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Method of purifying pramlintide

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

Active Publication Date: 2012-11-28
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
Comparison scheme
Effect test

Embodiment 1

[0017] 1. Sample treatment: Dissolve the crude peptide in ultrapure water to 10mg / mL, and use it after the sample is completely dissolved. Filter with a membrane filter, 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, and the diameter and length of the column are: 5cm×25cm. Mobile phase: A phase: 0.1% trifluoroacetic acid aqueous solution; B phase: acetonitrile. Flow rate: 70-80ml / min. Detection wavelength: 230nm. Gradient: B%: 25% to 45% (45min). The injection volume is 1.5-2.0g.

[0019] Purification process: Rinse the chromatographic column with more than 50% acetonitrile, then equilibrate and load the sample, and the sample volume is 1.5-2.0g. After linear gradient elution for 45 minutes, the target peak was collected, and the collected target peptide solution was concentrated by rotary evaporation under...

Embodiment 2

[0025] 1. Sample treatment: Dissolve the crude peptide in ultrapure water to 10mg / mL, and use it after the sample is completely dissolved. Filter with a membrane filter, 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, and the diameter and length of the column are: 15cm×25cm. Mobile phase: A phase: 0.1% trifluoroacetic acid aqueous solution; B phase: acetonitrile. Flow rate: 500-550ml / min. Detection wavelength: 230nm. Gradient: B%: 25% to 45% (45min). The injection volume is 15-20g.

[0027] Purification process: wash the chromatographic column with more than 50% acetonitrile, and then balance the sample, and the sample volume is 15-20g. After linear gradient elution for 45 minutes, the target peak was collected, and the collected target peptide solution was concentrated by rotary evaporation under reduced pr...

Embodiment 3

[0033] 1. Sample treatment: Dissolve the crude peptide in ultrapure water to 10mg / mL, and use it after the sample is completely dissolved. Filter with a membrane filter, 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, and the diameter and length of the column are: 30cm×25cm. Mobile phase: A phase: 0.1% trifluoroacetic acid aqueous solution; B phase: acetonitrile. Flow rate: 2000-2200ml / min. Detection wavelength: 230nm. Gradient: B%: 25% to 45% (45min). The injection volume is 65-75g.

[0035] Purification process: wash the chromatographic column with more than 50% acetonitrile, and then equilibrate the sample, and the sample volume is 65-75g. After linear gradient elution for 45 minutes, the target peak was collected, and the collected target peptide solution was concentrated by rotary evaporation under redu...

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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 (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 treating diabetes, which has good effect and few 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 report. Contents of the invention [0004] The purpose of the present invention is to provide a process suitable for industrialized purification of pramlintide, using reversed-phase high-performance liquid chromatography to purify pramlintide, with high purity and good yield, meeting the requirements ...

Claims

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Application Information

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