阅读说明：本技术 一种比伐卢定粗品的hplc纯化方法 (HPLC purification method of bivalirudin crude product ) 是由 陈艳明 韦怀福 南志远 于 2018-06-26 设计创作，主要内容包括：本发明描述了一种比伐卢定HPLC纯化方法,主要提供比伐卢定粗品HPLC纯化方法,目前比伐卢定纯化的纯度及收率较低,难以符合用于制备比伐卢定制剂的原料标准。本发明的技术方案是,通过优化比伐卢定粗品的浓度、HPLC流动相组成及梯度等纯化条件,最后得到了高纯度的比伐卢定,同时收率大大提高。(The invention describes a bivalirudin HPLC purification method, and mainly provides a bivalirudin crude product HPLC purification method. According to the technical scheme, purification conditions such as concentration of a bivalirudin crude product, HPLC mobile phase composition and gradient are optimized, so that high-purity bivalirudin is obtained finally, and meanwhile, the yield is greatly improved.)

1. An HPLC purification method of a bivalirudin crude product is characterized by comprising the following purification steps:

1) respectively preparing crude bivalirudin solutions with different concentrations by using purified water and 30-50% acetonitrile aqueous solution as solvents, carrying out ultrasonic treatment, filtering with a filter membrane, and taking filtrate for later use;

2) the mobile phase A is 0.01-0.1% trifluoroacetic acid water solution, the mobile phase B is 0.01-0.1% trifluoroacetic acid acetonitrile solution, a reverse phase high performance liquid system is utilized to purify the filtered bivalirudin crude product solution, a preparation column is a dynamic axial compression column, gradient elution separation and purification are carried out, an ultraviolet detector is adopted to detect a sample, and eluent containing target peak bivalirudin is collected;

3) and carrying out reduced pressure rotary evaporation and concentration on the separated and purified high-purity bivalirudin eluent, collecting a concentrated solution, and freeze-drying to obtain the bivalirudin API.

2. The method for preparing bivalirudin by HPLC purification according to claim 1, which is characterized in that: dissolving the crude bivalirudin in 50% acetonitrile as solvent.

3. The bivalirudin HPLC purification preparation method according to claim 1, wherein the HPLC purification preparation method comprises the following steps: the mobile phase A prepared by purification was 0.1% trifluoroacetic acid in water and the mobile phase B was 0.1% trifluoroacetic acid in acetonitrile.

4. The bivalirudin HPLC purification preparation method according to claim 1, wherein the HPLC purification preparation method comprises the following steps: the elution gradient was:

Technical Field

The invention relates to the field of medicines, in particular to a bivalirudin HPLC purification method, which specifically comprises the steps of preparing bivalirudin by using an acetonitrile-water-trifluoroacetic acid system and combining reversed-phase chromatographic filler purification, collecting a main peak, performing post-treatment, performing reduced pressure concentration, freezing and drying to obtain high-purity bivalirudin.

Background

Bivalirudin (trade name Angiomax) is an artificially synthesized anticoagulant drug, is applied by American Medicines company and is approved to be marketed in the United states by FDA in 12 months in 2000, belongs to a direct thrombin inhibitor, and the anticoagulant component of the Bivalirudin belongs to a peptide with 20 amino acid residues at the C end of a hirudin derivative, and directly inhibits thrombin by combining a catalyst site and a circulating anion output site of thrombin blood clots. Early clinical research shows that bivalirudin has definite anticoagulation effect, low bleeding event incidence rate and higher safety compared with the traditional heparin anticoagulation treatment, and the bivalirudin can be used for replacing heparin for patients receiving Percutaneous Coronary Intervention (PCI) and shows good treatment effect in other indications along with continuous research on bivalirudin clinical tests, so the future application prospect of the bivalirudin is very wide.

