CN113121675B

CN113121675B - Salt conversion method of GLP-1 analogue

Info

Publication number: CN113121675B
Application number: CN201911409945.8A
Authority: CN
Inventors: 黄嘉成; 尹传龙; 陶安进; 余品香
Original assignee: Hybio Pharmaceutical Wuhan Co ltd; Hybio Pharmaceutical Co Ltd
Current assignee: Hybio Pharmaceutical Wuhan Co ltd; Hybio Pharmaceutical Co Ltd
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2023-03-31
Anticipated expiration: 2039-12-31
Also published as: CN113121675A; WO2021135765A1

Abstract

The invention discloses a salt conversion method of a GLP-1 analogue, which comprises the following steps: 1) Sample preparation: preparing a purified fraction of the GLP-1 analog; 2) Sample dilution: diluting the purified fraction with deionized water; 3) And (3) concentrating a sample: concentrating with ultrafiltration membrane; 4) Transferring salt to a sample: performing salt conversion by using the ultrafiltration membrane in the step 3). The invention adopts ultrafiltration technology to carry out salt conversion, and has higher recovery rate than reverse salt conversion; the ultrafiltration technology is used for salt conversion, the obtained sample solution can be freeze-dried without decompression and concentration, the process is simple, and the overall efficiency is higher; the ultrafiltration technology is used for salt conversion, organic solvents are not needed, organic waste liquid is not generated, the cost is reduced, and meanwhile, the method is more environment-friendly.

Description

Salt conversion method of GLP-1 analogue

Technical Field

The invention belongs to the technical field of salt conversion methods, and particularly relates to a salt conversion method of a GLP-1 analogue.

Background

Glucagon-like peptide-1 (GLP-1) is a peptide hormone secreted by human intestinal epidermal cells, can promote insulin secretion, protect pancreatic beta cells, inhibit glucagon secretion and inhibit gastric emptying, has effects of lowering blood sugar and reducing weight, and is mainly used for treating type II diabetes. Because natural GLP-1 is unstable in a human body and is easily hydrolyzed by dipeptidyl peptidase IV (DPP-IV), a series of GPL-1 analogues are developed by drug research and development enterprises, the resistance of the compounds to DDP-IV is improved, and the half-life period is prolonged, such as somaglutide, liraglutide and the like.

For chemically synthesized GLP-1 analogues, salt conversion is mostly realized by reverse phase chromatography technology, such as patents CN104356224A, CN 109438569A, CN 107903318A and the like. The method comprises the following steps of preparing the soxhlet peptide in an acetate form by adopting a reverse phase chromatography technology in the patent CN104356224A, preparing the liraglutide in an acetate form by adopting a reverse phase chromatography technology in the patent CN 109438569A, and preparing the liraglutide in a phosphate form by adopting a reverse phase chromatography technology in the patent CN 107903318A. When the reverse phase chromatography is used for salt conversion, a large amount of organic solvent is required, a large amount of organic waste liquid is generated, on one hand, the use cost of the solvent and the treatment cost of the waste liquid are increased, and on the other hand, pharmaceutical enterprises are under greater environmental protection pressure; the samples after reverse phase chromatography salt conversion can be frozen and dried after decompression concentration and removal of most of organic solvents, so that the complexity of the process is increased and the efficiency is reduced; in addition, when reverse-phase salt conversion is carried out, dead adsorption action usually exists between a sample and a reverse-phase filler, and a phenomenon of peak-type tailing is easy to occur, so that the sample is difficult to be completely eluted, and finally, the recovery rate of reverse-phase salt conversion is low. In patent CN 108359006A, salt conversion was performed by gel chromatography to obtain salt-free form of somaglutide. But also uses more organic solvent and generates more organic waste liquid; the sample loading amount of the dextran gel salt conversion is small, the efficiency is low, and the amplification production is easy to limit; the sample solution after the salt conversion can be lyophilized after being subjected to vacuum concentration and most of organic solvent removal. The reverse phase chromatography and gel chromatography technologies can realize the salt conversion of the somaglutide and the liraglutide, but have certain disadvantages. In combination, reverse phase chromatography salt transfer and gel chromatography salt transfer are not advantageous in terms of recovery rate, cost, efficiency and environmental protection.

In order to improve the product recovery rate and production efficiency, reduce the production cost and environmental protection pressure, the invention uses the ultrafiltration technology to carry out salt conversion on the purified fraction of the GLP-1 analogue. In the whole ultrafiltration process, only the purified fraction of the GLP-1 analogue needs to be diluted by deionized water, and any organic solvent is not needed, so that the solvent cost is reduced, and the environmental protection pressure is also reduced; after the ultrafiltration is finished, the freeze drying can be directly carried out without carrying out reduced pressure concentration treatment, so that the process is simplified, and the efficiency is improved; meanwhile, the recovery rate of the sample in the process of transferring salt is improved.

