WO2018126524A1

WO2018126524A1 - Method for purifying areginine essence

Info

Publication number: WO2018126524A1
Application number: PCT/CN2017/075124
Authority: WO
Inventors: 郭添; 苏晨灿; 李乾; 张忠旗; 王惠嘉; 王万科; 韩广; 王慧; 高长波; 赵金礼; 杨小琳
Original assignee: 陕西慧康生物科技有限责任公司
Priority date: 2017-01-04
Filing date: 2017-02-28
Publication date: 2018-07-12
Also published as: AU2017100834A4; CN106632608A

Abstract

The present invention provides a method for purifying areginine essence, comprising: (1) resolving: resolving an areginine essence coarse product to be purified in acetic acid-sodium acetate solution, and filtering same to obtain a filtrate; (2) rough purification: loading the filtrate obtained in step (1) to a strong cation exchange column, performing gradient elution using a rough purification mobile phase, and collecting an eluent; and (3) desalination: loading the eluent collected in step (2) to a reverse polymer column, performing gradient elution using a desalting mobile phase, collecting the areginine essence solution, and performing vacuum concentration and freeze drying to obtain the areginine essence.

Description

Method for purifying a six-peptide

Technical field

The invention belongs to the technical field of polypeptide purification, and in particular, the invention relates to a method for purifying a hexapeptide.

Background technique

Six-peptide, also known as hexapeptide, has all the functions of botulinum toxin and is non-toxic. The six-peptide is an active polypeptide containing six amino acids. The amino acid sequence is Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2, which inhibits nerve conduction and prevents excessive muscle contraction, thereby preventing the formation of fine lines. As a skin wrinkle ingredient, it is widely used in various high-end cosmetics. The MAGIC CARE Magic Care Six-Peptide Flat Wrinkle Firming Solution, developed by the Swiss Legend Group Skincare Laboratory in 2010, is the cutting-edge representative of Liusheng Peptide Beauty Products. In addition, there are few literatures on the separation and purification methods of six peptides at home and abroad, and there is no literature to study the large-scale separation and purification methods related to hexapeptide.

Summary of the invention

The technical problem to be solved by the present invention is to overcome the shortcomings of the existing method for purifying the six peptides, and to provide a method for purifying the six peptides which is low in cost, high in purity and suitable for industrialization.

The purification method of the six peptides provided by the invention comprises:

(1) Dissolution: the crude hexapeptide to be purified is dissolved in an acetic acid-sodium acetate solution, and filtered to obtain a filtrate;

(2) Crude pure: the filtrate obtained in the step (1) is applied to a strong cation exchange column, and the eluate is collected by a gradient elution with a crude pure mobile phase;

(3) Desalting: The eluate collected in the step (2) was applied to a reverse phase polymer column, and a gradient elution with a desalted mobile phase was carried out to collect a six-peptide solution, and concentrated under reduced pressure and freeze-dried to obtain a six-peptide.

In the above purification method, in the step (1), the ratio of the crude hexapeptide to be purified to the acetic acid-sodium acetate solution is (0.5 g-100 g): (20 ml - 1000 ml), preferably, the crude hexapeptide to be purified The ratio with the acetic acid-sodium acetate solution was (0.5 g - 10 g): (20 ml - 100 ml).

In the above purification method, the acetic acid-sodium acetate solution is an acetic acid-sodium acetate solution having a concentration of 0.01 to 0.1 mol/L and a pH of 3.9 to 4.1.

In the foregoing purification method, the crude pure mobile phase consists of an initial buffer mobile phase and an elution buffer mobile phase, wherein the initial buffer mobile phase is an acetic acid-sodium acetate solution, and the elution buffer mobile phase Is an initial buffer mobile phase to which sodium chloride is added; preferably, the initial buffer mobile phase is an acetic acid-sodium acetate solution having a concentration of 0.01-0.1 mol/L and a pH of 3.9-4.1, the elution buffer The liquid mobile phase is the initial buffer mobile phase to which 0.5-1.5 mol/L sodium chloride is added.

In the foregoing purification method, the desalted mobile phase consists of a mobile phase A which is ultrapure water and a mobile phase B which is chromatographically pure methanol.

In the foregoing purification method, in the step (2), the crude pure mobile phase gradient is the initial buffer mobile phase: the elution buffer mobile phase is from 100:0 to (80-60): (20-40).

In the foregoing purification method, in the step (3), the desalting mobile phase gradient is the mobile phase A: the mobile phase B is from (100-95): (0-5) to (90-80): (10-20).