A large number of reports on the preparation method of bivalirudin are reported at home and abroad. The synthesis method comprises solid-phase synthesis, liquid-phase synthesis and solid-liquid phase synthesis, but the literature reports are less for a complete HPLC purification system method of the bivalirudin crude product. According to published documents, the purity of bivalirudin can only reach 98.5%, and the prepared bivalirudin API is close to the limit of impurities according to the regulation that related substances in the national drug standard YBH02852011 cannot exceed 1.5%, and the long-time storage within 2 years of the validity period easily causes unqualified inspection items of the related substances. Aiming at the problems of the bivalirudin raw material purification process, the invention provides an HPLC purification method of bivalirudin.

Disclosure of Invention

The invention aims to provide an HPLC purification method of bivalirudin. The purification preparation method solves the problems of low purity and low yield of the purified bivalirudin API at present.

The method comprises the steps of adjusting several different concentrations before the purification of the bivalirudin crude product, wherein the different concentrations are different in purification efficiency and bivalirudin purity; the invention is characterized in that the concentration of trifluoroacetic acid in the mobile phase is optimized, and the yield is obviously improved when the concentration of trifluoroacetic acid is 0.01-0.1%; the invention greatly improves the yield and the purity after the gradient elution method is optimized, and the purity can reach more than 99.5 percent.

Specifically, the purification method provided by the invention comprises the following steps:

(1) dissolving the crude product with purified water and 30-50% acetonitrile, filtering to prepare a crude product solution, and then injecting the crude product solution into a high performance liquid chromatograph.

(2) And (3) purification conditions: the mobile phase A is 0.01 to 0.1 percent of trifluoroacetic acid aqueous solution, the mobile phase B is 0.01 to 0.1 percent of trifluoroacetic acid acetonitrile solution, and the chromatographic column is YMC-Actas Triart Prep.

(3) And concentrating the eluent by rotary evaporation, and freeze-drying to obtain bivalirudin API.

Wherein, the conditions of the high performance liquid chromatography in the step (1) are as follows:

crude solution: preparing 2mg/ml crude product solution with purified water

The instrument comprises the following steps: waters preparative high performance liquid chromatograph, model: waters 2545

A chromatographic column: YMC-Actus Triart Prep C18-S, specification 250X 30mm, S-10 μm, 12nm,

in the step (2):

mobile phase A: 0.1% trifluoroacetic acid in water,

mobile phase B: 0.1 percent of trifluoroacetic acid acetonitrile solution,

monitoring wavelength: 215nm

Flow rate: 20ml/min

Elution gradient:

sample introduction amount: 50 mg/needle

Collecting main peak eluent, and detecting the purity of more than 99.0%.

Or the conditions of the high performance liquid chromatography in the step (1):

crude solution: preparing 2mg/ml crude product solution by using 30% acetonitrile

The instrument comprises the following steps: waters preparative HPLC, Waters 2545

A chromatographic column: YMC-Actus Triart Prep C18-S, specification 250X 30mm, S-10 μm, 12nm,

in the step (2):

mobile phase A: 0.1% trifluoroacetic acid in water,

mobile phase B: 0.1 percent of trifluoroacetic acid acetonitrile solution,

monitoring wavelength: 215nm

Flow rate: 20ml/min

Elution gradient:

sample introduction amount: 50 mg/needle

Collecting main peak eluent, and detecting that the purity of the main peak eluent is more than 99.0%.

Or the conditions of the high performance liquid chromatography in the step (1):

crude solution: preparing 2mg/ml crude product solution with purified water

The instrument comprises the following steps: waters preparative HPLC, Waters 2545

A chromatographic column: YMC-Actus Triart Prep C18-S, specification 250X 30mm, S-10 μm, 12nm,

in the step (2):

mobile phase A: 0.1% trifluoroacetic acid in water,

mobile phase B: 0.1 percent of trifluoroacetic acid acetonitrile solution,

monitoring wavelength: 215nm

Flow rate: 20ml/min

Elution gradient:

sample introduction amount: 50 mg/needle

Collecting main peak eluent, and detecting the purity of more than 99.5%.