Disclosure of Invention

In order to solve the problems raised in the background art described above, it is an object of the present invention to provide a method for salt transfer of a GLP-1 analog. The invention adopts ultrafiltration technology to carry out salt conversion on the purified fraction of the GLP-1 analogue, and finally converts the purified fraction into a salt-free form. After completing the salt conversion, the GLP-1 analogue refined peptide is obtained by directly freeze-drying.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a method for transferring salt of GLP-1 analogue, which is characterized by comprising the following steps:

1) Sample preparation: preparing a purified fraction of the GLP-1 analog;

2) Sample dilution: diluting the purified fraction with deionized water;

3) And (3) concentrating a sample: concentrating with ultrafiltration membrane;

4) Transferring the sample into salt: performing salt conversion by using the ultrafiltration membrane in the step 3).

Further, sample lyophilization is also included after 4): and (4) freeze-drying the ultrafiltration trapped fluid to obtain GLP-1 analogue solid powder.

Further, the purification fraction of the GLP-1 analogue in 1) is a purification fraction of the GLP-1 analogue which can be retained by an ultrafiltration membrane by any purification method.

Further, the sample dilution in 2) is: diluting the purified fraction with deionized water so that the concentration of the organic solvent in the solution is below the upper tolerance limit of the ultrafiltration membrane. In the purification of GLP-1 analogue, acetonitrile is mostly used, and the purified fraction is diluted with deionized water so that the concentration of acetonitrile in the solution is 5-10% (V/V).

Further, the sample concentration in 3) is carried out by using an ultrafiltration membrane with the molecular weight cutoff of 1K.

Further, the concentration multiple is 15-20 times; the pressure of the liquid inlet end is controlled to be 0.15-0.25 MPa, and the pressure of the reflux end is controlled to be less than 0.05 MPa.

Further, the sample in 4) is converted into salt: performing salt conversion by using the ultrafiltration membrane in the step 3), recording the volume of the concentrated sample solution as V, continuously supplementing deionized water into the concentrated sample solution, keeping the total volume V unchanged, and finishing ultrafiltration when the volume of the ultrafiltration permeating liquid is 8-10 times of V; the pressure of the liquid inlet end is controlled to be 0.15-0.25 MPa, and the pressure of the reflux end is controlled to be less than 0.05 MPa.

The chemically synthesized GLP-1 analogue can obtain refined peptide with the purity of more than or equal to 99.0 percent and the single impurity of less than or equal to 0.15 percent after purification and salt conversion. In the invention, the GLP-1 analogue fine peptide solution containing acid radical ions with the purity of more than or equal to 99.0 percent and the single impurity of less than or equal to 0.15 percent is obtained through early purification. And then, performing ultrafiltration salt conversion to remove acid radical ions in the solution to obtain the salt-free GLP-1 analogue with the purity of more than or equal to 99.0 percent and the single impurity of less than or equal to 0.15 percent.

The beneficial effects of the invention are: (1) The recovery rate of the salt conversion is higher than that of the reverse salt conversion by using the ultrafiltration technology; (2) The ultrafiltration technology is used for salt conversion, the obtained sample solution can be freeze-dried without decompression and concentration, the process is simple, and the overall efficiency is higher; (3) The ultrafiltration technology is used for salt conversion, organic solvents are not needed, organic waste liquid is not generated, the cost is reduced, and meanwhile, the method is more environment-friendly.

Drawings

FIG. 1 is a liquid chromatogram of a salt transfer sample from Somalutide ultrafiltration in example 1;

FIG. 2 is the sample mass spectrum of the salt conversion by ultrafiltration of the somaglutide of example 1;

FIG. 3 is a liquid chromatogram of a salt-converted sample from the ultrafiltration of somaglutide in example 2;

FIG. 4 is the sample quality spectrum of the salt conversion by ultrafiltration of the Somalutide of example 2;

FIG. 5 is a liquid chromatogram of a salt-converted sample from Somalutide ultrafiltration in example 3;

FIG. 6 is the sample mass spectrum of the salt conversion by ultrafiltration of the Somalutide in example 3;

FIG. 7 is the liquid chromatogram of the liraglutide ultrafiltration salt transfer sample in example 4;

FIG. 8 is the Liraglutide ultrafiltration salt conversion sample mass spectrum of example 4;

FIG. 9 is a liquid chromatogram of a liraglutide ultrafiltration salt transfer sample in example 5;

FIG. 10 is the Liraglutide ultrafiltration desalting sample mass spectrum of example 5;

FIG. 11 is a liquid chromatogram of a liraglutide ultrafiltration salt transfer sample of example 6;

FIG. 12 is a diagram of the Liraglutide ultrafiltration salt conversion sample mass spectrum of example 6;

FIG. 13 is a liquid chromatogram of a salt-converted sample from Somalutide ultrafiltration in comparative example 1;

FIG. 14 is a sample mass spectrum of the Somalutide ultrafiltration salt conversion sample of comparative example 1;

FIG. 15 is a liquid chromatogram of a Liraglutide ultrafiltration salt transfer sample of comparative example 2;

FIG. 16 is a mass spectrum of a liraglutide ultrafiltration salt conversion sample in comparative example 2;

FIG. 17 is a liquid chromatogram of a reverse phase salt-converted sample of somaglutide in comparative example 3;

FIG. 18 is a mass spectrum of a reverse phase salt conversion sample of somaglutide in comparative example 3;

FIG. 19 is a liquid chromatogram of a reverse phase salt-converted liraglutide sample from comparative example 4;

fig. 20 is a reverse phase salt conversion sample mass spectrum of liraglutide in comparative example 4.

