Linaclotide purifying method

A technology of linaclotide and purification method, which is applied in the field of linaclotide purification, can solve problems affecting product purity and content, impurity is difficult to remove, purification effect is not ideal, etc., and achieves simple operation, low impurity content, good The effect of economic and practical value

Inactive Publication Date: 2017-10-20
NANJING UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, when using reversed-phase high-performance liquid chromatography to purify linaclotide, the presence of many pairs of disulfide bonds in the molecule will lead to unsatisfactory purification results, and impurities such as polymers in the product are not easy to remove, affecting Product purity and content

Method used

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  • Linaclotide purifying method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] 1. Processing of crude linaclotide

[0027] Weigh 0.5g of crude linaclotide, add 10mL of pure water, mix evenly and place in an ultrasonic cleaner for about 10min to promote the dissolution of the sample, absorb the supernatant of the mixed system, and use a 0.22μm PVDF membrane to remove the insoluble Impurities.

[0028] 2. Purification of Linaclotide Samples

[0029] Use the anion exchange column SOURCE 15Q as the separation medium, after loading the crude product solution, use the 100mmol / L Tris-HCl buffer solution containing pH8.5 as the mobile phase A, and use the pH8.5 Tris-HCl buffer solution containing 1 mol / L NaCl The mobile phase B was used for gradient elution, and the eluate was collected; the system was flushed with mobile phase A first, and the sample was loaded after equilibration. The flow rate was 0.5mL / min, and the ratio of mobile phase A and mobile phase B was adjusted to 50 within 30 minutes. % for elution. After the sample peaks, adjust the prop...

Embodiment 2

[0033] 1. Processing of crude linaclotide

[0034] Weigh 0.5g of crude linaclotide, add 10mL of pure water, mix evenly and place in an ultrasonic cleaner for about 10min to promote the dissolution of the sample, absorb the supernatant of the mixed system, and use a 0.22μm PVDF membrane to remove the insoluble Impurities.

[0035] 2. Purification of Linaclotide Samples

[0036] Anion exchange column Q-SepharoseFast Flow was used as the separation medium. After loading the crude product solution, 80mmol / L Tris-HCl buffer solution containing pH8.2 was used as mobile phase A, and pH8.2 Tris-HCl buffer solution containing 0.8mol / L NaCl was used as mobile phase A. HCl buffer is mobile phase B for gradient elution, collect the eluate; first use mobile phase A to flush the system, and load the sample after equilibration, the flow rate is 0.5mL / min, adjust the ratio of mobile phase A and mobile phase B within 30min to 50% for elution. After the sample peaks, adjust the proportion of...

Embodiment 3

[0040] 1. Processing of crude linaclotide

[0041] Weigh 0.5g of crude linaclotide, add 10mL of pure water, mix evenly and place in an ultrasonic cleaner for about 10min to promote the dissolution of the sample, absorb the supernatant of the mixed system, and use a 0.22μm PVDF membrane to remove the insoluble Impurities.

[0042] 2. Purification of Linaclotide Samples

[0043]Use the anion exchange column SOURCE 15Q as the separation medium, after loading the crude product solution, use the 120mmol / L Tris-HCl buffer solution containing pH8.8 as the mobile phase A, and use the pH8.8 Tris-HCl buffer solution containing 1.2mol / L NaCl The mobile phase B was used for gradient elution, and the eluate was collected; the system was flushed with mobile phase A first, and the sample was loaded after equilibration. The flow rate was 0.5mL / min, and the ratio of mobile phase A and mobile phase B was adjusted to 50 within 30 minutes. % for elution. After the sample peaks, adjust the prop...

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Abstract

The invention mainly relates to a linaclotide purifying method and belongs to the technical field of bioseparation. According to the linaclotide purifying method, an ion exchange chromatography method and a high-performance liquid chromatography method are combined, an anion exchange column is adopted as a stationary phase, a Tris HCl buffering solution is adopted as a mobile phase A, a NaCl-contained Tris-HCl buffering solution is adopted as a mobile phase B, a gradient eluting method is adopted to treat a crude linaclotide solution, eluate is subjected to desalination and acetate changing by means of the high-performance liquid chromatography method, a C18 column is adopted as a separating medium, after a sample is loaded, gradient elution is performed by adopting an aqueous acetic acid solution as the mobile phase A and acetonitrile as the mobile phase B, and eluate is subjected to freeze-drying to obtain pure linaclotide. The linaclotide purifying method is simple to operate, low in separation cost, high in yield and suitable for large-scale linaclotide production; purity of the obtained pure linaclotide can reach to above 99%, so that the obtained pure linaclotide is low in impurity content and has favorable economic and practical values and an extensive application prospect.

Description

technical field [0001] The invention belongs to the technical field of medicinal chemistry, and in particular relates to a method for purifying linaclotide. Background technique [0002] Constipation refers to a common symptom of the digestive tract that refers to too few defecation times, or poor, laborious, difficult defecation, dry stool and small amount. Most patients with chronic constipation present with difficulty in defecation, dry stool, and only defecate once in a few days or even a week. During defecation, they may have cramping pain and a feeling of falling in the left abdomen. Some patients complain of bitter mouth, loss of appetite, abdominal distension, lower abdominal discomfort, and flatulence. More or more neurological symptoms such as dizziness, headache, fatigue, but generally not serious. Accompanied by severe abdominal pain, vomiting or blood in the stool, constipation caused by acute intestinal obstruction should be considered. General physical exami...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K1/18C07K1/16C07K1/14
CPCC07K7/08
Inventor 朱颐申姚忠朱本伟张爱明王华
Owner NANJING UNIV OF TECH
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