CN117603338A

CN117603338A - A kind of purification method of semaglutide

Info

Publication number: CN117603338A
Application number: CN202310571825.8A
Authority: CN
Inventors: 范晓阳; 吕亮; 刘彬; 杜慈; 汤传根
Original assignee: Nanjing Hanxin Pharmaceutical Technology Co ltd
Current assignee: Nanjing Hanxin Pharmaceutical Technology Co ltd
Priority date: 2022-08-22
Filing date: 2023-05-19
Publication date: 2024-02-27
Anticipated expiration: 2043-05-19
Also published as: CN117603338B

Abstract

The invention belongs to the technical field of medicines, and discloses a purification method of semaglutin. The invention completes the purification of the crude peptide of the semaglutin by a two-step reversed phase chromatography method, and finally obtains the semaglutin refined peptide with the purity of more than or equal to 99.80 percent and the maximum single impurity of less than or equal to 0.06 percent through freeze-drying. The first step of the invention adopts an acid system containing acetate for purification, which can greatly reduce the impurity content of the isomer; the second step adopts alkaline system purification of phosphate to effectively remove or reduce acetylated impurities and partial unknown pre-impurities, so that single impurities are controlled to be less than or equal to 0.06%; the purification process does not need a salt conversion or desalination step, is simpler and more convenient to operate, reduces the production period and is beneficial to industrial production.

Description

Purifying method of semagllutide

Technical Field

The invention relates to a purification method of polypeptide, in particular to a purification mode of semaglutin crude peptide, belonging to the technical field of medicine.

Background

Diabetes is an endocrine disease characterized by hyperglycemia, and can cause kidney injury, retinopathy, cardiovascular disease, etc. by a variety of factors. Diabetes is classified into type 1 and type 2 diabetes according to the pathogenesis, wherein type 2 diabetes accounts for more than 90% of the diabetes types. The pathogenesis of type 2 diabetes can be divided into two points: insulin resistance and insulin secretion defects. Most type 2 diabetes mellitus is a hyperglycemia disease which is usually caused on the basis of insulin resistance, and as the content of sugar in the body is increased, islet beta cells generate sugar toxicity, so that the glucose oxidation process and glucose signal transduction are damaged, and finally, insulin secretion of a patient is defective, so that the blood sugar is continuously improved. Therefore, the current clinical treatment of type 2 diabetes mainly adopts methods of oral hypoglycemic agents or injection of long-acting insulin. With the excellent effect of human glucagon-1 (GLP-1) and analogues thereof on treating type 2 diabetes, the human glucagon-1 (GLP-1) and analogues thereof occupy more important position in the field of novel medicines for treating diabetes. GLP-1 is a gastrointestinal hormone containing 37 amino acid residues, and can be combined with a pancreatic GLP-1 receptor to enable the GLP-1 receptor to be in a glucose concentration dependent mode, so that islet beta cells are effectively stimulated, insulin secretion is promoted, damaged functions of the islet beta cells are recovered, synthesis and release of glucagon can be inhibited, and finally, the effect of reducing blood sugar is achieved. However, natural GLP-1 can be rapidly hydrolyzed and inactivated by plasma enzyme dipeptidyl peptidase IV (DPP-IV), and the half-life period is about 2 minutes, so that the difficulty of clinical application of the natural GLP-1 is increased. Therefore, modification of GLP-1 structure, while maintaining the same pharmacological activity, and prolongation of half-life are important points of research on the novel GLP-1 analogue drug.

GLP-1 analogs that have been currently approved by the FDA for the treatment of type 2 diabetes mellitus include exenatide injection, liraglutide injection, semraglutide tablet, and the like. The semaglutin is a GLP-1 analogue produced by the Norand Norde company through a gene recombination technology, the homology with GLP-1 reaches 94%, and compared with the GLP-1, the semaglutin has the following modification on the structure: 1) Ala8 on the peptide chain is replaced by Aib8, so that the hydrolysis site of dipeptidyl peptidase IV can be covered to prevent degradation by enzyme; 2) Lys26 is grafted with a modified fatty chain comprising 2 8-amino-3, 6-dioxaoctanoic acid structural units, 1 glutamic acid structure and octadecanedioic acid, and the obtained long fatty chain can be tightly combined with albumin, so that the kidney clearance rate is reduced; 3) Lys34 is replaced with Arg34. Modification at 3 positions makes the half-life of semaglutin as long as 165h.