Detailed Description

For a better understanding of the present invention, the following examples are given to illustrate the present invention, but the present invention is not limited to the following examples.

The meanings of abbreviations used in the specification and claims are listed in the following table:

in the examples of the present application, GLP-1 analogs are exemplified by somagluteptide or liraglutide.

Example 1: salt conversion of somaglutide

1) Crude soxhlet peptide was dissolved at a concentration of about 30mg/ml with 20mM, pH =8.0, disodium phosphate buffer (pH adjusted with phosphate and sodium hydroxide) and passed through a 0.45 μm microfiltration membrane. 20mM disodium hydrogen phosphate buffer (pH adjusted with sodium hydroxide and phosphoric acid) with pH =8.0 was used as an equilibrium solution A phase, and an A phase containing 1.0M NaCl was used as a B phase; 100% of phase A has strong balanceAnion exchange columns (100 x 150mm,10 μm,

) 5 CV of; after balancing, loading the sample, wherein the loading amount is 80g; after sampling, eluting 5 CV by 100% phase A, and then performing linear gradient elution by phase A and phase B (phase B is 0-100%, elution is 60 min), wherein the flow rate is 500.0ml/min, and the detection wavelength is 280nm; collecting the main peak to obtain the first-step purification fraction of the Somaltul peptide. The first purified fraction was used as sample and an octaalkylsilane-bonded silica gel packing was used as stationary phase (150 x 250mm,10 μm;, in;)>

) Performing gradient linear elution (26-46% of organic phase and 100min of elution time) by using 0.05% phosphoric acid as a water phase and acetonitrile as an organic phase, wherein the flow rate is 550ml/min, and the detection wavelength is 280nm; collecting target peaks to obtain the somaglutide fraction with purity not less than 99.0% and single impurity not more than 0.15%. Taking 4.0L of the purified fraction as a sample to be subjected to ultrafiltration and salt conversion, wherein the concentration of acetonitrile is 35% and the concentration of the somaglutide is 2.18g/L.

2) Sample dilution: according to the sample: deionized water (V: V) =1: deionized water was added at a ratio of 3 such that the acetonitrile concentration in the sample was 8.75% by volume, 16.0L.

3) And (3) concentrating a sample: concentrating with ultrafiltration membrane with molecular weight cutoff of 1K. The pressure of the liquid inlet end is controlled to be 0.15MPa, and the pressure of the reflux end is controlled to be 0.02MPa. The concentration multiple was 20 times, and the volume of the sample after concentration was 0.8L.

4) Transferring salt to a sample: performing salt conversion by using the ultrafiltration membrane in the step 3), and continuously supplementing deionized water into the concentrated sample solution, and keeping the total volume of 0.8L unchanged. The pressure of the liquid inlet end is controlled to be 0.15MPa, and the pressure of the reflux end is controlled to be 0.02MPa. When the volume of the ultrafiltration permeate was 7.2L, the ultrafiltration was terminated.

5) Sample freeze-drying: freeze-drying the ultrafiltration trapped fluid to obtain 8.51g of solid somaglutide powder, and the salt conversion recovery rate is 97.59%. The liquid chromatogram of the Somalglu ultrafiltration salt conversion sample is shown in figure 1, the quality chromatogram of the Somalglu ultrafiltration salt conversion sample is shown in figure 2, and the purity of the converted Somalglu solution is more than or equal to 99.0 percent and the single impurity content is less than or equal to 0.15 percent; the conductivity statistics of the salt conversion process by the soxhlet ultrafiltration are shown in table 1.

TABLE 1 Suomalu peptide Ultrafiltration Transsalt Process conductivity statistics

Note: v is the volume of the sample after concentration and before ultrafiltration for salt conversion.

Example 2: salt conversion of somaglutide

1) Sample preparation: the crude soymaglutide peptide was dissolved at a concentration of about 35mg/ml in 30mM, pH =9.0 Tris buffer (pH adjusted with hydrochloric acid) and passed through a 0.45 μm microfiltration membrane. Using 30mM Tris buffer (pH adjusted by hydrochloric acid) with pH =9.0 as an equilibrium solution A phase, and using an A phase containing 1.0M NaCl as a B phase; 100% of a phase equilibrium strong anion exchange column (100 x 150mm,15 μm,

) 5 CV of; after balancing, loading the sample, wherein the loading amount is 60g; after sampling, eluting 5 CV phases by 100% of phase A, and then performing linear gradient elution by phase A and phase B (phase B is eluted for 50min from 0-100%), wherein the flow rate is 500.0ml/min, and the detection wavelength is 280nm; collecting the main peak to obtain the first-step purification fraction of the somaglutide. The first purified fraction was used as sample and an octaalkylsilane-bonded silica gel packing was used as stationary phase (150 x 250mm,10 μm;, in;)>

) Performing gradient linear elution (organic phase 23-43% and elution time 60 min) with 0.15% phosphoric acid as water phase and acetonitrile as organic phase, wherein the flow rate is 550ml/min, and the detection wavelength is 280nm; collecting target peaks to obtain the somaglutide fraction with purity not less than 99.0% and single impurity not more than 0.15%. Taking 2.0L of the purified fraction as sample to be ultrafiltered and converted into salt, wherein acetonitrile concentration is 32.0%, and Soma isThe concentration of the luminal peptide is 2.42g/L.

