence worldwide. According to the latest data released by the World Health Organization, the number of diabetic patients in the world has reached 180 million in 2007, and the incidence rate is still increasing year by year. According to epidemiological statistics, there were nearly 92 million diabetic patients in my country in 2010. Because diabetes devises every system of the whole body, it even induces many fatal complications, seriously affects people's working ability, threatens people's life safety, and poses great harm to people's health. Diabetes is mainly divided into type I and type II, and the latter accounts for more than 90% of the total number of diabetic patients. [0003] Liraglutide is a long-acting glucagon-like peptide 1 (GLP-1) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/605C07K1/16C07K1/06C07K1/04
CPCC07K14/605B01D15/166B01D15/1871C07K1/16
Inventor 覃亮政潘俊锋马亚平袁建成
Owner HYBIO PHARMA
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