At present, the preparation methods of the semaglutin mainly comprise two main types: 1. after Arg34GLP-1 (9-37) peptide chain is obtained by a biological recombination method, non-natural amino acid dipeptide and a side chain are added by a chemical synthesis method; 2. directly adding amino acid step by chemical method to synthesize straight peptide chain, and adding side chain. Since more impurities such as side reaction impurities, isomer impurities, racemic impurities, amino acid deletions or added impurities are easily generated in the chemical synthesis process. In order to effectively remove the above impurities, the crude peptide of semaglutin is usually purified by reverse phase chromatography. In the patent CN 110845602a, three reverse phase purification and one thin film evaporation are required to obtain a semaglutin sample with purity greater than 99% and maximum single impurity less than 0.2%, and the purification steps are more, wherein multiple stationary phases are required for the reverse phase purification, and new thin film evaporation equipment is required to be put into the device, so that the equipment cost is increased; the use of two organic solvents, acetonitrile and isopropanol, in the mobile phase of elution in patent CN 105777872B can lead to an increased risk of residual organic solvents in the sample, and a single-step salt conversion process is required to obtain a semaglutinin sample with purity of 99.38% and single impurity of less than 0.15% after two-step reverse phase purification; in the patent CN 111848777A, the semaglutin sample with the purity of more than 99 percent is obtained after two-step reversed-phase purification, desalination, concentration and freeze-drying, the maximum single impurity level is not mentioned, and the desalination is carried out by adopting an ultrafiltration membrane, so that the operation time is long, and the industrial large-scale production is not facilitated.

Based on the problems existing in the prior art, on the premise of ensuring the purity, the yield and the maximum single impurity content of the semaglutin, a purification method capable of reducing the complexity of the process, reducing the period and being beneficial to industrial production is found, and the method is a problem to be solved urgently.

Disclosure of Invention

The invention aims to provide a purification method of semaglutin. In the invention, a two-step reversed phase chromatography purification mode is adopted for the crude peptide of the semaglutin, and the semaglutin refined peptide with the purity of more than or equal to 99.80 percent and the single impurity of less than or equal to 0.06 percent is finally obtained through freeze-drying.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

a method for purifying semaglutin by two-step reverse phase high performance liquid chromatography comprising the steps of:

(1) The first step is purification: acid buffer solution containing acetate and ion pairing agent and organic solvent are used as mobile phase, and reversed-phase filler is used as stationary phase;

(2) And (3) purifying: the alkaline buffer solution containing phosphate and the organic solvent are used as mobile phases, and the reverse phase filler is used as a stationary phase.

As an embodiment of the present invention, the acidic buffer containing acetate and ion pairing agent is selected from any one of ammonium acetate-trifluoroacetic acid (TFA) buffer, sodium acetate-trifluoroacetic acid buffer, potassium acetate-trifluoroacetic acid buffer.

The invention adopts acetate buffer to prepare other buffers (such as formate buffer), has better buffer capacity, and can keep the buffer capacity when being matched with strong acid TFA for use; whereas TFA would destroy the buffer capacity of the system when formate buffer is used in combination with TFA.

As an embodiment of the present invention, the mass concentration of TFA in the ammonium acetate-trifluoroacetic acid (TFA) buffer, sodium acetate-trifluoroacetic acid buffer, potassium acetate-trifluoroacetic acid buffer is 0.001% to 1%, optionally 0.025% to 0.1%, such as further optionally 0.05%; the acetate concentration is 0.1-500mM, optionally 10-50mM, such as further optionally 30mM.

As one embodiment of the invention, the acetate-containing acidic buffer has a pH of < 5.0, optionally 2.0-3.0, such as further optionally 2.5; acetic acid is preferably used to adjust the pH, as it does not introduce new anions, does not disrupt the buffering capacity of the system and does not affect the concentration of the buffer.

As one embodiment of the invention, the mobile phase used in the first purification step is subjected to linear gradient elution by taking a mixed solution of an acid buffer solution containing acetate and an ion pairing agent and acetonitrile as a mobile phase RP-A1 and an aqueous solution containing acetonitrile as a mobile phase RP-B, wherein the initial gradient of the mobile phase RP-B is 50% -60%, and optionally 55%; the termination gradient of mobile phase RP-B is 70% -80%, alternatively 75%.

In one embodiment of the present invention, the phosphate-containing alkaline buffer is selected from any one of sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.

As one embodiment of the invention, the buffer salt concentration is 0.1-500mM, optionally 10-50mM, such as further optionally 20mM; the pH of the alkaline buffer solution containing phosphate is more than 7.0, and is optionally 7.5-8.5, such as further optionally 8.0; the pH is preferably adjusted using phosphoric acid, since it does not introduce new anions, does not destroy the buffer capacity of the system and does not affect the concentration of the buffer.

As one embodiment of the invention, the mobile phase used in the second purification step adopts a mixed solution of phosphate-containing alkaline buffer solution and acetonitrile as a mobile phase RP-A2, and adopts an aqueous solution containing acetonitrile as a mobile phase RP-B, wherein the initial gradient of the mobile phase RP-B is 30% -40%, such as 38%; the termination gradient of mobile phase RP-B is 41% to 53%, such as optionally 48%.

As an embodiment of the present invention, the organic solvent used in the first purification step and the second purification step is an aqueous solution containing acetonitrile, such as an aqueous solution (mass concentration) optionally containing 10% to 60% acetonitrile.