2) Sample dilution: according to the sample solution: deionized water (V: V) =1:5 deionized water was added to give a sample solution of 5.33% acetonitrile by volume of 12.0L.

3) And (3) concentrating a sample: concentrating with ultrafiltration membrane with molecular weight cutoff of 1K. The pressure of the liquid inlet end is controlled to be 0.20MPa, and the pressure of the reflux end is controlled to be 0.03MPa. The concentration factor was 18 times, and the sample volume after concentration was 0.67L.

4) Transferring the sample into salt: performing salt conversion by using the ultrafiltration membrane in the step 3), and continuously supplementing deionized water into the concentrated sample solution, and keeping the total volume to be 0.67L unchanged. The pressure of the liquid inlet end is controlled to be 0.20MPa, and the pressure of the reflux end is controlled to be 0.03MPa. When the volume of the ultrafiltration permeate was 5.36L, the ultrafiltration was terminated.

5) Sample freeze-drying: freeze-drying the ultrafiltration retentate to obtain 4.70g of soxhlet peptide solid powder, and the salt conversion recovery rate is 97.11%. The liquid chromatogram of the Somallu peptide ultrafiltration salt conversion sample is shown in figure 3, the quality chromatogram of the Somallu peptide ultrafiltration salt conversion sample is shown in figure 4, and the purity of the Somallu peptide solution after salt conversion is more than or equal to 99.0 percent and less than or equal to 0.15 percent; the conductivity statistics of the salt conversion process by the soxhlet ultrafiltration are shown in table 2.

TABLE 2 Suomalu peptide Ultrafiltration Transsalt Process conductivity statistics

Example 3: salt conversion of somaglutide

1) Sample preparation: the crude soxhlet peptide was dissolved at a concentration of about 20mg/ml in 15mM, pH =7.0 diammonium phosphate buffer (pH adjusted with phosphoric acid and ammonia), and passed through a 0.45 μm microfiltration membrane. Using 15mM diammonium phosphate buffer (pH adjusted by phosphoric acid and ammonia water) with pH =7.0 as an equilibrium solution A phase, and using an A phase containing 1.0M NaCl as a B phase; 100% of a phase equilibrium strong anion exchange column (100 x 150mm,30 μm,

) 5 CV's; after balancing, loading the sample, wherein the loading amount is 100g; after sampling, eluting 5 CV by 100% phase A, and then performing linear gradient elution by phase A and phase B (phase B is 0-100%, elution is 40 min), wherein the flow rate is 500.0ml/min, and the detection wavelength is 280nm; collecting the main peak to obtain the first-step purification fraction of the somaglutide. The first purified fraction was used as sample and an octaalkylsilane-bonded silica gel packing was used as stationary phase (150 x 250mm,10 μm;, in;)>

) Performing gradient linear elution (25-45% of organic phase and 80min of elution time) by taking 0.10% phosphoric acid as a water phase and acetonitrile as an organic phase, wherein the flow rate is 550ml/min, and the detection wavelength is 280nm; collecting target peak to obtain the fraction of the Somalutide with the purity more than or equal to 99.0 percent and the single impurity less than or equal to 0.15 percent. 8.0L of the purified fraction was taken as a sample to be subjected to ultrafiltration and salt transfer, wherein the concentration of acetonitrile was 34.0%, and the concentration of somaglutide was 2.29g/L.

2) Sample dilution: according to the sample solution: deionized water (V: V) =1: deionized water was added at a ratio of 4 such that the acetonitrile concentration in the sample solution was 6.80% and the volume was 40.0L.

3) And (3) concentrating a sample: concentrating with ultrafiltration membrane with molecular weight cutoff of 1K. The pressure at the liquid inlet end is controlled to be 0.25MPa, and the pressure at the reflux end is controlled to be 0.04MPa. The concentration factor was 15 times, and the sample volume after concentration was 2.67L.

4) Transferring the sample into salt: performing salt conversion by using the ultrafiltration membrane in the step 3), and continuously supplementing deionized water into the concentrated sample solution, and keeping the total volume to be 2.67L unchanged. The pressure at the liquid inlet end is controlled to be 0.25MPa, and the pressure at the reflux end is controlled to be 0.04MPa. When the volume of the ultrafiltration permeating liquid is 26.7L, the ultrafiltration is finished.

