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Chromatographic method for effectively improving purification yield of synthetic peptide

A purification method and peptide synthesis technology, which is applied in the field of preparation and purification of liraglutide, can solve the problems of small loading, low purity, and low efficiency of peptide products, and achieve the effect of improving the total yield of purification and reducing purification steps

Active Publication Date: 2019-12-06
SINOPEP ALLSINO BIOPHARMACEUTICAL CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The specific implementation principle is to increase the filling height of a single chromatographic column filler, improve the separation of impurities in the synthetic peptide by a single chromatographic column, and improve the sample purification yield. Technical problems such as low purity and low efficiency; the above strategies are suitable for dynamic axial compression columns

Method used

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  • Chromatographic method for effectively improving purification yield of synthetic peptide
  • Chromatographic method for effectively improving purification yield of synthetic peptide
  • Chromatographic method for effectively improving purification yield of synthetic peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1 Purification of liraglutide

[0071] 1.1 column bed height: 300mm

[0072] Take crude target liraglutide

[0073] Sample treatment: Dissolve 150.0 g of liraglutide sample in aqueous acetonitrile solution, and filter through a 0.22 μm filter membrane after complete dissolution. Collect the filtered liraglutide crude peptide aqueous solution for later use.

[0074] The first step of HPLC purification

[0075] Chromatographic conditions: use octadecyl or octyl bonded silica gel packing stationary phase (100mm×250mm, 8-20μm) as chromatographic column; use 0.1% ammonium sulfate (take 1000ml water, add 1ml ammonium sulfate, mix well, use Ammonia water adjusted the pH value to 3.3) as the mobile phase A; acetonitrile as the mobile phase B; the flow rate was 20 mL per minute, multiple gradient elution, the detection wavelength was 230 nm; the sample volume per needle was 15.0 g.

[0076] The elution gradient in the following table was used for elution.

[0077...

Embodiment 2

[0110] The purification of embodiment 2 semaglutide

[0111] 2.1 Column bed height: 300mm

[0112] Take crude semaglutide

[0113] Sample treatment: Dissolve a sample containing 12.0 g of semaglutide in an aqueous acetonitrile solution, and filter through a 0.22 μm filter membrane after complete dissolution. Collect the filtered semaglutide crude peptide aqueous solution for later use.

[0114] The first step of HPLC purification

[0115] Chromatographic conditions: use octadecyl or octyl bonded silica gel packing stationary phase (50mm×300mm, 10 μm) as the chromatographic column; use 0.1% TFA (take 1000ml water, adjust the pH value to 2.3 with ammonia water) as the mobile phase A; Acetonitrile is used as the mobile phase B; the flow rate is 20mL per minute, multiple gradient elution, the detection wavelength is 230nm; the sample volume per needle is 3.0g.

[0116] The elution gradient in the following table was used for elution.

[0117]

[0118] Fractions of semaglutid...

Embodiment 3

[0152] crude liraglutide

[0153] Sample treatment: Dissolve 150.0 g of liraglutide sample in aqueous acetonitrile solution, and filter through a 0.22 μm filter membrane after complete dissolution. Collect the filtered liraglutide crude peptide aqueous solution for later use.

[0154] The first step of HPLC purification

[0155] Chromatographic conditions: use octadecyl or octyl bonded silica gel packing stationary phase (100mm×300mm, 8-20μm) as chromatographic column; use 0.1% ammonium sulfate (take 1000ml water, add 1ml ammonium sulfate, mix well, use Ammonia water adjusted the pH value to 3.3) as the mobile phase A; acetonitrile as the mobile phase B; the flow rate was 20 mL per minute, multiple gradient elution, the detection wavelength was 230 nm; the sample volume per needle was 15.0 g.

[0156] The elution gradient in the following table was used for elution.

[0157]

[0158] Fractions of liraglutide samples with a purity greater than 95.0% were collected. Use a...

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Abstract

The invention belongs to the field of pharmaceutical preparation, and relates to a chromatographic method for efficiently improving the purification yield of synthetic peptide, in particular to a method for improving the purification yield of liraglutide. According to the method for improving the purification yield of the liraglutide, impurities whose physicochemical properties have small differences from that of a target substance such asachiral enantiomeric impurities can be effectively removed, and the method is particularly suitable for purification of samples with complex impurity spectra; and meanwhile, the technical problems such as small loading quantity of peptide products, large individual impurities, low purity, and low efficiency can also be solved.

Description

technical field [0001] The invention belongs to the field of medicine preparation, in particular to a preparation and purification method of liraglutide. Background technique [0002] Liraglutide is the first long-acting human glucagon-like peptide-1 (GLP-1) analog developed by Novo Nordisk, Denmark, with 97% homology to GLP-1. %, its peptide sequence is: H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(γ - Glu-Palmitoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH; liraglutide can lower blood sugar, promote islet cell regeneration, slightly prolong gastric emptying, etc. This effect has broad application prospects. 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 effectively lower blood sugar and may have a variety of cardiovascular hazards factors are protective. Liraglutide injection ...

Claims

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Application Information

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IPC IPC(8): C07K14/605C07K1/36C07K1/34C07K1/20B01D15/18B01D15/32B01D15/42
CPCB01D15/1871B01D15/325B01D15/426C07K14/605
Inventor 赵呈青谷海涛肖英
Owner SINOPEP ALLSINO BIOPHARMACEUTICAL CO LTD
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