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Atosiban purification and separation method

A technology of purification, separation and atosiban is applied in the field of polypeptide drug preparation to achieve the effects of improving safety, improving purification yield and reducing production cost

Inactive Publication Date: 2017-11-03
ZHEJIANG PEPTITES BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

to Asp 5 - Isolation of atosiban impurity has not been reported

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1. Take 100 g of atosiban crude product, dissolve each gram of atosiban crude product in 200 ml of 20v / v% acetonitrile aqueous solution, and ultrasonically treat it. After the crude product is completely dissolved, filter it with a filter membrane with a pore size of 0.45 μm, and collect the filtrate for later use .

[0044] 2. Use mobile phase A to balance the reversed-phase chromatographic column, mobile phase A: 0.6 wt% sodium dihydrogen phosphate aqueous solution (adjust the pH value to 5.5 with sodium hydroxide), flow rate 800ml / min. The detection wavelength is 220nm. The specifications of the reversed-phase chromatographic column are: 20*25cm, and the stationary phase in the column is a reversed-phase C18 filler.

[0045] 3. Load the filtrate from step 1 into a reversed-phase chromatographic column.

[0046] 4. Elute with a gradient of B (acetonitrile)%: 25-65% (60min), and collect the eluted peaks.

[0047] 5. Concentrate the collected target peptide solution ...

Embodiment 2

[0051] 1. Take 80g of atosiban crude product, dissolve each gram of atosiban crude product in 200ml 20v / v% acetonitrile aqueous solution, ultrasonically treat, after the crude product is completely dissolved, filter it with a 0.45μm filter membrane, and collect the filtrate for later use .

[0052] 2. Use mobile phase A to equilibrate the reversed-phase chromatographic column, mobile phase A: 1.0 wt% sodium dihydrogen phosphate aqueous solution (adjust pH value to 4.5 with phosphoric acid), flow rate 600ml / min. The detection wavelength is 220nm. The specifications of the reversed-phase chromatographic column are: 20*25cm, and the stationary phase in the column is a reversed-phase C18 filler.

[0053] 3. Load the filtrate from step 1 into a reversed-phase chromatographic column.

[0054] 4. Elute with a gradient of B (acetonitrile)%: 21-61% (60min), and collect the eluted peaks.

[0055] 5. Concentrate the collected target peptide solution with a purity of more than 99.7% by...

Embodiment 3

[0059] 1. Take 60g of atosiban crude product, dissolve each gram of atosiban crude product in 200ml of 20v / v% acetonitrile aqueous solution, ultrasonicate, after the sample is completely dissolved, filter with a 0.45 μm filter membrane with a pore size, and collect the filtrate for later use.

[0060] 2. Use mobile phase A to equilibrate the reversed-phase chromatographic column, mobile phase A: 0.7wt% potassium dihydrogen phosphate aqueous solution (potassium hydroxide adjusts the pH value to 5.0), and the flow rate is 1000ml / min. The detection wavelength is 220nm. The specifications of the reversed-phase chromatographic column are: 20*25cm, and the stationary phase in the column is a reversed-phase C18 filler.

[0061] 3. Load the filtrate from step 1 into a reversed-phase chromatographic column.

[0062] 4. Elute with a gradient of B (acetonitrile)%: 25-65% (60min), and collect the eluted peaks.

[0063] 5. Concentrate the collected target peptide solution with a purity o...

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PUM

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Abstract

The invention discloses an atosiban purification and separation method. The method includes steps: dissolving a crude product of atosiban in acetonitrile water solution, and filtering through a filter membrane to obtain crude solution for standby application; adopting a mobile phase A which is sodium dihydrogen phosphate or monopotassium phosphate aqueous solution for balancing a reversed phase column; loading the crude solution into the reversed phase column; adopting the mobile phase A and a mobile phase B which is acetonitrile for gradient eluting, and collecting eluting peaks; subjecting collected target peptide solution with purity higher than 99.7% to vacuum rotary evaporation and concentration at a temperature not higher than 35 DEG C; converting concentrated liquid into peptide solution of acetate; subjecting the peptide solution of acetate to vacuum rotary evaporation and concentration at a temperature not higher than 35 DEG C, and performing freeze drying to obtain a finished product of atosiban. The atosiban purification and separation method has advantages that toxic impurities including Asp5-atosiban and Gly9-OH-atosiban in atosiban can be separated completely, medicine safety is improved while product yield is increased, and acquired samples are high in stability and applicable to industrial production.

Description

technical field [0001] The invention relates to the technical field of polypeptide medicine preparation, in particular to a method for purifying and separating atosiban. Background technique [0002] Atosiban is a synthetic 9-peptide, the English name is: Atosiban, which is a cyclic polypeptide, and its sequence is as follows: [0003] c[Mpa-D-Tyr(Et)-Ile-Thr-Asn-Cys]-Pro-Orn-Gly-NH2 [0004] Atosiban is a breakthrough product in obstetrics and gynecology. It is an oxytocin analogue, and it is an oxytocin competitive antagonist of receptors on the decidua and fetal membranes in the uterus. It can directly interact with oxytocin Competing for oxytocin receptors, inhibiting the combination of oxytocin and oxytocin receptors, thereby directly inhibiting the action of oxytocin on the uterus and inhibiting uterine contraction; it can also inhibit the hydrolysis of phosphatidylinositol, block the generation of second messengers and Ca 2+ activities, thereby indirectly inhibiting...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/16C07K1/36C07K1/20C07K1/34
Inventor 刘志国李雪豪陈晓航
Owner ZHEJIANG PEPTITES BIOTECH CO LTD
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