Method for purifying sermaglutide

A technology of semaglutide and semaglutide crude peptide, which is applied in the field of peptide purification, can solve the problems of low yield and low purity of semaglutide samples, and achieve the effect of improving purity and yield

Active Publication Date: 2018-10-12
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 semaglutide will produce a variety of impurities that affect the purity and yield of the sample, and the semaglutide sample obtained by separation through the existing purification technology has low purity and low yield; the present invention solves such problems

Method used

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  • Method for purifying sermaglutide
  • Method for purifying sermaglutide
  • Method for purifying sermaglutide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Take crude semaglutide

[0051] Sample treatment: Dissolve a sample containing 3 g of semaglutide crude peptide (crude peptide: 4.6 g) in aqueous acetonitrile, and filter it with a 0.22 μm filter membrane after complete dissolution. Collect the filtered semaglutide crude peptide aqueous solution for later use.

[0052] The first step of HPLC purification

[0053] Chromatographic conditions: chromatographic column with tetraalkylsilane bonded silica gel filler as stationary phase (30mm×250mm, 10μm); 0.2% phosphoric acid (take 1000ml water, add 2ml phosphoric acid, mix well, adjust the pH value to 2.3 with ammonia water) The mobile phase is A; acetonitrile is used as the mobile phase B; the flow rate is 20 mL per minute; the detection wavelength is 230 nm; the sample volume per needle is 0.6 g. The elution gradient in the following table was used for elution.

[0054]

[0055] Fractions of semaglutide samples with a purity greater than 95% were collected. Use a rot...

Embodiment 2

[0062] Take crude semaglutide

[0063] Sample treatment: Dissolve a sample containing 8g of semaglutide crude peptide (crude peptide: 12.2g) in aqueous acetonitrile, and filter through a 0.22μm filter membrane after complete dissolution. Collect the filtered crude peptide aqueous solution for later use.

[0064] The first step of HPLC purification

[0065] Chromatographic conditions: chromatographic column with tetraalkylsilane bonded silica gel filler as stationary phase (50mm×250mm, 10μm); 0.2% phosphoric acid (take 1000ml water, add 2ml phosphoric acid, mix well, adjust the pH value to 2.3 with ammonia water) The mobile phase is A; acetonitrile is used as the mobile phase B; the flow rate is 60 mL per minute; the detection wavelength is 230 nm; the loading amount is 1.6 g. The elution gradient in the following table was used for elution.

[0066]

[0067] Fractions of semaglutide samples with a purity greater than 95% were collected. Use a rotary evaporator with a wa...

Embodiment 3

[0073] Take crude semaglutide

[0074] Sample treatment: Dissolve a sample containing 32 g of crude semaglutide peptide (crude peptide: 48.8 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.

[0075] The first step of HPLC purification

[0076] Chromatographic conditions: chromatographic column with tetraalkylsilane bonded silica gel filler as stationary phase (100mm×250mm, 10μm); 0.2% phosphoric acid (take 1000ml water, add 2ml phosphoric acid, mix well, adjust the pH value to 2.3 with ammonia water) Mobile phase A; acetonitrile as mobile phase B; flow rate of 200mL per minute; detection wavelength of 230nm; sample loading of 6.4g. The elution gradient in the table below was used for elution.

[0077]

[0078] Fractions of samples containing semaglutide were collected. Use a rotary evaporator with a water bath temperature of 30-35°C and a vacuum below -...

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Abstract

The invention discloses a method for purifying sermaglutide. The method comprises the following steps that in the first step, a sermaglutide crude product is pre-treated, and a sermaglutide crude peptide aqueous solution is obtained; in the second step, a tetralkylsilane bonded silica gel filler serves as a fixed phase, phosphoric acid serves as a mobile phase A, acetonitrile serves as a mobile phase B, and first HPLC (high performance liquid chromatography) purification is conducted so as to remove impurities of sermaglutide segment; in the third step, a solvent is removed, and a first step sample solution of sermaglutide is obtained; in the fourth step, an octane silane bonded silica gel filler serves as a fixed phase, an ammonium acetate solution serves as a mobile phase A, acetonitrileserves as a mobile phase B, and secondary HPLC purification is conducted so as to remove impurities having the similar physical and chemical properties as sermaglutide; and in the fifth step, the solvent is removed to obtain a second step sample solution of sermaglutide, and a sermaglutide sample solution is obtained. HPLC purification is conducted twice, the purity of the sample obtained in thefirst HPLC purification process and the secondary HPLC purification process is 92% and 99% respectively, and thus the purity and the yield of semaglutide are improved.

Description

technical field [0001] The invention relates to the field of peptide purification, in particular to a method for purifying semaglutide. Background technique [0002] Chinese name: semaglutide [0003] English name: Sermaglutide [0004] The peptide sequence is: [0005] H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Octadecanedioic-γ-Glu-PEG2- PEG2)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH; [0006] Semaglutide 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). Compared with semaglutide, the modified semaglutide has enhanced hydrophilicity, inhibits the hydrolysis of DPP-4 enzyme, prolongs the biological half-life, lowers blood sugar for a long time, promotes islet cell regeneration, prolongs gastric emptying and other functions Semaglutide has various effects such as lowering blood sugar, promoting regeneration of islet cells, slightly prolon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/605C07K1/16
CPCC07K14/605
Inventor 赵呈青肖英陈烨
Owner HANGZHOU SINOPEP ALLSINO PHARMA TECH DEV CO LTD
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