5) Sample freeze-drying: freeze-drying the ultrafiltration retentate to obtain 17.84g of soxhlet peptide solid powder, and the salt conversion recovery rate is 97.38%. The liquid chromatogram of the Somalglu ultrafiltration salt conversion sample is shown in FIG. 5, the quality chromatogram of the Somalglu ultrafiltration salt conversion sample is shown in FIG. 6, and the purity of the converted Somalglu solution is more than or equal to 99.0%, and the single impurity content is less than or equal to 0.15%; the conductivity statistics of the salt conversion process by the soxhlet ultrafiltration are shown in table 3.

TABLE 3 Suomalu peptide Ultrafiltration salt conversion Process conductivity statistics

Example 4: liraglutide transsalification

1) Sample preparation: the crude liraglutide was dissolved at a concentration of about 30mg/ml in 30mM, pH =8.5 ammonium bicarbonate buffer (pH adjusted with formic acid and ammonia) and filtered through a 0.45 μm microfiltration membrane. Using 30mM ammonium bicarbonate buffer solution (pH adjusted by formic acid and ammonia water) with pH =8.5 as an equilibrium solution A phase, and using an A phase containing 1.0M NaCl as a B phase; 100% of the a phase equilibrated strong anion exchange column (100 x 150mm,15 μm,

) 5 CV of; after balancing, loading the sample, wherein the loading amount is 50g; after sampling, eluting 5 CV by 100% phase A, and then performing linear gradient elution by phase A and phase B (phase B is 0-100%, elution is 70 min), wherein the flow rate is 500.0ml/min, and the detection wavelength is 280nm; collecting the main peak to obtain the first-step purified fraction of the liraglutide. The fraction purified in the first step was used as a sample and an octaalkylsilane-bonded silica filler as a stationary phase (150X 250mm,10 μm;,/H;)>

) Performing gradient linear elution (the organic phase is 28-48 percent and the elution time is 90 min) by taking 0.15 percent phosphoric acid as a water phase and acetonitrile as an organic phase, wherein the flow rate is 550ml/min, and the detection wavelength is 280nm; collecting the target peak to obtain the liraglutide fraction with the purity of more than or equal to 99.0 percent and the single impurity of less than or equal to 0.15 percent. Taking 4.0L of the purified fraction as a sample to be subjected to ultrafiltration and salt conversion, wherein the concentration of acetonitrile is 42.0 percent, and the concentration of liraglutide is 1.69g/L.

2) Sample dilution: according to the sample solution: deionized water (V: V) =1: deionized water was added at a ratio of 4 to give a sample solution having an acetonitrile concentration of 8.40% and a volume of 20.0L.

3) And (3) concentrating a sample: concentrating with ultrafiltration membrane with molecular weight cutoff of 1K. The pressure of the liquid inlet end is controlled to be 0.15MPa, and the pressure of the reflux end is controlled to be 0.02MPa. The concentration factor was 16 times, and the sample volume after concentration was 1.25L.

4) Transferring the sample into salt: performing salt conversion by using the ultrafiltration membrane in the step 3), continuously supplementing deionized water into the concentrated sample solution, and keeping the total volume to be 1.25L unchanged. The pressure of the liquid inlet end is controlled to be 0.15MPa, and the pressure of the reflux end is controlled to be 0.02MPa. And finishing the ultrafiltration when the volume of the ultrafiltration permeating liquid is 11.25L.

5) Sample freeze-drying: and (4) freeze-drying the ultrafiltration trapped fluid to obtain 6.51g of liraglutide solid powder, wherein the salt conversion recovery rate is 96.30%. The liquid chromatogram of the liraglutide ultrafiltration salt conversion sample is shown in figure 7, the quality chromatogram of the liraglutide ultrafiltration salt conversion sample is shown in figure 8, and the purity of the liraglutide solution after salt conversion is more than or equal to 99.0 percent and the single impurity content is less than or equal to 0.15 percent; the conductivity statistics for the liraglutide ultrafiltration salt transfer process are shown in table 4.

TABLE 4 Liraglutide Ultrafiltration Transsalt Process conductivity statistics

Example 5: liraglutide transsalification

1) Sample preparation: the crude liraglutide was dissolved at a concentration of about 25mg/ml in 25mM, pH =8.0 dibasic potassium phosphate buffer (pH adjusted with phosphate and potassium hydroxide) and filtered through a 0.45 μm microfiltration membrane. Using 25mM dipotassium hydrogen phosphate buffer (pH adjusted by phosphoric acid and potassium hydroxide) with pH =8.0 as an equilibrium solution A phase, and using an A phase containing 1.0M NaCl as a B phase; 100% of a phase equilibrium strong anion exchange column (100 x 150mm,30 μm,

) 5 CV of; after balancing, loading the sample, wherein the loading amount is 60g; after sampling, eluting 5 CV by 100% phase A, then performing linear gradient elution by phase A and phase B (phase B is 0-100%, elution is 50 min), the flow rate is 500.0ml/min, and the detection wavelength is 280nm; collecting mainAnd (4) obtaining the first-step purified fraction of the liraglutide. The fraction purified in the first step was used as a sample and an octaalkylsilane-bonded silica filler as a stationary phase (150X 250mm,10 μm;,/H;)>

) Performing gradient linear elution (organic phase 29-49% and elution time 80 min) with 0.10% phosphoric acid as water phase and acetonitrile as organic phase, wherein the flow rate is 550ml/min, and the detection wavelength is 280nm; collecting the target peak to obtain the liraglutide fraction with the purity of more than or equal to 99.0 percent and the single impurity of less than or equal to 0.15 percent. 6.0L of the purified fraction was taken as a sample to be subjected to ultrafiltration for salt transfer, wherein the concentration of acetonitrile was 43.0% and the concentration of liraglutide was 1.78g/L.