In the foregoing purification method, the crude pure mobile phase consists of an initial buffer mobile phase and an elution buffer mobile phase, wherein the initial buffer mobile phase is an acetic acid-sodium acetate solution, and the elution buffer mobile phase Is a high concentration, high pH acetic acid-sodium acetate buffer; preferably, the initial buffer mobile phase is a sodium acetate-sodium acetate solution having a concentration of 0.01-0.1 mol/L and a pH of 3.9-4.1, The elution buffer mobile phase consists of phase A and phase B. The phase A is an acetic acid-sodium acetate solution with a concentration of 0.01-0.1 mol/L and a pH of 5.5-6.2, and the phase B is a concentration of 0.5-1.5 mol/L. An acetic acid-sodium acetate solution having a pH of 5.5-6.2.

In the foregoing purification method, in the step (3), the desalting mobile phase gradient is the mobile phase A: the mobile phase B is from (100-95): (0-5) to (90-80): (5-10).

With the technical solution of the present invention, at least the following beneficial effects are obtained:

1. The method of the invention combines a strong cation exchange column with a reverse phase polymer column, first uses a strong cation exchange column to crudely purify the six peptides, removes most impurities, and then uses a reversed phase polymer column. Desalting to obtain a further purified hexapeptide, greatly improving the purification efficiency.

2. The method of the invention saves the cost of the mobile phase throughout the purification process, and the purification process is environmentally friendly.

3. The two columns are alternately used in the method of the invention, which effectively compensates for the difficulty in completely separating the impurities of different structures and different chemical properties in the crude peptide by a single column, and the obtained six peptides have high purity (greater than 95%) and high yield.

DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a mass spectrum of a hexapeptide obtained by purification according to the method of the first embodiment of the present invention.

Figure 2 is a high performance liquid chromatogram of the hexapeptide obtained by the method of the first embodiment of the present invention.

Figure 3 is a mass spectrum of a hexapeptide obtained by purification according to the method of the second embodiment of the present invention.

Figure 4 is a high performance liquid chromatogram of the hexapeptide obtained by the method of the second embodiment of the present invention.

detailed description

The present invention will be described in detail by the following detailed description of the preferred embodiments of the invention, and the invention is not limited thereto.

At present, in view of the purification method of the six peptides, there is a problem that the cost is high but the effect is not satisfactory. To solve the problem, the present invention provides a method for separating and purifying the six peptides by ion exchange high performance liquid chromatography, which in turn Including the following steps:

(3) Desalting: The eluate collected in step (2) is loaded onto a reversed-phase polymer column, and the desalting stream is used. The mobile phase was subjected to gradient elution, and the six peptide solution was collected, concentrated under reduced pressure and freeze-dried to obtain a six-peptide.

In the step (1), the crude hexapeptide to be purified is prepared by a solid phase synthesis method, and when the crude hexapeptide to be purified is dissolved in an acetic acid-sodium acetate solution, preferably, according to the hexapeptide to be purified The ratio of the crude product to the acetic acid-sodium acetate solution is (0.5 g-100 g): (20 ml - 1000 ml) for dissolution, more preferably, the ratio of the crude hexapeptide to be purified to the acetic acid-sodium acetate solution is (0.5 g - 10 g): (20 ml - 100 ml) was dissolved. After dissolving the crude hexapeptide to be purified in an acetic acid-sodium acetate solution, it is preferred to adjust the pH to 3.9-4.1 with 1 mol/L sodium hydroxide, sonicate, and then filter with a filter (for example, a 0.45 μm filter). And collect the filtrate for use.

In the step (2), the column uses a strong cation exchange column, and the so-called strong cation exchange column refers to a novel polymeric sulfonic acid cation exchange ligand bonded to the surface of the high purity inert silica gel. The structure is a stable polymeric envelope structure that is more efficient than conventional polymer ion exchange columns and has better mechanical strength and reproducibility. In the present invention, the strong cation exchange column is a strong cation exchange column with agarose microspheres, wherein the agarose microspheres can be SP high flow agarose microspheres (SP Bio-sepFF) with a particle size of 50. -160 μm, which is commercially available, for example, SP high flow agarose microspheres produced by Xi'an Jiaotong Biotechnology Co., Ltd. can be used in the present invention.

In the step (3), the column uses a reverse phase polymer column, and the so-called reverse phase polymer column refers to a type of polymer column in which the polarity of the stationary phase is weaker than that of the fluidity. In the present invention, the reversed-phase polymer column used is a reversed-phase polymer column having a filler of styrene-divinylbenzene type filler, for example, the filler is SBC MCI GEI F-type reversed phase chromatography packing material, and the particle diameter is 30-50 μm. The SBC MCI GEI F type reverse phase chromatography packing produced by, for example, Chengdu Kepu Biotechnology Co., Ltd. can be used in the present invention by conventional commercial purchase.

The crude pure mobile phase used in the step (2) consists of the initial buffer mobile phase and the elution buffer mobile phase, and the desalted mobile phase used in the step (3) consists of the mobile phase A and the mobile phase B.

