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Liraglutide purifying method

A technology of liraglutide and liraglutide crude peptide, which is applied in the field of peptide purification, can solve the problems of low purity and low yield, and achieve the effect of solving the problem of low purity

Active Publication Date: 2018-11-13
HANGZHOU SINOPEP ALLSINO PHARMA TECH DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Solid-phase synthesis of liraglutide will produce impurities such as "poly-Gly", "gly-deficient" and epimers (such as D-His) that affect the purity and yield of the sample. Through the existing purification technology The isolated liraglutide sample has low purity and low yield; the present invention solves such problems

Method used

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  • Liraglutide purifying method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] crude liraglutide

[0056] Sample treatment: Dissolve a sample containing 2.9 g of crude liraglutide (crude product weight: 4 g) in aqueous acetonitrile, and filter through a 0.22 μm filter membrane after complete dissolution. Collect the filtered liraglutide crude peptide aqueous solution for later use.

[0057] The first step of HPLC purification

[0058] Chromatographic conditions: use octaalkylsilane bonded silica gel packing stationary phase (30mm×250mm, 10μm) as chromatographic column; use 0.5% phosphoric acid (take 1000ml water, add 5ml phosphoric acid, mix well, adjust the pH value to 7.0 with triethylamine ) is mobile phase A; acetonitrile is mobile phase B; flow rate is 20mL per minute; detection wavelength is 230nm;

[0059] The elution gradient in the following table was used for elution.

[0060]

[0061] Fractions of liraglutide samples with a purity greater than 90% were collected. Use a rotary evaporator with a water bath temperature of 30-35°C an...

Embodiment 2

[0067] crude liraglutide

[0068] Sample treatment: Dissolve 8 g of crude liraglutide (crude product weight: 11.2 g) in aqueous acetonitrile, and filter through a 0.22 μm filter membrane after complete dissolution. Collect the filtered crude peptide aqueous solution for later use.

[0069] The first step of HPLC purification

[0070] Chromatographic conditions: a chromatographic column with octaalkylsilane bonded silica gel packing as the stationary phase (50mm×250mm, 10μm); use 0.5% phosphoric acid (take 1000ml water, add 5ml phosphoric acid, mix well, adjust the pH value to 7.0) is the mobile phase A; acetonitrile is used as the mobile phase B; the flow rate is 60 mL per minute; the detection wavelength is 230 nm; the sample volume per needle is 1.6 g. The elution gradient in the following table was used for elution.

[0071]

[0072] Fractions of liraglutide samples with a purity greater than 90% were collected. Use a rotary evaporator with a water bath temperature o...

Embodiment 3

[0078] crude liraglutide

[0079] Sample treatment: Dissolve 32 g of crude liraglutide (crude product weight: 44.9 g) in aqueous acetonitrile, and filter through a 0.22 μm filter membrane after complete dissolution. Collect the filtered crude peptide aqueous solution for later use.

[0080] The first step of HPLC purification

[0081] Chromatographic conditions: a chromatographic column with octaalkylsilane bonded silica gel packing as the stationary phase (100mm×250mm, 10 μm); use 0.5% phosphoric acid (take 1000ml water, add 5ml phosphoric acid, mix well, adjust the pH value to 7.0) is the mobile phase A; acetonitrile is the mobile phase B; the flow rate is 200mL per minute; the detection wavelength is 230nm; the sample volume per needle is 6.4g. The elution gradient in the following table was used for elution.

[0082]

[0083] Fractions of liraglutide samples with a purity greater than 90% were collected. Use a rotary evaporator with a water bath temperature of 30-35...

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Abstract

The invention discloses a liraglutide purifying method. The liraglutide purifying method comprises the following steps that 1, a liraglutide coarse product is pretreated to obtain a liraglutide crudepeptide water solution; 2, first-time HPLC purification for removing fragment impurities is performed; 3, a solvent is removed, and a first-step liraglutide sample solution is obtained; 4, second-timeHPLC purification for removing multi-Gly-sequence peptides, Gly-sequence-deleted peptides and epimers produced during synthesis is performed; 5, part of solvent is removed to obtain a second-step liraglutide sample solution, and a PH value is regulated to 6.5 so as to obtain a final liraglutide sample solution. The multi-Gly-sequence peptides, Gly-sequence-deleted peptides, epimers and other impurities produced during synthesis can be removed, and the purity and yield of liraglutide samples are improved.

Description

technical field [0001] The invention relates to the field of peptide purification, in particular to a purification method for solid-phase synthesis of liraglutide. Background technique [0002] Liraglutide, English name: Liraglutide. [0003] The peptide sequence is: [0004] H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly--Gln- [0005] Ala-Ala-Lys(Pal-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH [0006] Liraglutide is the first long-acting GLP-1 analogue developed by Novo Nordisk, Denmark. It is 97% homologous to human glucagon-like peptide-1 (GLP-1). Liraglutide has various effects such as lowering blood sugar, promoting regeneration of islet cells, and slightly prolonging gastric emptying, and has broad application prospects. [0007] Liraglutide, as a new generation of incretin-based hypoglycemic drugs, not only has a long acting time, but also fully retains multiple physiological activities of natural GLP-1, which can safely and effectiv...

Claims

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Application Information

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IPC IPC(8): C07K14/605C07K1/16
CPCC07K14/605
Inventor 谷海涛赵呈青肖英陈烨
Owner HANGZHOU SINOPEP ALLSINO PHARMA TECH DEV CO LTD
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