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Liraglutide preparation method

A technology of liraglutide and peptide fragments, which is applied in the field of polypeptide drug preparation, can solve the problems of complicated operation, difficult purification of crude products, low product yield and the like, and achieves simple operation, wide practical value and application prospect, and high product yield. Effect

Inactive Publication Date: 2013-09-04
CHENGDU SHENGNUO BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0013] The technical problem to be solved by the present invention is that the existing method is cumbersome to operate, the crude product is not easy to purify, and the product yield is low

Method used

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  • Liraglutide preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Synthesis of Liraglutide Protected Peptide

[0063] The peptide sequence of GLP1(7-37) is:

[0064] R-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-

[0065] Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-

[0066] Leu-Val-Arg-Gly-Arg-Gly-OH;

[0067] where R is Fmoc.

[0068] Take 3.6g of GLP1(7-37), dissolve it with 50% acetonitrile aqueous solution, adjust the pH to 10.5 with DIEA, add 1.6g of N α -PAL-γ-Glu(OtBu)-ONSu in acetonitrile aqueous solution, while maintaining pH 10.5 with DIEA, stirred for 1.5 hours, added 50% acetonitrile aqueous solution containing 0.6g Gly, continued to stir for 0.5 hours, evaporated under reduced pressure Acetonitrile, centrifuged to remove N a -Pal-Glu-(ONSu)-OtBu, adjust pH 5.0, get liraglutide protected peptide.

Embodiment 2

[0069] Example 2 Preparation of Liraglutide Crude Product

[0070] The liraglutide protected peptide prepared in Example 1 was first treated with a DMF (10mL / g liraglutide protected peptide) solution containing 25% (V) piperidine for 25 minutes, and the resulting solid was added to a volume ratio of TFA: water = 95: 5 in the lysis reagent (lysis reagent 10mL / g resin), stir evenly, stir and react at room temperature for 3 hours, filter the reaction mixture with a sand core funnel, collect the filtrate, wash the resin 3 times with a small amount of TFA, and combine After the filtrate was concentrated under reduced pressure, anhydrous diethyl ether was added to precipitate, and then the precipitate was washed with anhydrous diethyl ether for 3 times, and the off-white powder obtained after drying was the crude liraglutide with a purity of 92.3%.

Embodiment 3

[0071] Example 3 Purification of Liraglutide Crude Product

[0072] Take the crude liraglutide obtained in Example 2, add water and stir, adjust the pH to 8.5 with ammonia water until completely dissolved, and filter the solution with a 0.45 μm mixed microporous membrane for purification. Purification is carried out by high performance liquid chromatography, the chromatographic filler for purification is 10 μm reversed-phase C18, the mobile phase system is 0.1% TFA / water solution-0.1% TFA / acetonitrile solution, and the flow rate of the 77mm*250mm chromatographic column is 90mL / min. Gradient system elution, cyclic sample injection and purification, take the crude product solution and load it on the chromatographic column, start the mobile phase elution, collect the main peak and evaporate the acetonitrile to obtain the concentrated liquid of liraglutide purification intermediate.

[0073] Take the concentrated liquid of the purified intermediate of liraglutide, and filter it wi...

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Abstract

The invention belongs to the technical field of polypeptide medicament preparation methods, particularly relates to a liraglutide preparation method, and aims to solve the technical problems of complex operation process, high purification difficulty of a crude product and low product yield in the existing method. The technical scheme for solving the technical problems is as follows: the liraglutide preparation method comprises the following steps: performing coupling reaction on an N-protected GLP1(7-37) peptide segment and N-alpha-PAL-gamma-Glu(OtBu)-ONSu under the action of alkali to obtain a liraglutide-protected peptide, removing the N-protection, and performing acidolysis to obtain a liraglutide crude product; and purifying to obtain a liraglutide pure product, wherein the sequence of the used GLP1(7-37) peptide segment is as follows: R-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH; and R is Fmoc, Dde or ivDde. The new liraglutide preparation method provided by the invention has the advantages that the method is simple to operate, the crude product is easy to purify and the product is high in yield.

Description

technical field [0001] The invention belongs to the technical field of polypeptide drug preparation methods, in particular to a method for preparing liraglutide. Background technique [0002] Liraglutide has the following structure: [0003] His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser- [0004] Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (N α -PAL-γ-Glu)- [0005] Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH. [0006] Liraglutide is a GLP-1 (glucagon-like peptide) analog with 97% sequence homology to human GLP-1, which can bind and activate the GLP-1 receptor. The GLP-1 receptor is the target of natural GLP-1, an endogenous incretin hormone that promotes glucose-dependent insulin secretion from pancreatic β-cells. Different from Natural GLP-1, the pharmacokinetic and pharmacodynamic characteristics of liraglutide in humans are suitable for once-daily dosing regimen. After subcutaneous injection, the mechanism of prolonged action time includes: self-association to slow absorp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/605C07K1/06
Inventor 曾德志郭德文王晓莉文永均
Owner CHENGDU SHENGNUO BIOPHARM
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