WO2020147421A1

WO2020147421A1 - Sugammadex isolation and purification method

Info

Publication number: WO2020147421A1
Application number: PCT/CN2019/120176
Authority: WO
Inventors: 江必旺; 王希; 李易
Original assignee: 苏州纳微科技股份有限公司
Priority date: 2019-01-18
Filing date: 2019-11-22
Publication date: 2020-07-23
Also published as: CN109517094A

Abstract

Provided is a high-efficiency sugammadex isolation and purification method, comprising the following steps: dissolving and filtering crude sugammadex extract; loading the described filtered solution into a weak anion exchange resin and performing chromatography; eluting a target product using saline solution as a mobile phase; collecting in segments solution having a target peak value, gathering component liquids meeting requirements. The present method is used for deep purification of sugammadex, only requiring one chromatography purification step to be able to meet the requirements of sugammadex purity greater than 99.0% and individual impurities less than 0.1%, having a high and stable purification yield; using an aqueous solution of sodium acetate and an aqueous solution of sodium chloride as mobile phases, and not using an organic solvent, the mobile phase is safe, does not contaminate and is relatively low-cost; the isolation method is simple and convenient, and may be used for large-scale production, greatly decreasing production costs.

Description

A method for separating and purifying sugammadex

Technical field

The invention relates to the field of medicine purification, in particular to a method for efficiently separating and purifying sugammadex.

Background technique

Sugammadex is a selective antagonist of steroidal muscle relaxants, which is used to quickly reverse the neuromuscular blockade caused by the anesthetics rocuronium and vecuronium during surgery in adult patients. Shu Glucose is a synthetic modified γ-cyclodextrin composed of a lipophilic core and a hydrophilic outer end. The administration of sugammadex via intravenous injection can significantly improve the recovery of muscle relaxation after surgery. The rapid reversal of the neuromuscular blockade of steroidal muscle relaxants in a short time is of positive significance for improving the safety of general anesthesia.

The structural formula is as follows:

At present, domestic and foreign patents, literature and other reports on sugammadex are mostly synthetic methods and clinical patents, and there are few reports on separation and purification methods. CN201810153493.0 provides a method for separating and purifying sugammadex, which describes the separation and purification using C18 reversed-phase column chromatography, and the mobile phase is ultrapure water and acetonitrile. The method uses a solvent as an organic reagent, which is harmful. Therefore, it is necessary to do further research on the separation, purification and preparation of sugammadex.

Summary of the invention

The purpose of the present invention is to provide a method for efficiently separating and purifying sugammadex. It only needs one step of chromatographic purification to meet the requirements of sugammadex purity greater than 99.0% and single impurities less than 0.1%, and the purification yield is high and stable. At the same time, the invention uses a pure water phase, no organic solvent, low cost, environmental protection and green, and is suitable for industrial separation and purification.

In order to achieve the above objective, the technical scheme of the present invention is as follows: a method for separating and purifying sugammadex, including the following steps: dissolving and filtering the crude sugammadex extract; and removing the filtered sugammadex solution Load the sample to a weak anion exchange resin chromatography column for chromatography; use a salt solution as the mobile phase to elute the sugammadex solution; collect the target peak sugammadex solution in sections to determine the components that meet the requirements The solution was collected to obtain purified sugammadex.

Preferably, the mobile phase is: Phase A: 20mM sodium acetate with pH=5.5; Phase B: 20mM sodium acetate with pH=5.5+1M NaCl.

Preferably, the microsphere model of the weak anion exchange resin is UniDEAE.

Preferably, before loading the sample, the weak anion exchange resin chromatography column is equilibrated, first with 40%-50% phase B for impurity removal, and then with 100% phase A for equilibrium.

Preferably, the gradient elution process is to wash with phase A first, and then perform gradient elution with A and B relative to the main peak.

Preferably, in the phase B solution, the elution gradient concentration ranges from 0% to 32%.

Preferably, the gradient elution process is as follows: in the first stage, wash with phase A; in the second stage, use A and B phase gradient to elute the main peak, wherein the concentration of phase B is increased from 0% to 4%; and third In the fourth stage, the concentration of phase B is increased from 8% to 20%; in the fifth stage, the concentration of phase B is increased from 20% to 32%.

Preferably, the purity of the crude sugammadex extract is 90%-95%.

Preferably, the purity of the purified sugammadex is above 99.0%.

Preferably, the yield of the purified sugammadex is above 59.0%.