2) Sample dilution: according to the sample solution: deionized water (V: V) =1:5 deionized water was added so that the sample solution had an acetonitrile concentration of 7.17% and a volume of 36.0L.

3) And (3) concentrating a sample: concentrating with ultrafiltration membrane with molecular weight cutoff of 1K. The pressure at the liquid inlet end is controlled to be 0.25MPa, and the pressure at the reflux end is controlled to be 0.04MPa. The concentration factor was 15 times, and the sample volume after concentration was 2.4L.

4) Transferring salt to a sample: performing salt conversion by using the ultrafiltration membrane in the step 3), continuously supplementing deionized water into the concentrated sample solution, and keeping the total volume to be 2.4L unchanged. The pressure at the liquid inlet end is controlled to be 0.25MPa, and the pressure at the reflux end is controlled to be 0.04MPa. And finishing the ultrafiltration when the volume of the ultrafiltration permeating liquid is 19.2L.

5) Sample freeze-drying: freeze-drying the ultrafiltration trapped fluid to obtain 10.27g of liraglutide solid powder, wherein the salt conversion recovery rate is 96.16%. The liquid chromatogram of the liraglutide ultrafiltration salt conversion sample is shown in fig. 9, the quality chromatogram of the liraglutide ultrafiltration salt conversion sample is shown in fig. 10, and the purity of the liraglutide solution after salt conversion is more than or equal to 99.0 percent and the single impurity content is less than or equal to 0.15 percent; the conductivity statistics for the liraglutide ultrafiltration salt transfer process are shown in table 5.

TABLE 5 Liraglutide Ultrafiltration Transsalt Process conductivity statistics

Example 6: liraglutide transsalification

1) Sample preparation: the crude liraglutide was dissolved at a concentration of about 35mg/ml in 10mM ammonium acetate buffer pH =9.0 (pH adjusted with acetic acid and ammonia) and filtered through a 0.45 μm microfiltration membrane. Using 10mM ammonium acetate buffer solution (pH adjusted by acetic acid and ammonia water) with pH =9.0 as an equilibrium solution A phase, and using an A phase containing 1.0M NaCl as a B phase; 100% of the a phase equilibrated strong anion exchange column (100 x 150mm,10 μm,

) 5 CV's; after balancing, loading the sample, wherein the loading amount is 70g; after sampling, eluting 5 CV by 100% phase A, and then performing linear gradient elution by phase A and phase B (phase B is 0-100%, elution is 60 min), wherein the flow rate is 500.0ml/min, and the detection wavelength is 280nm; collecting the main peak to obtain the first-step purified fraction of the liraglutide. The fraction purified in the first step was used as a sample and an octaalkylsilane-bonded silica filler as a stationary phase (150X 250mm,10 μm;,/H;)>

) Performing gradient linear elution (organic phase 31-51% and elution time 100 min) with 0.05% phosphoric acid as water phase and acetonitrile as organic phase, wherein the flow rate is 550ml/min, and the detection wavelength is 280nm; collecting the target peak to obtain the liraglutide fraction with the purity of more than or equal to 99.0 percent and the single impurity of less than or equal to 0.15 percent. Taking 5.0L of the purified fraction as a sample to be subjected to ultrafiltration and salt conversion, wherein the concentration of acetonitrile is 45.0%, and the concentration of liraglutide is 1.58g/L.

2) Sample dilution: according to the sample solution: deionized water (V: V) =1: deionized water was added at a ratio of 6 to give a sample solution with an acetonitrile concentration of 6.43% and a volume of 35.0L.

3) And (3) concentrating a sample: concentrating with ultrafiltration membrane with molecular weight cutoff of 1K. The pressure of the liquid inlet end is controlled to be 0.20MPa, and the pressure of the reflux end is controlled to be 0.03MPa. The concentration multiple was 20 times, and the volume of the sample after concentration was 1.75L.

4) Transferring the sample into salt: performing salt conversion by using the ultrafiltration membrane in the step 3), continuously supplementing deionized water into the concentrated sample solution, and keeping the total volume to be 1.75L unchanged. The pressure of the liquid inlet end is controlled to be 0.20MPa, and the pressure of the reflux end is controlled to be 0.03MPa. And finishing the ultrafiltration when the volume of the ultrafiltration permeating liquid is 17.5L.