In a first embodiment of the present invention, the initial buffer mobile phase used in the step (2) is an acetic acid-sodium acetate solution, and the elution buffer mobile phase is an initial buffer mobile phase to which sodium chloride is added; Ground, the initial buffer mobile phase is acetic acid-sodium acetate solution with a concentration of 0.01-0.1 mol/L and a pH of 3.9-4.1. The elution buffer mobile phase is the initial buffer for adding 0.5-1.5 mol/L sodium chloride. Liquid mobile phase. The mobile phase A used in the step (3) is ultrapure water, and the mobile phase B is chromatographically pure methanol.

In step (2), before loading the filtrate collected in step (1) onto the strong cation exchange column, the strong cation exchange column is first equilibrated with the initial buffer mobile phase to a pH of the detector effluent of 3.9-4.1, conductance. The rate is constant. After loading the filtrate onto a strong cation exchange column, the gradient buffer phase was eluted from the initial buffer mobile phase: elution buffer mobile phase gradient from 100:0 to (80-60): (20-40). For example, the elution process can be divided into different stages, for example, 0-60 minutes is the first stage, 60-90 minutes is the second stage, 90-150 minutes is the third stage, or 0-80 minutes is the first stage. Stage, 80-120 minutes is the second stage, 120-240 minutes is the third stage, or 0-110 minutes is the first stage, 110-170 minutes is the second stage, 170-340 minutes is the third stage, or 0 - 120 minutes for the first stage, 120-180 minutes for the second stage, 180-340 minutes for the third stage, the gradient of the elution process may be 100% of the initial buffer mobile phase in the first stage, initial in the second stage Buffer mobile phase: elution buffer mobile phase from (100-70): (0-30) to (70-65): (30-35), third stage initial buffer mobile phase: elution buffer flow The phase is (70-65): (30-35) constant current. Gradient elution and collection of the eluent.

In step (3), the reverse phase polymer column is first equilibrated with mobile phase A prior to loading the eluate collected in step (2) onto the reverse phase polymer column. After loading the eluent to the reversed-phase polymer column, the gradient is carried out according to the gradient of (100-95): (0-5) to (90-80): (10-20) according to mobile phase A: mobile phase B Elution. For example, the elution process can be divided into different stages, for example, 0-45 minutes is the first stage, 45-100 minutes is the second stage, or 0-50 minutes is the first stage, and 50-100 minutes is the second stage, and 50-100 minutes is the second stage. Stage, or 0-80 minutes for the first stage, 80-160 minutes for the second stage, or 0-100 minutes for the first stage, 100-200 minutes for the second stage, and the gradient of the elution process may be the first stage Mobile phase A: mobile phase B from (100-95): (0-5) to (90-80): (10-20), second phase mobile phase A: mobile phase B is (90-80): ( 10-20) Constant current. Gradient elution, collecting the peptide solution of the peak of interest, and concentrating the desalted target peptide solution under reduced pressure at less than 40 ° C, concentrating to about 50 mg-100 mg/mL, and lyophilizing, thereby obtaining a purity of 95% or more of the six peptides. The mass spectrum of the solid powder is shown in Figure 1, and the high performance liquid chromatogram is shown in Figure 2.

In a second embodiment of the present invention, the initial buffer mobile phase used in the step (2) is an acetic acid-sodium acetate solution, and the elution buffer mobile phase is a high concentration, high pH acetic acid-acetic acid. Sodium buffer; preferably, the initial buffer mobile phase is a sodium acetate-sodium acetate solution having a concentration of 0.01-0.1 mol/L and a pH of 3.9-4.1, and the elution buffer mobile phase is composed of phase A and B. Phase composition, Phase A is an acetic acid-sodium acetate solution having a concentration of 0.01-0.1 mol/L and a pH of 5.5-6.2, and Phase B is an acetic acid-sodium acetate solution having a concentration of 0.5-1.5 mol/L and a pH of 5.5-6.2. The mobile phase A used in the step (3) is ultrapure water, and the mobile phase B is chromatographically pure methanol.

In step (3), the reverse phase polymer column is first equilibrated with mobile phase A prior to loading the eluate collected in step (2) onto the reverse phase polymer column. After loading the eluent to the reversed-phase polymer column, the gradient is carried out according to the gradient of (100-95): (0-5) to (90-80): (10-20) according to mobile phase A: mobile phase B Elution. For example, the elution process can be divided into different stages, for example, 0-45 minutes is the first stage, 45-100 minutes is the second stage, or 0-50 minutes is the first stage, and 50-100 minutes is the second stage, and 50-100 minutes is the second stage. Stage, or 0-80 minutes for the first stage, 80-160 minutes for the second stage, or 0-100 minutes for the first stage, 100-200 minutes for the second stage, and the gradient of the elution process may be the first stage Mobile phase A: mobile phase B from (100-98): (0-2) to (95-90): (5-10), second phase mobile phase A: mobile phase B is (95-90): ( 5-10) Constant current. Gradient elution, collecting the peptide solution of the peak of interest, and concentrating the desalted target peptide solution under reduced pressure at less than 40 ° C, concentrating to about 50 mg-100 mg/mL, and lyophilizing, thereby obtaining a purity of 95% or more of the six peptides. The solid powder has a mass spectrum as shown in Fig. 3, and a high performance liquid chromatogram as shown in Fig. 4.

