Preparation method of oxytocin

A technique of oxytocin and chromatography, applied in the field of preparation of oxytocin, can solve the problems of dilute sample concentration, bulky volume, low yield and the like, and achieve the effect of optimizing the production process

Active Publication Date: 2017-05-17
SPH NO 1 BIOCHEM & PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is to provide a preparation method of oxytocin in order to overcome the defects of complex preparation process, low yield, dilute sample concentration and bulky volume of oxytocin in the prior art

Method used

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  • Preparation method of oxytocin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 (HPLC method detects the purity and quantification of oxytocin precursor crude product and purified intermediate solution)

[0030] Instrument: Agilent 1200 HPLC

[0031] Separation column: Waters XBridge-C18, 4.6×150mm, 5μm

[0032] Mobile phase: A is 50% by volume acetonitrile aqueous solution, B is 0.02M KH 2 PO 4 pH 3.0 aqueous solution, flow rate is 1.0mL / min, detection wavelength is 220nm, detected at room temperature, elution gradient is shown in the table below, percentages are volume percentages.

[0033] Elution step Elution time eluent 1 0~15min 35%A+65%B 2 15~25min 35%A+65%B→100%A 3 25~28min 100％A 4 28.0~28.1min 100%A→35%A+65%B 5 28.1~32min 35%A+65%B

Embodiment 2

[0034] Embodiment 2 (50mm inner diameter L&L4002 preparative column packing)

[0035] Using Load&Lock dynamic axial compression and static locking technology, the filler is silica gel C18 (Kromasil C18), with a pore size of 10nm, a particle size of 10μm, a column packing density of 0.66g / mL, and a column bed pressure of 133bar, using a Varian chromatography packing system, 325g Dry powder filler, 650mL of isopropanol is stirred and homogenized, poured into L&L4002 preparative column with an inner diameter of 50mm, the compression ratio is 1.5:1, and the carrier gas is N 2 , adjust the carrier gas pressure so that the oil pressure gauge pressure is 200bar, and dynamically compress the column bed to a height of 25.5cm, as a preparative column for reverse-phase cyclization, reverse-phase purification and reverse-phase desalination schemes.

Embodiment 3

[0036] Example 3 Reversed-phase cyclization, reverse-phase purification and reverse-phase desalination of raw material of oxytocin precursor crude product

[0037] Instrument: Varian SD-1 high pressure liquid phase preparation system

[0038] Chromatographic column: Example 2 self-packed preparative column Load&Lock4002 50×260mm, C18 10μm 10nm

[0039] The crude product of oxytocin precursor is the crude product of oxytocin precursor obtained by cracking and drying by solid-phase synthesis method, and its structural formula is Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH 2 of trifluoroacetate.

[0040] The crude oxytocin precursor solution is a 5 g / L solution formed by dissolving the crude oxytocin precursor in 5% acetonitrile aqueous solution.

[0041] Mobile phase A1 is purified water, A2 is 0.03% H by volume 2 o 2 NaOH aqueous solution with a pH of 7.5, the mobile phase B is acetonitrile, and the mobile phase C is the crude oxytocin precursor solution. According to the method...

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Abstract

The invention discloses a preparation method of oxytocin. The preparation method comprises steps as follows: an oxytocin precursor crude product solution is sequentially subjected to reversed-phase cyclization, reversed-phase purification and reversed-phase desalination with high-performance reversed-phase liquid chromatography; filler used in the high-performance reversed-phase liquid chromatography method is silica gel C18; an oxytocin precursor crude product contains two free sulfydryl groups. The reversed-phase adsorbing cyclization, purification and desalination one-step method is applied in the preparation method for preparation of a polypeptide pure product, the production process is optimized, and the preparation method is suitable for industrial continuous production.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals. More specifically, the present invention relates to a preparation method of oxytocin. Background technique [0002] Oxytocin, also known as oxytocin, English name is Oxytocin, structural formula: [0003] [0004] The molecular formula is: C 43 h 66 N 12 o 12 S 2 , the molecular weight is 1007.2 [0005] Oxytocin is used to induce labor, oxytocin, postpartum and post-abortion uterine bleeding caused by uterine atony or poor abdominal contraction; understand placental reserve function (oxytocin provocation test); nasal drops can promote milk discharge. Oxytocin can indirectly stimulate the contraction of uterine smooth muscle, simulating the uterine contraction of normal childbirth, leading to cervical dilatation, and the uterine response to oxytocin gradually increases during pregnancy and reaches its peak at full term. Oxytocin can also stimulate the contraction of the smooth muscle ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/16C07K1/20
Inventor 江锡铭朱鑫磊杨柳青丁金国黄臻辉
Owner SPH NO 1 BIOCHEM & PHARMA CO LTD
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