Or the conditions of the high performance liquid chromatography in the step (1):

crude solution: preparing 2mg/ml crude product solution by using 50% acetonitrile

The instrument comprises the following steps: waters preparative HPLC, Waters 2545

A chromatographic column: YMC-Actus Triart Prep C18-S, specification 250X 30mm, S-10 μm, 12nm,

in the step (2):

mobile phase A: 0.01 percent of trifluoroacetic acid aqueous solution,

mobile phase B: 0.01 percent of trifluoroacetic acid acetonitrile solution,

monitoring wavelength: 215nm

Flow rate: 20ml/min

Elution gradient:

sample introduction amount: 50 mg/needle

Collecting main peak eluent, and detecting the purity of more than 99.0%.

Wherein, the conditions of the high performance liquid chromatography in the step (2) are as follows:

crude solution: preparing 2mg/ml crude product solution by using 50% acetonitrile

The instrument comprises the following steps: waters preparative HPLC, Waters 2545

A chromatographic column: YMC-Actus Triart Prep C18-S, specification 250X 30mm, S-10 μm, 12nm,

in the step (2):

mobile phase A: 0.03 percent of trifluoroacetic acid aqueous solution,

mobile phase B: 0.03 percent of trifluoroacetic acid acetonitrile solution,

monitoring wavelength: 215nm

Flow rate: 20ml/min

Elution gradient:

sample introduction amount: 50 mg/needle

Collecting main peak eluent, and detecting the purity of more than 99.0%.

Wherein, the conditions of the high performance liquid chromatography in the step (2) are as follows:

crude solution: preparing 2mg/ml crude product solution by using 50% acetonitrile

The instrument comprises the following steps: waters preparative HPLC, Waters 2545

A chromatographic column: YMC-Actus Triart Prep C18-S, specification 250X 30mm, S-10 μm, 12nm,

in the step (2):

mobile phase A: 0.05% trifluoroacetic acid in water,

mobile phase B: 0.05 percent of trifluoroacetic acid acetonitrile solution,

monitoring wavelength: 215nm

Flow rate: 20ml/min

Elution gradient:

sample introduction amount: 50 mg/needle

Collecting main peak eluent, and detecting the purity to be 99.0%.

Or the conditions of the high performance liquid chromatography in the step (1):

crude solution: preparing 2mg/ml crude product solution by using 50% acetonitrile

The instrument comprises the following steps: waters preparative HPLC, Waters 2545

A chromatographic column: YMC-Actus Triart Prep C18-S, specification 250X 30mm, S-10 μm, 12nm,

in the step (2):

mobile phase A: 0.1% trifluoroacetic acid in water,

mobile phase B: 0.1 percent of trifluoroacetic acid acetonitrile solution,

monitoring wavelength: 215nm

Flow rate: 20ml/min

Elution gradient:

sample introduction amount: 50 mg/needle

Collecting main peak eluent, and detecting the purity to be more than 99.8%.

Or the conditions of the high performance liquid chromatography in the step (1):

crude solution: preparing 2mg/ml crude product solution by using 50% acetonitrile

The instrument comprises the following steps: waters preparative HPLC, Waters 2545

A chromatographic column: YMC-Actus Triart Prep C18-S, specification 250X 30mm, S-10 μm, 12nm,

in the step (2):

mobile phase A: 0.1% trifluoroacetic acid in water,

mobile phase B: 0.1 percent of trifluoroacetic acid acetonitrile solution,

monitoring wavelength: 215nm

Flow rate: 40ml/min

Elution gradient:

sample introduction amount: 50 mg/needle

Collecting main peak eluent, and detecting the purity of more than 99.0%.

Drawings

FIG. 1: detection result of related substances of bivalirudin crude product

FIG. 2: example 1 detection results of bivalirudin related substances after purification

FIG. 3: example 2 detection results of bivalirudin related substances after purification

FIG. 4: example 4 detection results of bivalirudin related substances after purification

Detailed Description

The present invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for the purpose of illustration and should not be taken as limiting the invention as detailed in the claims.