5) Sample freeze-drying: freeze-drying the ultrafiltration trapped fluid to obtain 7.57g of liraglutide solid powder, wherein the salt conversion recovery rate is 95.82%. The liquid chromatogram of the liraglutide ultrafiltration salt conversion sample is shown in fig. 11, the quality chromatogram of the liraglutide ultrafiltration salt conversion sample is shown in fig. 12, and the purity of the liraglutide solution after salt conversion is more than or equal to 99.0 percent and the single impurity content is less than or equal to 0.15 percent; the conductivity statistics for the liraglutide ultrafiltration salt transfer process are shown in table 6.

TABLE 6 Liraglutide Ultrafiltration Transsalt Process conductivity statistics

Comparative example 1: ultrafiltration salt conversion of Somalutide (Ultrafiltration membrane with molecular weight cut-off of 2K)

1) Sample preparation: the purification procedure was as in example 1. Taking 4.0L of the purified fraction as a sample to be subjected to ultrafiltration and salt conversion, wherein the concentration of acetonitrile is 35%, and the concentration of the somaglutide is 2.18g/L.

2) Sample dilution: according to the sample solution: deionized water (V: V) =1: deionized water was added at a ratio of 3 such that the acetonitrile concentration in the sample solution was 8.75% by volume, 16.0L.

3) And (3) concentrating a sample: concentrating with ultrafiltration membrane with molecular weight cutoff of 2K. The pressure at the liquid inlet end is controlled to be 0.15MPa, and the pressure at the reflux end is controlled to be 0.02MPa. The concentration factor was 20 times, and the sample volume after concentration was 0.8L.

4) Transferring the sample into salt: performing salt conversion by using the ultrafiltration membrane in the step 3), continuously supplementing deionized water into the concentrated sample solution, and keeping the total volume to be 0.8L. The pressure of the liquid inlet end is controlled to be 0.15MPa, and the pressure of the reflux end is controlled to be 0.02MPa. And finishing the ultrafiltration when the volume of the ultrafiltration permeating liquid is 7.2L.

5) Sample freeze-drying: and (3) freeze-drying the ultrafiltration retentate to obtain 7.71g of soxhlet peptide solid powder, wherein the salt conversion recovery rate is 88.42%. The liquid chromatogram of the somagluteptide ultrafiltration salt conversion sample is shown in fig. 13, the mass chromatogram of the somagluteptide ultrafiltration salt conversion sample is shown in fig. 14, and the purity of the somagluteptide solution after salt conversion is more than or equal to 99.0 percent and less than or equal to 0.15 percent; the conductivity statistics of the somaglutide ultrafiltration salt transfer process are shown in table 7.

TABLE 7 Suomalu peptide Ultrafiltration salt conversion Process conductivity statistics

Comparative example 2: ultrafiltration salt transfer of liraglutide (Ultrafiltration membrane with cut-off molecular weight of 2K)

1) Sample preparation: the purification procedure was as in example 4. Taking 4.0L of the purified fraction as a sample to be subjected to ultrafiltration and salt conversion, wherein the concentration of acetonitrile is 42.0 percent, and the concentration of liraglutide is 1.69g/L.

3) And (3) concentrating a sample: concentrating with ultrafiltration membrane with molecular weight cutoff of 2K. The pressure of the liquid inlet end is controlled to be 0.15MPa, and the pressure of the reflux end is controlled to be 0.02MPa. The concentration times were 16 times, and the sample volume after concentration was 1.25L.

4) Transferring the sample into salt: performing salt conversion by using the ultrafiltration membrane in the step 3), continuously supplementing deionized water into the concentrated sample solution, and keeping the total volume to be 1.25L unchanged. The pressure at the liquid inlet end is controlled to be 0.15MPa, and the pressure at the reflux end is controlled to be 0.02MPa. And finishing the ultrafiltration when the volume of the ultrafiltration permeating liquid is 11.25L.

5) Sample freeze-drying: and (4) freeze-drying the ultrafiltration trapped fluid to obtain 5.81g of liraglutide solid powder, wherein the salt conversion recovery rate is 85.95%. The liquid chromatogram of the liraglutide ultrafiltration salt conversion sample is shown in figure 15, the quality chromatogram of the liraglutide ultrafiltration salt conversion sample is shown in figure 16, and the purity of the liraglutide solution after salt conversion is more than or equal to 99.0 percent and the single impurity content is less than or equal to 0.15 percent; the conductivity statistics for the liraglutide ultrafiltration salt transfer process are shown in table 8.

TABLE 8 Liraglutide Ultrafiltration Transsalt Process conductivity statistics

Comparative example 3: reverse phase salt conversion of somaglutide

1) Sample preparation: the purification procedure was as in example 1. Taking 4.0L of the purified fraction as a sample to be reverse-salted, wherein: the concentration of acetonitrile is 35 percent, and the concentration of the somaglutide is 2.18g/L.

2) Sample dilution: according to the sample solution: deionized water (V: V) =1:1, 4.0L of deionized water was added, and after diluting the sample, the salt transfer was performed.