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the invention is not limited thereto Some embodiments. Unless otherwise indicated, the materials and apparatus used in the examples are commercially available and are conventionally used in the art as long as they meet the experimental needs.

Example 1

Dissolve

Weigh 0.5g of solid phase synthesis of the six peptides, dissolved in 20ml, pH=4, 0.01mol/L acetic acid-sodium acetate aqueous solution (concentration is about 25mg/mL), adjust pH=3.9 with 1mol/L sodium hydroxide. 4.1, sonicated, filtered through a 0.45 μm filter, and the filtrate was collected for use.

2. Crude

Purification conditions: Column: The packing was SP high flow agarose microsphere Bio-sepFF, a strong cation exchange column with a particle size of 50-160 μm, and the column packing volume was 50 mL. The initial buffer mobile phase was: 0.01 mol/L pH = 3.9-4.1 acetic acid-sodium acetate aqueous solution, and the elution buffer mobile phase was the initial buffer mobile phase to which 1 mol/L of sodium chloride was added. The flow rate was 5 mL/min. Detection wavelength: 215 nm. Gradient: 0 to 60 minutes initial buffer mobile phase 100%, 60 to 90 minutes initial buffer mobile phase: elution buffer mobile phase from 100:0 to 70:30, 90 to 150 minutes for the initial buffer mobile phase: The elution buffer mobile phase was a constant flow of 70:30.

Purification process: The cation exchange column was equilibrated with 100% initial buffer mobile phase to pH 3.9-4.1 of the detector effluent, and the conductivity was constant. A linear gradient elution was performed to collect the peptide solution of the peak of interest for use.

3. Desalting

Purification conditions: Column: The filler was SBC MCI GEI F type 30-50 μm reverse phase chromatography packing F type 30-50 μm, and the column packing volume was 50 mL. The mobile phase A is: ultrapure water, and the mobile phase B is: chromatographically pure methanol. The flow rate was 7 mL/min. Detection wavelength: 215 nm. Gradient: 0 to 50 minutes mobile phase A: mobile phase B from 100:0 to 90:10, 50 to 100 minutes for mobile phase A: mobile phase B is 90:10 constant current.

Purification process: The polymer column was equilibrated with 100% mobile phase A ultrapure water and loaded. Linear gradient elution, the peptide solution of the peak of interest is collected, and the desalted target peptide solution is concentrated under reduced pressure at less than 40 ° C, concentrated to about 50 mg-100 mg / mL, and then lyophilized to obtain a purity of 97.4% hydrochloric acid type six peptide Qualified solid powder.

Example 2

Dissolve

Weigh 1.0g of the solid peptide obtained from the solid phase synthesis, dissolve it with 50ml, pH=4, 0.01mol/L acetic acid/sodium acetate aqueous solution (concentration is about 20mg/mL), adjust pH=3.9 with 1mol/L sodium hydroxide. 4.1, sonicated, filtered through a 0.45 μm filter, and the filtrate was collected for use.

2. Crude

Purification conditions: Column: The packing was SP high flow agarose microsphere Bio-sepFF, a strong cation exchange column with a particle size of 50-160 μm, and the column packing volume was 150 mL. The initial buffer mobile phase was: 0.01 mol/L pH = 3.9-4.1 acetic acid-sodium acetate aqueous solution, and the elution buffer mobile phase was the initial buffer mobile phase to which 1 mol/L of sodium chloride was added. The flow rate was 10 mL/min. Detection wavelength: 215 nm. Gradient: 0 to 80 minutes initial buffer mobile phase 100%, 80 to 120 minutes initial buffer mobile phase: elution buffer mobile phase from 100:0 to 70:30, 120 to 240 minutes for the initial buffer mobile phase: The elution buffer mobile phase was a constant flow of 70:30.

3. Desalting

Purification conditions: Column: The packing was SBC MCI GEI F type 30-50 μm reverse phase chromatography packing F type 30-50 μm, and the column packing volume was 150 mL. The mobile phase A is: ultrapure water, and the mobile phase B is: chromatographically pure methanol. The flow rate was 20 mL/min. Detection wavelength: 215 nm. Gradient: 0 to 80 minutes mobile phase A: mobile phase B from 100:0 to 90:10, 80 to 160 minutes for mobile phase A: mobile phase B is 90:10 constant current.

