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Purification method of linaclotide

A technology of linaclotide and purification method, which is applied in the field of purification of polypeptide drugs, can solve the problems of low total product yield, difficulty in removing impurities, and low purity, and achieve high yield, good multimer impurities, and low cost Effect

Active Publication Date: 2015-12-16
HANGZHOU HEZE PHARMA TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The technical problem to be solved by the present invention is that existing methods purify polypeptides containing multiple pairs of disulfide bonds, specifically linaclotide, which is difficult to remove impurities, and the total yield of the product is low and the purity is low.

Method used

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  • Purification method of linaclotide
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  • Purification method of linaclotide

Examples

Experimental program
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Effect test

Embodiment 1

[0078] Example 1: Solid phase synthesis of linaclotide

[0079] Weigh 66.67g of 2-CTC resin with a degree of substitution of 1.50mmol / g, add it to the solid phase reaction column, add it to the solid phase reaction column, wash once with DCM, and swell the resin with DCM for 30 minutes, then take 229.76g of Fmoc -Tyr(tBu)-OH (500mmol) was dissolved in DMF, activated by adding 124ml DIEA (750mmol) in an ice-water bath, then added to the above-mentioned reaction column equipped with resin, reacted for 2 hours, and then blocked by adding 1000ml of anhydrous methanol for 1 hour. Washed 3 times with DMF to obtain Fmoc-Tyr(tBu)-CTC resin. Use a mixed solution of DMF:pyridine with a volume ratio of 4:1 to remove Fmoc protection, then wash with DMF for 6 times, weigh 175.72g Fmoc-Cys(Trt)-OH (300mmol), add 40.52gHOBt (300mmol) at a volume ratio of 1 : 1 mixed solution of DCM and DMF, add 46ml DIC (300mmol) under ice-water bath to activate, then add to the above-mentioned reaction c...

Embodiment 2

[0080] Example 2: Cleavage of Linaclotide

[0081] Weigh 33.09g of linaclotide linear peptide resin and add it to a 1000mL three-necked round-bottomed flask. EDT, PhSMe, H 2 Prepare 330ml of lysate for O and TFA, add the lysate to the above resin, and react at 20~30°C for 2 hours. The reaction solution was added to glacial ether for precipitation for 1 hour, centrifuged, washed with anhydrous ether for 6 times, and vacuum-dried to obtain 14.56 g of linaclotide linear peptide.

Embodiment 3

[0082] Example 3: Cyclization of Linaclotide

[0083] Add 120g (1mol) sodium dihydrogen phosphate and 1.91g (20mmol) guanidine hydrochloride to 10L solution (water: acetonitrile = 85:15) respectively, stir and dissolve, then add 10.2g linaclotide linear peptide, 25-30℃ After stirring and reacting for 24 hours, it was set aside. The purity of the crude linaclotide was 64.5% (HPLC detection), the polymer impurity content was 2.96% (gel chromatography detection), and the synthesis yield was 45.2%.

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Abstract

The invention relates to a purification method of linaclotide. The purification method is characterized by comprising following steps: (1), pH of a solution of a linaclotide crude product is regulated by trifluoroacetic acid to be 3.5 plus or minus 0.2; (2), gradients are set according to volume fraction, 50% of a flowing phase B is used for washing a reverse phase filler column for 10 min, and 5% of the flowing phase B is adopted to perform isocratic elution balancing for 10 min; (3), the solution in the step (1) is loaded to reverse phase filler; (4), gradients are set according to volume fraction, the initial-state flowing phase B of the elution gradients is 5% and kept for 5 min, the proportion of the flowing phase B is increased to 16% in one minute, the proportion of the flowing phase B is increased to 26% in 80 minutes, and elution fractions are collected; (5), the elution fractions are transformed into salt and subjected to concentration and freeze-drying, and fine linaclotide is obtained.

Description

technical field [0001] The invention relates to a purification method of polypeptide drugs, in particular to a purification method of linaclotide. Background technique [0002] Linaclotide (linaclotide) is a guanylate cyclase C (GC-C) agonist developed by Ironwood Company of the United States. It was approved by the US FDA on August 30, 2012, and its trade name is Linzess. The drug comes in capsule form for the treatment of irritable bowel syndrome with constipation (IBS-C) and chronic idiopathic constipation (CIC), and it is the first drug for the treatment of constipation with such a mechanism of action. [0003] Linaclotide is a 14-amino acid peptide with the sequence Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH(1-6), (2 -10), (5-13)-tris(disulfide), white to off-white amorphous powder, slightly soluble in water and aqueous sodium chloride (0.9%). [0004] Linaclotide, a guanylate cyclase C agonist, results in increased intracellular and extracellular cycl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K1/20
Inventor 倪晟朱明月姜维斌赵航周亮
Owner HANGZHOU HEZE PHARMA TECH
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