UniDEAE is a weak anion exchange chromatography medium. Its matrix is monodisperse polyacrylate, and the surface of the matrix is also bonded with tertiary amine functional groups. The polyacrylate matrix has very weak hydrophobicity and can separate and purify sugammadex in the pure water phase. At the same time, it has the characteristics of a polymer matrix and has good alkali resistance and can be used in relatively high alkaline conditions. Activated chromatographic media under flushing has more excellent impact resistance, alkali resistance and aging resistance, far better than traditional silica gel matrix reversed-phase chromatography packing, so it has a long service life.

At the same time, UniDEAE chromatography media has strictly controlled particle size and pore structure (particle size is 36μm, pore size

, As shown in Figure 1, the microscopic scanning electron microscope image of the chromatographic medium). Sugammadex is a synthetic modified γ-cyclodextrin, composed of a lipophilic core and a hydrophilic outer end. The functional groups and matrix properties of UniDEAE can bind and separate Sugammadex well and can be used as Sugammadex. The chromatographic medium for sugar separation and purification has a good pertinence.

The invention uses sodium acetate aqueous solution and sodium chloride aqueous solution as mobile phases, and can complete the elution of the main peak of sugammadex with 20-22 column volumes without using organic solvents, so the mobile phase used in the method of the invention is safe and pollution-free And the cost is lower.

The invention adopts UniDEAE-30S as a stationary phase, through gradient elution, the purity of sugammadex is higher than 99.0%, the single impurities are less than 0.1%, and the yield can reach 59.0%, which is suitable for the deep purification of sugammadex.

Therefore, the method of the present invention separates and purifies sugammadex, not only has high purity, high yield and stability, but also has simple and convenient operation, the used stationary phase can be reused, the used mobile phase is environmentally friendly and harmless, and the cost is greatly reduced.

BRIEF DESCRIPTION

Figure 1 is a scanning electron micrograph of UniDEAE microspheres used in Example 1.

Fig. 2 shows the HPLC analysis of the crude sugammadex extract before purification in Example 1.

Figure 3 shows the high performance liquid chromatography analysis of sugammadex purified in Example 1.

detailed description

The technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to these embodiments.

A method for the separation and purification of sugammadex, comprising the following steps: dissolving and filtering the crude sugammadex extract; loading the filtered sugammadex solution to a weak anion exchange resin chromatography column. Chromatography; using a salt solution as the mobile phase for elution to the sugammadex solution; collect the target peak sugammadex solution in sections, and summarize the components that meet the requirements to obtain purified sugammadex.

Preferably, the microsphere model of the weak anion exchange resin is UniDEAE.

Preferably, the microsphere model of the weak anion exchange resin is UniDEAE-30S.

Preferably, the purity of the crude sugammadex extract is 90%-95%.

Preferably, the purity of the purified sugammadex is above 99.0%.

Preferably, the yield of the purified sugammadex is above 59.0%.

Example 1

Take the crude sugammadex extract with a purity of 93.11% and dissolve it with water. The sugammadex content in the solution is 50 mg/mL. After the solution is clarified, filter with a 0.45μm filter membrane and collect the filtrate for later use. A 4.6×250mm chromatographic column, UniDEAE-30S microspheres (produced by Suzhou Nanomicro Technology Co., Ltd.) is used as the chromatographic column packing, and the packed column volume is 4.2ml. Gradient elution was performed with 20mM sodium acetate solution (phase A) and 20mM sodium acetate+1M NaCl (phase B) as mobile phases. Perform pre-column pretreatment on the chromatography column, first use 50% B phase for impurity removal, and then use 100% A phase for equilibrium. The flow rate is controlled at 0.4ml/min. The gradient elution process is as follows: first rinse with 100% A phase for 15 minutes, then increase the elution concentration gradient from 0% B to 4% B within 15-20 minutes, and increase the concentration gradient from 4% B to 4% B within 20-40 minutes 8%B, the concentration gradient increases from 8%B to 20%B within 40-120 minutes, and the concentration gradient increases from 20%B to 32%B within 120-200 minutes. The solution of the target peak is collected in sections, and the components that meet the requirements are summarized. After HPLC analysis, the purity of sugammadex in the eluent is 99.658%, the single impurities are less than 0.1%, and the yield is 59.5%.

Figure 2 shows the HPLC analysis of the crude extract of Shugenglucose before purification, and it can be seen that there are certain impurities. Figure 3 shows the purified Shugenglucose HPLC analysis. It can be seen that there are very few impurities and very small peaks.