Purification process: The polymer column was equilibrated with 100% mobile phase A ultrapure water and loaded. Linear gradient elution, the peptide solution of the peak of interest is collected, and the desalted peptide solution is concentrated under reduced pressure at less than 40 ° C, concentrated to about 50 mg-100 mg / mL, and then lyophilized to obtain a purity of 97% hydrochloric acid type six peptide Qualified solid powder.

Example 3

Dissolve

Weigh 30.0 g of the solid phase synthesis of the six peptides, dissolved in 300 ml, pH=4, 0.01 mol/L acetic acid-sodium acetate aqueous solution (concentration is about 100 mg/mL), and adjusted to pH=3.9 with 1 mol/L sodium hydroxide. 4.1, Ultrasonic treatment, filtration through a 0.45 μm filter, and collection of the filtrate for use.

2. Crude

Purification conditions: column: the packing is SP high flow agarose microsphere Bio-sepFF, a strong cation exchange column with a particle size of 50-160 μm, and the column packing volume is 2500 mL. The initial buffer mobile phase was: 0.01 M pH = 3.9-4.1 acetic acid-sodium acetate aqueous solution, and the elution buffer mobile phase was the initial buffer mobile phase to which 1 mol/L sodium chloride was added. The flow rate was 25 mL/min. Detection wavelength: 215 nm. Gradient: 0 to 110 minutes initial buffer mobile phase 100%, 110 to 170 minutes initial buffer mobile phase: elution buffer mobile phase from 75:25 to 70:30, 170 to 340 minutes as initial buffer mobile phase: The elution buffer mobile phase was a constant flow of 70:30.

3. Desalting

Purification conditions: Column: The packing was SBC MCI GEI F type 30-50 μm reverse phase chromatography packing F type 30-50 μm, and the column packing volume was 300 mL. The mobile phase A is: ultrapure water, and the mobile phase B is: chromatographically pure methanol. The flow rate was 30 mL/min. Detection wavelength: 215 nm. Gradient: 0 to 45 minutes mobile phase A: mobile phase B from 100:0 to 90:10, 45 to 100 minutes for mobile phase A: mobile phase B is 90:10 constant current.

Purification process: The polymer column was equilibrated with 100% mobile phase A ultrapure water and loaded. Linear gradient elution, the peptide solution of the peak of interest is collected, and the desalted target peptide solution is concentrated under reduced pressure at less than 40 ° C, concentrated to about 50 mg-100 mg / mL, and then lyophilized to obtain a purity of 96.2% hydrochloric acid type six peptide Qualified solid powder.

Example 4

Dissolve

60.0 g of the solid phase synthesis of the six peptides was weighed and dissolved in 600 ml, pH=4, 0.01 mol/L acetic acid-sodium acetate aqueous solution (concentration: about 100 mg/mL), and adjusted to pH=3.9 with 1 mol/L sodium hydroxide. 4.1, sonicated, filtered through a 0.45 μm filter, and the filtrate was collected for use.

2. Crude

Purification conditions: column: packing is SP high flow agarose microsphere Bio-sepFF, particle size A strong cation exchange column of 50-160 μm with a column packing volume of 2500 mL. The initial buffer mobile phase was: 0.01 mol/L pH = 3.9-4.1 acetic acid-sodium acetate aqueous solution, and the elution buffer mobile phase was the initial buffer mobile phase to which 1 mol/L of sodium chloride was added. The flow rate was 25 mL/min. Detection wavelength: 215 nm. Gradient: 0 to 110 minutes initial buffer mobile phase 100%, 110 to 170 minutes initial buffer mobile phase: elution buffer mobile phase from 70:30 to 65:35, 170 to 340 minutes as initial buffer mobile phase: The elution buffer mobile phase was a 65:35 constant current.

3. Desalting

Purification conditions: Column: The filler was SBC MCI GEI F type 30-50 μm reverse phase chromatography packing F type 30-50 μm, and the column packing volume was 3000 mL. The mobile phase A is: ultrapure water, and the mobile phase B is: chromatographically pure methanol. The flow rate was 50 mL/min. Detection wavelength: 215 nm. Gradient: 0 to 100 minutes mobile phase A: mobile phase B from 100:0 to 90:15, 100 to 200 minutes for mobile phase A: mobile phase B is 90:15 constant current.

Purification process: The polymer column was equilibrated with 100% mobile phase A ultrapure water and loaded. Linear gradient elution, the peptide solution of the peak of interest is collected, and the desalted target peptide solution is concentrated under reduced pressure at less than 40 ° C, concentrated to about 50 mg-100 mg / mL, and then lyophilized to obtain a purity 96% hydrochloric acid type six peptide Qualified solid powder.

