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Method of purifying pramlintide

A pramlintide, the purpose of the technology, applied in the field of HPLC, can solve the problem of no large-scale production, to achieve the effect of good yield and high purity

Active Publication Date: 2009-09-09
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the published literature and patents, there is no large-scale production and high yield purification process report

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0017] 1. Sample processing: The crude peptide was dissolved in ultrapure water to 10 mg / mL, and the sample was completely dissolved before using Filter through a membrane and collect the filtrate for later use.

[0018] 2. The first purification: purification conditions: chromatographic column: a chromatographic column with octadecylsilane-bonded silica gel as the stationary phase, the diameter and length of the column are: 5cm×25cm. Mobile phase: Phase A: 0.1% aqueous trifluoroacetic acid; Phase B: acetonitrile. Flow rate: 70-80ml / min. Detection wavelength: 230nm. Gradient: B%: 25% to 45% (45 min). The injection volume is 1.5-2.0 g.

[0019] Purification process: Rinse the chromatographic column with more than 50% acetonitrile, and load the sample after equilibration, and the sample load is 1.5-2.0g. Linear gradient elution was carried out for 45 min, the target peak was collected, and the collected target peptide solution was concentrated to about 50-60 mg / mL by rotar...

Embodiment 2

[0025] 1. Sample processing: The crude peptide was dissolved in ultrapure water to 10 mg / mL, and the sample was completely dissolved before using Filter through a membrane and collect the filtrate for later use.

[0026] 2. The first purification: purification conditions: chromatographic column: a chromatographic column with octadecylsilane-bonded silica gel as the stationary phase, the diameter and length of the column are: 15cm×25cm. Mobile phase: Phase A: 0.1% aqueous trifluoroacetic acid; Phase B: acetonitrile. Flow rate: 500-550ml / min. Detection wavelength: 230nm. Gradient: B%: 25% to 45% (45 min). The injection volume is 15-20g.

[0027] Purification process: Rinse the chromatographic column with more than 50% acetonitrile, and load the sample after equilibration, and the sample load is 15-20g. Linear gradient elution was carried out for 45 min, the target peak was collected, and the collected target peptide solution was concentrated to about 50-60 mg / mL by rotary ...

Embodiment 3

[0033] 1. Sample processing: The crude peptide was dissolved in ultrapure water to 10 mg / mL, and the sample was completely dissolved before using Filter through a membrane and collect the filtrate for later use.

[0034] 2. The first purification: purification conditions: chromatographic column: a chromatographic column with octadecylsilane-bonded silica gel as the stationary phase, the diameter and length of the column are: 30cm×25cm. Mobile phase: Phase A: 0.1% aqueous trifluoroacetic acid; Phase B: acetonitrile. Flow rate: 2000-2200ml / min. Detection wavelength: 230nm. Gradient: B%: 25% to 45% (45 min). The injection volume is 65-75g.

[0035] Purification process: Rinse the chromatographic column with more than 50% acetonitrile, and load the sample after equilibration, and the sample load is 65-75g. Linear gradient elution was carried out for 45 min, the target peak was collected, and the collected target peptide solution was concentrated to about 50-60 mg / mL by rotar...

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Abstract

The invention discloses a method of purifying pramlintide, belonging to the technical field of HPLC and comprising the steps: 1) after a synthesized coarse peptide is dissolved, a fixed phase serving as reverse silica gel column and mobile phases trifluoroacetic acid aqueous solution as phase A and chromatogram pure acetonitrile as phase B, are used to carry out gradient elution and purification to collect peptide solution of target peak; 2) after the target peptide solution purified in the step 1) is concentrated, and the fixed phase as reverse silica gel column and mobile phases phosphate aqueous solution as phase A and chromatogram pure acetonitrile as phase B, are used to carry out gradient elution and purification to collect peptide solution of target peak; and 3) the phosphate is converted into acetate by anion exchange salt conversion method. The invention purifies pramlintide by the reverse phase high-efficiency liquid phase chromatography and anion exchange method, with high purity and good yield, and provides a process suitable for purifying pramlintide in bulk to reach the industrial requirement.

Description

technical field [0001] The invention belongs to the technical field of HPLC, in particular to a method for large-scale purification of Pramlintide. technical background [0002] Pramlintide Chinese preparation name is Pramlintide acetate, English name: Pramlintide acetate, molecular formula: C 171 H 267 N 51 O 53 S 2 · x(C 2 H 4 O 2 ) · x(H 2 O), molecular weight: 3949.39, CAS accession number: 196078-30-5. [0003] Pramlintide is a polypeptide drug for the treatment of diabetes, which has good effect and small side effects, and has a good market prospect. In the published literature and patents, there is no large-scale production and high yield purification process reported. SUMMARY OF THE INVENTION [0004] The object of the present invention is to provide a process method suitable for industrialized purification of pramlintide, using reversed-phase high performance liquid chromatography to purify pramlintide, with high purity and good yield, meeting the requ...

Claims

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Application Information

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IPC IPC(8): C07K14/575C07K1/20C07K1/18A61P3/10
Inventor 康旭覃亮政马亚平袁建成
Owner HYBIO PHARMA
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