Method for preparing vasopressin deamidization impurities

A technology for deamidation and impurities, applied in the field of biopharmaceuticals, can solve the problems of dilute sample concentration, low yield and large volume, and achieve the effect of optimizing the production process

Active Publication Date: 2017-05-24
SPH NO 1 BIOCHEM & PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a method for deamidating impurities of vasopressin in order to overcome the defects of complex preparation process, low yield, dilute sample concentration and large volume in the prior art. Preparation

Method used

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  • Method for preparing vasopressin deamidization impurities
  • Method for preparing vasopressin deamidization impurities

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 (HPLC method to detect the crude product of vasopressin deamidation impurity precursor and the purity of the purified product solution)

[0031] Instrument: Waters 2695 / 2489 high performance liquid chromatography

[0032] Separation column: Agilent XDB-C18 4.6×250mm, 5μm

[0033] Mobile phase: A is 0.1% TFA aqueous solution in volume percentage, B is 0.1% TFA-50% acetonitrile aqueous solution in volume percentage (TFA is trifluoroacetic acid)

[0034] The flow rate is 1.0mL / min, the detection wavelength is 214nm, and it is detected at room temperature. The elution gradient is shown in the table below, and the percentages are volume percentages.

[0035] Elution step Elution time eluent 1 0~5min 95%A+5%B 2 5~25min 95%A+5%B→50%A+50%B 3 25~30min 100％B 4 30~35min 95%A+5%B

Embodiment 2

[0036] Embodiment 2 (75mm inner diameter L&L4003 preparative column packing)

[0037] Using Load&Lock dynamic axial compression and static locking technology, the filler is styrene-divinylbenzene copolymer (reversed phase packing Agilent PLRP-S), the pore size is 10nm, the particle size is 10μm, and the column packing density is 0.33g / mL. Bed pressure 650psi, using Varian chromatography packing system, 370g dry powder filler, 2L methanol after stirring and homogenizing, poured into the L&L4003 preparative column with an inner diameter of 75mm, the compression ratio is 3:1, and the carrier gas is N 2 , adjust the carrier gas pressure so that the pressure of the oil pressure gauge is 2000psi, and the dynamic axial compression reaches a column bed height of 26cm.

Embodiment 3

[0038] Example 3 Reverse-phase cyclization, reverse-phase purification and reverse-phase desalting of vasopressin deamidation impurity precursor crude raw material

[0039] Instrument: Varian SD-1 high pressure liquid phase preparation system

[0040] Chromatographic column: self-packed preparative column Load&Lock4003 75×260mm in Example 2, PLRP-S 10μm 10nm

[0041] The crude product of vasopressin deamidation impurity precursor is the crude product of vasopressin deamidation impurity precursor obtained by cleavage and drying by solid phase synthesis method, and its structural formula is Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly of trifluoroacetate.

[0042] The crude vasopressin deamidation impurity precursor solution is a 5 g / L solution formed by dissolving the crude vasopressin deamidation impurity precursor in 5% acetonitrile aqueous solution.

[0043] Mobile phase A1 is purified water, A2 is 0.03 volume percent H 2 o 2pH is 7.5 NaOH aqueous solution, the mobile phase B is ...

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Abstract

The invention discloses a method for preparing vasopressin deamidization impurities. The preparation method comprises the steps of adopting efficient liquid phase reversed phase chromatography to cause vasopressin deamidization impurity precursor crude product solution to sequentially undergo reverse phase cyclization, reverse phase purification and reverse phase desalting. Filler of the efficient liquid phase reversed phase chromatography is styrene divinylbenzene copolymer. The vasopressin deamidization impurity precursor crude product contains two free sulfhydryl groups. The reverse phase adsorption method cyclization, purification and desalting are applied creatively, the cyclization, purification and desalting problem is solved in one time, the production process is optimized, and the 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 method for preparing vasopressin deamidation impurities. Background technique [0002] Vasopressin is a synthetic polypeptide consisting of nine amino acid residues with a chemical structure of The theoretical molecular weight of vasopressin is 1084.24. Vasopressin tannin is an antidiuretic hormone drug, which can promote the reabsorption of water in the distal renal tubule and collecting duct and has an antidiuretic effect. Its preparation is clinically used for the treatment of diabetes insipidus. [0003] At present, most of the domestic vasopressin deamidation impurities first adopt solid-phase synthesis to protect the vasopressin deamidation impurity resin, and then undergo cracking and drying to obtain the crude product of the vasopressin deamidation impurity precursor (Cys-Tyr-Phe-Gln-Asn-Cys- Trifluoroacetate of Pro-Arg-Gly), high...

Claims

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Application Information

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IPC IPC(8): C07K7/16C07K1/20
Inventor 江锡铭丁金国黄臻辉
Owner SPH NO 1 BIOCHEM & PHARMA CO LTD
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