Example 5

Dissolve

Weigh 100.0g of the six-peptide obtained by solid phase synthesis, dissolved in 1000ml, pH=4, 0.01mol/L acetic acid-sodium acetate aqueous solution (concentration is about 100mg/mL), and adjusted to pH=3.9 with 1mol/L sodium hydroxide. 4.1, sonicated, filtered through a 0.45 μm filter, and the filtrate was collected for use.

2. Crude

Purification conditions: column: the packing is SP high flow agarose microsphere Bio-sepFF, a strong cation exchange column with a particle size of 50-160 μm, and the column packing volume is 2500 mL. The initial buffer mobile phase was: 0.01 mol/L pH = 3.9-4.1 acetic acid-sodium acetate aqueous solution, and the elution buffer mobile phase was the initial buffer mobile phase to which 1 mol/L of sodium chloride was added. The flow rate was 25 mL/min. Detection wavelength: 215 nm. Ladder Degree: 0 to 120 minutes Initial buffer mobile phase 100%, 120 to 180 minutes Initial buffer mobile phase: Elution buffer mobile phase from 70:30 to 65:35, 180 to 340 minutes as initial buffer mobile phase: The elution buffer mobile phase was a 65:35 constant current.

3. Desalting

Example 6

Dissolve

2. Crude

Purification conditions: Column: The packing was SP high flow agarose microsphere Bio-sepFF, a strong cation exchange column with a particle size of 50-160 μm, and the column packing volume was 50 mL. The initial buffer mobile phase is: 0.01 mol/L pH=3.9-4.1 acetic acid-sodium acetate aqueous solution, and the elution buffer mobile phase is phase A: acetic acid-sodium acetate having a concentration of 0.01 mol/L and a pH of 5.5-6.2. Solution, phase B: acetic acid-sodium acetate solution having a concentration of 1 mol/L and a pH of 5.5-6.2, flow rate 5 mL/min. Detection wavelength: 215 nm. Gradient: 0 to 60 minutes initial buffer mobile phase 100%, 60 to 90 minutes initial buffer mobile phase: elution buffer mobile phase from 100:0 to 70:30, 90 to 150 minutes for the initial buffer mobile phase: Elution The buffer mobile phase is a constant flow of 70:30.

3. Desalting

Purification process: The polymer column was equilibrated with 100% mobile phase A ultrapure water and loaded. Linear gradient elution, the peptide solution of the peak of interest is collected, and the desalted target peptide solution is concentrated under reduced pressure at less than 40 ° C, concentrated to about 50 mg-100 mg / mL, and then lyophilized to obtain a purity of 97% acetic acid type six peptide Qualified solid powder.

Example 7

Dissolve

2. Crude

Purification conditions: Column: The packing was SP high flow agarose microsphere Bio-sepFF, a strong cation exchange column with a particle size of 50-160 μm, and the column packing volume was 150 mL. The initial buffer mobile phase is: 0.01 mol/L pH=3.9-4.1 acetic acid-sodium acetate aqueous solution, and the eluted mobile phase is phase A: acetic acid-sodium acetate solution having a concentration of 0.01 mol/L and a pH of 5.5-6.2. Phase B: acetic acid-sodium acetate solution having a concentration of 1.0 mol/L and a pH of 5.5-6.2, and a flow rate of 10 mL/min. Detection wavelength: 215 nm. Gradient: 0 to 80 minutes initial buffer mobile phase 100%, 80 to 120 minutes initial buffer mobile phase: elution buffer mobile phase from 100:0 to 70:30, 120 to 240 minutes for the initial buffer mobile phase: The elution buffer mobile phase was a constant flow of 70:30.

Purification process: equilibration of the cation exchange column with 100% initial buffer mobile phase to the detector stream The pH of the liquid was 3.9-4.1, and the conductivity was constant. A linear gradient elution was performed to collect the peptide solution of the peak of interest for use.

3. Desalting

Purification process: The polymer column was equilibrated with 100% mobile phase A ultrapure water and loaded. Linear gradient elution, the peptide solution of the peak of interest is collected, and the desalted target peptide solution is concentrated under reduced pressure at less than 40 ° C, concentrated to about 50 mg-100 mg / mL, and then lyophilized to obtain a purity of 96.5% acetic acid type six peptide Qualified solid powder.

Example 8

Dissolve

Weigh 30.0 g of the solid phase synthesis of the six peptides, dissolved in 300 ml, pH=4, 0.01 mol/L acetic acid-sodium acetate aqueous solution (concentration is about 100 mg/mL), and adjusted to pH=3.9 with 1 mol/L sodium hydroxide. 4.1, sonicated, filtered through a 0.45 μm filter, and the filtrate was collected for use.