3) Transferring salt to a sample: stationary phase C4 preparative columns (100 x 250mm,10 μm,

) (ii) a The mobile phase A is deionized water, and the mobile phase B is acetonitrile; the detection wavelength is 280nm; the flow rate is 220mL/min; gradient: phase B: 10 → 10%, 0-30 min;10 → 60 percent, 30-31 min;60 → 60 percent, 31-50 min; and (4) loading, transferring salt, and collecting a target peak.

4) And (3) concentrating the sample under reduced pressure: and carrying out rotary evaporation concentration on the collected target peak, wherein the rotary evaporation temperature is 30 ℃, and most of acetonitrile is removed. The concentration of the sample after concentration was about 18mg/ml.

5) Sample freeze-drying: and freeze-drying the concentrated sample solution to obtain 7.89g of solid soxhlet peptide powder, wherein the salt conversion recovery rate is 90.48%. The liquid chromatogram of the salt-converted somaglutide reverse phase sample is shown in FIG. 17, the mass chromatogram of the salt-converted somaglutide reverse phase sample is shown in FIG. 18, and the purity of the salt-converted somaglutide solution is more than or equal to 99.0%, and the single impurity content is less than or equal to 0.15%; the conductivity statistics of the reverse phase salt conversion process of somaglutide are shown in table 9.

TABLE 9 Suomaluride reverse phase salt transfer elution Process conductivity statistics

Note: the elution time is 35-45 min, which is the peak time of the soxhlet peptide during reverse salt conversion.

Comparative example 4: reverse phase trans-salt of liraglutide

1) Sample preparation: the purification procedure was as in example 4. 4.0L of the purified fraction was taken as a sample to be reverse-salted, wherein the concentration of acetonitrile was 42.0% and the concentration of liraglutide was 1.69g/L.

3) Transferring the sample into salt: stationary phase the column was prepared for C4 (100 x 250mm,10 μm,

) (ii) a The mobile phase A is deionized water, and the mobile phase B is acetonitrile; the detection wavelength is 280nm; the flow rate is 220mL/min; gradient: phase B: 10 → 10%, 0-30 min;10 → 70 percent, 30-31 min;70 → 70%, 31-50 min; and (4) loading, transferring salt, and collecting a target peak. />

4) And (3) concentrating the sample under reduced pressure: and carrying out rotary evaporation concentration on the collected target peak, wherein the rotary evaporation temperature is 30 ℃, and most of acetonitrile is removed. The concentration of the sample after concentration was about 15mg/ml.

5) Sample freeze-drying: and freeze-drying the concentrated sample solution to obtain 6.04g of liraglutide solid powder, wherein the salt conversion recovery rate is 89.35%. The liquid chromatogram of the liraglutide reverse phase salt conversion sample is shown in figure 19, the quality chromatogram of the liraglutide reverse phase salt conversion sample is shown in figure 20, and the purity of the liraglutide solution after salt conversion is more than or equal to 99.0 percent and the single impurity content is less than or equal to 0.15 percent; the conductivity statistics for the reverse phase desalting of liraglutide are shown in table 10.

TABLE 10 Liraglutide reverse phase salt transfer elution Process conductivity statistics

Note: the elution time is 35-45 min, which is the time of the peak appearance of the liraglutide during reverse phase salt conversion.

The above description is only a specific embodiment of the present invention, and not all embodiments, and any equivalent modifications of the technical solutions of the present invention, which are made by a person skilled in the art through reading the present specification, are covered by the claims of the present invention.

Claims

1. A method for transferring salt of GLP-1 analogue, which is characterized by comprising the following steps:

1) Sample preparation: preparing a purified fraction of the GLP-1 analog;

2) Sample dilution: diluting the purified fraction with deionized water;

the sample dilution was: diluting the purified fraction with deionized water to make the concentration of the organic solvent in the solution below the tolerance upper limit of the ultrafiltration membrane;

3) And (3) concentrating a sample: concentrating with ultrafiltration membrane; the molecular weight cut-off of the ultrafiltration membrane is 1K;

the concentration multiple is 15 to 20 times; the pressure of the liquid inlet end is controlled to be 0.15-0.25 MPa, and the pressure of the reflux end is controlled to be below 0.05 MPa;

4) Transferring the sample into salt: performing salt conversion by using the ultrafiltration membrane in the step 3), recording the volume of the concentrated sample solution as V, continuously supplementing deionized water into the concentrated sample solution, keeping the total volume V unchanged, and finishing ultrafiltration when the volume of the ultrafiltration permeating liquid is 8-10 times of V; the pressure of the liquid inlet end is controlled to be 0.15-0.25 MPa, and the pressure of the reflux end is controlled to be less than 0.05 MPa.

2. The method for the transcaltation of a GLP-1 analog according to claim 1, further comprising, after 4), sample lyophilization: and (4) freeze-drying the ultrafiltration trapped fluid to obtain GLP-1 analogue solid powder.

3. The method for converting a salt of a GLP-1 analog according to claim 1, wherein the purified fraction of the GLP-1 analog in 1) is a purified fraction of the GLP-1 analog which can be retained by an ultrafiltration membrane by any purification method.