Example 2

Take the crude sugammadex extract with a purity of 92.55% and dissolve it with water. The sugammadex content in the solution is 50 mg/mL. After the solution is clarified, filter with a 0.45μm filter membrane and collect the filtrate for later use. A 4.6×250mm chromatographic column is used, UniQ-30S microspheres are used as the chromatographic column packing, and the column volume is 4.2ml. Use 20mM sodium acetate solution (A phase) and 20mM sodium acetate + 1M NaCl (B phase) as mobile phases for gradient elution, pre-column the column pre-column, first use 50% B phase for impurity removal, and then use 100 %A phase is balanced. The flow rate is controlled at 0.4ml/min. The gradient elution process is as follows: first rinse with 100% phase A for 15 minutes, then increase the concentration gradient from 0% B to 4% B within 15-20 minutes, and increase the concentration gradient from 4% B to 8% within 20-40 minutes B, the concentration gradient increases from 8% B to 20% B within 40-120 minutes, and the concentration gradient increases from 20% B to 32% B within 120-200 minutes. The solution of the target peak is collected in sections, and the components that meet the requirements are summarized. After high performance liquid chromatography analysis, the purity of sugammadex in the eluent is 99.2%, the single impurities are less than 0.1%, and the yield is 23.3%.

Example 3

Take the crude sugammadex extract with a purity of 92.55% and dissolve it with water. The sugammadex content in the solution is 50 mg/mL. After the solution is clarified, filter with a 0.45μm filter membrane and collect the filtrate for later use. A 4.6×250mm chromatographic column is used, UniDEAE-30S microspheres are used as the column packing, and the column volume is 4.2ml. Use 20mM sodium acetate solution (A phase) and 20mM sodium acetate + 1M NaCl (B phase) as mobile phases for gradient elution, pre-column the column pre-column, first use 50% B phase for impurity removal, and then use 100 %A phase is balanced. The flow rate is controlled at 0.4ml/min. The gradient elution process is that 10% B is increased to 40% B and eluted 20 column volumes. Collect the solution of the target peak in sections, and summarize the components that meet the requirements. After high performance liquid chromatography analysis, the purity of the sugammadex in the eluent is 98.310%, and the single impurity content exceeds 0.1%, which is not qualified. section.

The invention is used for the deep purification of sugammadex, only one step of chromatographic purification can meet the requirement of sugammadex with a single impurities of less than 0.1%, and the purification yield is high and stable. The invention uses sodium acetate aqueous solution and sodium chloride aqueous solution as mobile phases, does not use organic solvents, and the mobile phases used are safe, pollution-free and low in cost. At the same time, the separation method of the invention is simple and convenient, can be used for large-scale production, and greatly reduces production costs.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, a number of modifications and improvements can be made, all of which belong to the present invention. The scope of protection of the invention.

Claims

A method for separating and purifying sugammadex, which is characterized by comprising the following steps: dissolving and filtering the crude sugammadex extract; loading the filtered sugammadex solution onto a weak anion exchange resin Perform chromatography; use salt solution as the mobile phase to perform gradient elution with respect to the sugammadex solution; collect the target peak sugammadex solution in sections, and summarize the components that meet the requirements to obtain purified sugammadex sugar.
The method for separating and purifying sugammadex according to claim 1, wherein the mobile phase is: phase A: 20mM sodium acetate with pH=5.5; phase B: 20mM sodium acetate with pH=5.5+1M NaCl.
The method for separating and purifying sugammadex according to claim 1, wherein the product model of the weak anion exchange resin is UniDEAE.
The method for separating and purifying sugammadex glucose according to claim 1, characterized in that, before loading the sample, the weak anion exchange resin chromatography column is equilibrated, and 40%-50% phase B is used for impurity removal. Then balance with 100% A phase.
The method for separating and purifying sugammadex glucose according to claim 1, wherein the gradient elution process is first washing with phase A, and then performing gradient elution with A and B relative to the main peak.
The method for separating and purifying sugammadex according to claim 5, wherein in the phase B solution, the elution gradient concentration ranges from 0% to 32%.
The method for separating and purifying sugammadex according to claim 5, characterized in that the gradient elution process is, in the first stage, washing with phase A; in the second stage, eluting the main peak with A and B phase gradients , Among them, the concentration of phase B increased from 0% to 4%; the third phase, the concentration of phase B increased from 4% to 8%; the fourth phase, the concentration of phase B increased from 8% to 20%; the fifth phase , The concentration of phase B increased from 20% to 32%.
The method for separating and purifying sugammadex according to any one of claims 1 to 7, wherein the purity of the crude sugammadex extract is 90%-95%.
The method for separating and purifying sugammadex according to any one of claims 1 to 7, wherein the purity of the purified sugammadex is 99.0% or more.
The method for separating and purifying sugammadex according to any one of claims 1 to 7, wherein the yield of the purified sugammadex is more than 59.0%.