2. Crude

Purification conditions: column: the packing is SP high flow agarose microsphere Bio-sepFF, a strong cation exchange column with a particle size of 50-160 μm, and the column packing volume is 2500 mL. The initial buffer mobile phase is: 0.01M pH=3.9-4.1 acetic acid-sodium acetate aqueous solution, and the eluted mobile phase is phase A: acetic acid-sodium acetate solution with a concentration of 0.01 mol/L and a pH of 5.5-6.2, phase B : acetic acid-sodium acetate solution having a concentration of 1.0 mol/L and a pH of 5.5-6.2, and a flow rate of 25 mL/min. Detection wavelength: 215 nm. Gradient: 0 to 110 minutes initial buffer mobile phase 100%, 110 to 170 minutes initial buffer mobile phase: elution buffer mobile phase from 75:25 to 70:30, 170 to 340 minutes as initial buffer mobile phase: elution The buffer mobile phase is a constant flow of 70:30.

3. Desalting

Purification process: The polymer column was equilibrated with 100% mobile phase A ultrapure water and loaded. Linear gradient elution, the peptide solution of the peak of interest is collected, and the desalted peptide solution is concentrated under reduced pressure at less than 40 ° C, concentrated to about 50 mg-100 mg / mL, and then lyophilized to obtain a purity of 96.3% acetic acid type six peptide Qualified solid powder.

Example 9

Dissolve

2. Crude

Purification conditions: column: the packing is SP high flow agarose microsphere Bio-sepFF, a strong cation exchange column with a particle size of 50-160 μm, and the column packing volume is 2500 mL. The initial buffer mobile phase is: 0.01 mol/L pH=3.9-4.1 acetic acid-sodium acetate aqueous solution, and the eluted mobile phase is phase A: acetic acid-sodium acetate solution having a concentration of 0.01 mol/L and a pH of 5.5-6.2. Phase B: acetic acid-sodium acetate solution having a concentration of 1.0 mol/L and a pH of 5.5-6.2, and a flow rate of 25 mL/min. Detection wavelength: 215 nm. Gradient: 0 to 110 minutes initial buffer mobile phase 100%, 110 to 170 minutes initial buffer mobile phase: elution buffer mobile phase from 70:30 to 65:35, 170 to 340 minutes as initial buffer mobile phase: The elution buffer mobile phase was a 65:35 constant current.

3. Desalting

Purification conditions: column: packing is SBC MCI GEI F type 30-50μm reversed phase chromatography The material F type is 30-50 μm, and the column filling volume is 3000 mL. The mobile phase A is: ultrapure water, and the mobile phase B is: chromatographically pure methanol. The flow rate was 50 mL/min. Detection wavelength: 215 nm. Gradient: 0 to 100 minutes mobile phase A: mobile phase B from 100:0 to 90:15, 100 to 200 minutes for mobile phase A: mobile phase B is 90:15 constant current.

Purification process: The polymer column was equilibrated with 100% mobile phase A ultrapure water and loaded. Linear gradient elution, the peptide solution of the peak of interest is collected, and the desalted target peptide solution is concentrated under reduced pressure at less than 40 ° C, concentrated to about 50 mg-100 mg / mL, and then lyophilized to obtain a purity 96% acetic acid type six peptide Qualified solid powder.

Example 10

1. Dissolution: Weigh 100.0g of solid phase synthesis of the six peptides, dissolved in 1000ml, pH=4, 0.01mol/L acetic acid-sodium acetate aqueous solution (concentration is about 100mg/mL), with 1mol/L sodium hydroxide The pH was adjusted to 3.9-4.1, sonicated, filtered through a 0.45 μm filter, and the filtrate was collected for use.

2. Crude

Purification conditions: column: the packing is SP high flow agarose microsphere Bio-sepFF, a strong cation exchange column with a particle size of 50-160 μm, and the column packing volume is 2500 mL. The initial buffer mobile phase is: 0.01 mol/L pH=3.9-4.1 acetic acid-sodium acetate aqueous solution, and the eluted mobile phase is phase A: acetic acid-sodium acetate solution having a concentration of 0.1 mol/L and a pH of 5.5-6.2. Phase B: acetic acid-sodium acetate solution having a concentration of 1.0 mol/L and a pH of 5.5-6.2, and a flow rate of 25 mL/min. Detection wavelength: 215 nm. Gradient: 0 to 120 minutes initial buffer mobile phase 100%, 120 to 180 minutes initial buffer mobile phase: elution buffer mobile phase from 70:30 to 65:35, 180 to 340 minutes as initial buffer mobile phase: The elution buffer mobile phase was a 65:35 constant current.

3. Desalting

Purification conditions: Column: The filler was SBC MCI GEI F type 30-50 μm reverse phase chromatography packing F type 30-50 μm, and the column packing volume was 3000 mL. The mobile phase A is: ultrapure water, and the mobile phase B is: chromatographically pure methanol. The flow rate was 50 mL/min. Detection wavelength: 215 nm. Gradient: 0 to 100 minutes mobile phase A: mobile phase B from 100:0 to 90:15, 100 to 200 minutes for mobile phase A: flow Phase B is a constant flow of 90:15.

