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Purification method of semaglutide

A technology of semaglutide, purification method, applied in the field of peptide purification

Pending Publication Date: 2021-01-29
SHENZHEN JYMED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved in the present invention is to provide a method for purifying semaglutide to reduce the simplex to less than 0.1% and solve the problem of semaglutide D-His at the same time. 1 The problem of impurity purification

Method used

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  • Purification method of semaglutide
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  • Purification method of semaglutide

Examples

Experimental program
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Effect test

Embodiment 1

[0033] (1) Sample pretreatment:

[0034] Take 2.0 g of crude semaglutide (containing about 0.8 g of semaglutide) sample and dissolve it in 1% ammonia solution. After completely dissolving, adjust the pH of the solution to 7.5 with phosphoric acid, and then bathe in water at 45°C to 50°C for 1 ~2h, filter with a 0.45um filter membrane, and collect the filtrate as an aqueous solution of crude semaglutide for future use.

[0035] (2) One-step purification:

[0036] One-step purification of crude semaglutide aqueous solution, chromatographic conditions: Preparative chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30*250mm. Mobile phase A: 20mM / L ammonium chloride, aqueous solution adjusted to pH=7 with ammonia water, mobile phase B: acetonitrile; detection wavelength is 230nm, flow rate is 25ml / min, sample volume is 0.8g, elution gradient is: 0~ 5min: B% is 10%-26%; 5-120min: B% is 26%-46%.

[0037] Collect semaglutide fraction...

Embodiment 2

[0047] (1) Sample pretreatment:

[0048] Take 2.7g of crude semaglutide (containing about 1.08g of semaglutide) sample and dissolve it in 5% ammonia solution. After completely dissolving, use phosphoric acid to adjust the pH of the solution to 9, and then bathe in water at 45°C-50°C for 1 ~2h, filter with a 0.45um filter membrane, and collect the filtrate as an aqueous solution of crude semaglutide for future use.

[0049] (2) One-step purification:

[0050] One-step purification of crude semaglutide aqueous solution, chromatographic conditions: Preparative chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30*250mm. Mobile phase A: 100mM / L ammonium chloride, ammonia water to adjust pH=9 aqueous solution, mobile phase B: acetonitrile; detection wavelength is 230nm, flow rate is 25ml / min, sample volume is 1.08g, elution gradient is: 0~ 5min: B% is 10%-26%; 5-120min: B% is 26%-46%.

[0051] Collect semaglutide fraction samples ...

Embodiment 3

[0061] (1) Sample pretreatment:

[0062] Take 2.3g of crude semaglutide (containing about 0.92g of semaglutide) sample and dissolve it in 2% ammonia solution. After completely dissolving, adjust the pH of the solution to 8 with phosphoric acid, and then bathe in water at 45°C-50°C for 1 ~2h, filter with a 0.45um filter membrane, and collect the filtrate as an aqueous solution of crude semaglutide for future use.

[0063] (2) One-step purification:

[0064] One-step purification of crude semaglutide aqueous solution, chromatographic conditions: Preparative chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30*250mm. Mobile phase A: 50mM / L ammonium chloride, aqueous solution adjusted to pH=8 with ammonia water, mobile phase B: acetonitrile; detection wavelength is 230nm, flow rate is 25ml / min, sample volume is 0.92g, elution gradient is: 0~ 5min: B% is 10%-26%; 5-120min: B% is 26%-46%.

[0065] Collect semaglutide fraction samp...

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Abstract

The invention discloses a purification method of semaglutide. The method comprises the following steps: (1) dissolving a crude semaglutide product in dilute ammonia water, carrying out water bath andfiltering successively, and collecting a filtrate; (2) performing gradient elution by taking a reverse-phase silica gel filler as a stationary phase, a salt solution with a pH value regulated to 7-9 by ammonia water as a mobile phase A and acetonitrile as a mobile phase B, collecting fractions, and performing rotary evaporation to remove a part of the solvent to obtain a primarily-purified solution; (3) carrying out gradient elution by taking the reversed-phase silica gel filler as the stationary phase, a saline solution with a pH value adjusted to 2-3 by phosphoric acid as the mobile phase Aand a mixed solution of acetonitrile and isopropanol as the mobile phase B, collecting fractions, and carrying out rotary evaporation to remove a part of the solvents so as to obtain a secondarily-purified solution; and (4) carrying out gradient elution by taking the reverse-phase silica gel filler as the stationary phase, an aqueous solution of ammonium bicarbonate as the mobile phase A and acetonitrile as the mobile phase B, collecting fractions, carrying out rotary evaporation, and carrying out freeze-drying so as to obtain saltless refined peptide of semaglutide, wherein the saltless refined peptide has purity of greater than 99.5% and an individual impurity content of less than 0.1%. The method is simple and convenient to operate and facilitates large-scale preparation of semaglutide.

Description

technical field [0001] This field relates to a method for purifying peptides, in particular to a method for purifying semaglutide. [0002] technical background [0003] Semaglutide, whose English name is Semaglutide, is a new type of long-acting glucagon-like peptide-1 (GLP-1) analog developed and produced by Novo Nordisk, Denmark, for the treatment of type II diabetes. Semaglutide has the effects of lowering blood sugar, losing weight and protecting cardiovascular system, and was recommended by the FDA for approval on October 18, 2017. After the Lys side chain of semaglutide is modified by PEG, Glu and octadecanedicarboxylic acid, the hydrophilicity is greatly improved, and the binding force with albumin is enhanced; at the same time, after the Ala at the second position of the N-terminus is mutated into Aib, the effective It avoids being inactivated by DPP-IV enzymatic hydrolysis, and its half-life reaches 40 hours. Patients only need to inject it once a week. The oral do...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/605C07K1/20
CPCC07K14/605
Inventor 姚林蔡志伟李新宇张利香邱心敏
Owner SHENZHEN JYMED TECH
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