Purification process: The polymer column was equilibrated with 100% mobile phase A ultrapure water and loaded. Linear gradient elution, the peptide solution of the peak of interest is collected, and the desalted peptide solution is concentrated under reduced pressure at less than 40 ° C, concentrated to about 50 mg-100 mg / mL, and then lyophilized to obtain a purity of 95.8% acetic acid type six peptide Qualified solid powder.

The invention has been described above in terms of a preferred embodiment, but it should be understood by those skilled in the art that the invention is not intended to limit the scope of the invention. It should be noted that variations and permutations equivalent to those of the embodiments are intended to be included within the scope of the appended claims. Therefore, the scope of the invention should be determined by the scope defined in the claims.

Claims

A method for purifying a hexapeptide, characterized in that it comprises:

(1) Dissolution: the crude hexapeptide to be purified is dissolved in an acetic acid-sodium acetate solution, and filtered to obtain a filtrate;

(2) Crude pure: the filtrate obtained in the step (1) is applied to a strong cation exchange column, and the eluate is collected by a gradient elution with a crude pure mobile phase;

(3) Desalting: The eluate collected in the step (2) was applied to a reverse phase polymer column, and a gradient elution with a desalted mobile phase was carried out to collect a six-peptide solution, and concentrated under reduced pressure and freeze-dried to obtain a six-peptide.
The purification method according to claim 1, wherein in the step (1), the ratio of the crude hexapeptide to be purified to the acetic acid-sodium acetate solution is (0.5 g - 100 g): (20 ml - 1000 ml), preferably The ratio of the crude hexapeptide to be purified to the acetic acid-sodium acetate solution was (0.5 g to 10 g): (20 ml - 100 ml).
The purification method according to claim 1, wherein the acetic acid-sodium acetate solution is an acetic acid-sodium acetate solution having a concentration of 0.01 to 0.1 mol/L and a pH of 3.9 to 4.1.
The purification method according to any one of claims 1 to 3, wherein the crude pure mobile phase consists of an initial buffer mobile phase and an elution buffer mobile phase, wherein the initial buffer mobile phase is An acetic acid-sodium acetate solution, the elution buffer mobile phase is an initial buffer mobile phase to which sodium chloride is added; preferably, the initial buffer mobile phase has a concentration of 0.01-0.1 mol/L and a pH of 3.9. -4.1 acetic acid-sodium acetate solution, the elution buffer mobile phase is the initial buffer mobile phase to which 0.5-1.5 mol/L sodium chloride is added.
The purification method according to any one of claims 1 to 3, wherein the desalted mobile phase is composed of a mobile phase A and a mobile phase B, wherein the mobile phase A is ultrapure water, and the mobile phase B is chromatographically pure methanol.
The purification method according to claim 4 or 5, wherein in the step (2), the crude pure mobile phase gradient is an initial buffer mobile phase: the elution buffer mobile phase is from 100:0 to (80- 60): (20-40).
The purification method according to claim 4 or 5, wherein in the step (3), The desalination mobile phase gradient is mobile phase A: mobile phase B consists of (100-95): (0-5) to (90-80): (10-20).
The purification method according to any one of claims 1 to 3, wherein the crude pure mobile phase consists of an initial buffer mobile phase and an elution buffer mobile phase, wherein the initial buffer mobile phase is An acetic acid-sodium acetate solution, the elution buffer mobile phase is a high concentration, high pH acetic acid-sodium acetate buffer; preferably, the initial buffer mobile phase is at a concentration of 0.01-0.1 mol/L, pH The value is 3.9-4.1 acetic acid-sodium acetate solution, the elution buffer mobile phase consists of phase A and phase B, and phase A is acetic acid-acetic acid with a concentration of 0.01-0.1 mol/L and a pH of 5.5-6.2. The sodium solution, phase B is an acetic acid-sodium acetate solution having a concentration of 0.5-1.5 mol/L and a pH of 5.5-6.2.
The purification method according to any one of claims 1 to 3, wherein the desalted mobile phase is composed of a mobile phase A and a mobile phase B, wherein the mobile phase A is ultrapure water, and the mobile phase B is chromatographically pure methanol.
The purification method according to claim 8 or 9, wherein in the step (2), the crude pure mobile phase gradient is an initial buffer mobile phase: the elution buffer mobile phase is from 100:0 to (80- 60): (20-40).
The purification method according to claim 8 or 9, wherein in the step (3), the desalting mobile phase gradient is a mobile phase A: the mobile phase B is from (100-95): (0-5) to ( 90-80): (5-10).