As an embodiment of the present invention, the reversed phase packing used in the first purification and the second purification is selected from a stationary phase made of porous silica particles or silica gel. For example, the stationary phase used in the present application may be made of porous silica particles having chemically bonded straight alkyl chains of 4 to 18 carbon atoms. For example a straight alkyl chain containing four (C4), eight (C8), twelve (C12) or eighteen (C18) carbon atoms, i.e. a butyl, octyl, dodecyl or octadecyl moiety. More specifically, octaalkyl-bonded silica gel or octadecyl-bonded silica gel, such as one or more types selected from the group consisting of Sepax BR-C18, sepax GP-C18, unisil 15-100C 18, sepax Bio-C8 (2), BR-C18 (2), HPLCONE 8C18-100AA, HPLCONE 8C18K, HPLCONE 8C8K, YMC-C18 or YMC-C8, more specifically, one of BR-C18 (2) or YMC-C18 packing is preferred as the first purification step, and one of HPLCONE 8C18K or YMC-C18 packing is preferred as the second purification step.

As an embodiment of the present invention, a mixed solvent composed of an alkaline solution containing Tris (Tris) hydroxymethyl aminomethane) and an organic solvent is used to dissolve the crude peptide of semaglutin before the first purification step. The mass ratio of the mixed solvent for dissolution to the semaglutin crude peptide is 30-70:1. after the step of dissolving the crude peptide of semaglutin, a filtration operation is optionally added to remove insoluble particles.

As an embodiment of the present invention, the crude peptide of semaglutin is dissolved using an alkaline solution containing Tris (Tris) and an organic solvent, more preferably a mixed solution containing Tris aqueous solution and acetonitrile, before purification. The concentration of Tris is 0.1-1000mM, such as preferably 10-100mM, such as further alternatively 20-80mM, or 30-70mM, or 40-60mM. The acetonitrile is present in a mass concentration of 5-20%, such as further optionally 10%. The pH of the alkaline solution containing Tris is more than or equal to 7.5, and is optionally 8.0-9.0, and is further optionally 8.5; preferably, hydrochloric acid is used to adjust the pH.

As an embodiment of the present invention, the semaglutinin is isolated after purification by a method selected from one or a combination of lyophilization, addition of anti-solvent crystallization and isoelectric precipitation.

As a more specific embodiment of the present invention, a purification method of semaglutin, purified by crude peptide pretreatment and two-step reversed-phase high performance liquid chromatography, comprises the steps of:

(1) Pretreatment of crude peptide: dissolving the crude peptide of semaglutin by using an alkaline solution containing Tris (Tris (hydroxymethyl aminomethane) and acetonitrile;

(2) The first step is purification: octadecylsilane chemically bonded silica filler is used as a stationary phase, a buffer salt solution containing ammonium acetate-TFA and acetonitrile is used as a mobile phase RP-A1, an acetonitrile solution is used as a mobile phase RP-B, linear gradient elution is carried out, and main peak section components are collected;

(3) And (3) purifying: octadecylsilane chemically bonded silica filler is used as a stationary phase, a buffer salt solution containing phosphate and acetonitrile is used as a mobile phase RP-A2, an acetonitrile solution is used as a mobile phase RP-B, linear gradient elution is carried out, and a main peak component is collected.

As an embodiment of the present invention, the drying mode of the purified semaglutin can be selected from freeze drying or spray drying.

As one embodiment of the invention, before the first purification step uses mobile phase RP-A1 and mobile phase RP-B for elution, a solvent for crude peptide pretreatment, such as an alkaline solution containing Tris (Tris-hydroxymethyl aminomethane) and acetonitrile mixed solution, is optionally used for balancing the chromatographic column so as to keep the pH value of a mobile phase system in the chromatographic column consistent with that of a sample; then using an acetonitrile equilibrium chromatographic column with the concentration of 5-15% to remove the salt introduced in the equilibrium chromatographic column in the last step; finally, the column was equilibrated with mobile phase RP-A1.

As an embodiment of the invention, the second purification step uses a mobile phase RP-A2 equilibrium chromatography column optionally before elution with mobile phases RP-A2 and RP-B.

In one embodiment of the present invention, the detection wavelength of the reverse phase high performance liquid phase method is 250 to 300nm, preferably 280nm.

In the present invention, the solvent for dissolution, the eluting mobile phase (buffer salt type, acid-base type), the concentration, the pH, the gradient elution, etc. are all obtained by experimental screening, and the optimal system is determined by comparing the purity, the yield, the effect of removing impurities, etc. The purification mobile phase system used in the invention has an effect of removing impurities in the crude peptide of the semaglutin and has better yield than other systems.

The crude peptide of the semaglutin used in the purification method can be obtained by a fermentation recombination and solid phase synthesis (coupling one by one or fragment coupling) method.

Compared with the prior art, the purification method of the semaglutin has the following beneficial effects:

(1) The invention completes the purification of the crude peptide of the semaglutin through two-step reversed-phase chromatography, the purity of the semaglutin refined peptide obtained after freeze-drying is more than or equal to 99.80 percent, and the maximum single impurity content is less than or equal to 0.06 percent, which is greatly improved compared with the prior art.

(2) The first step of the invention adopts an acid system containing acetate to purify, which can greatly reduce the impurity content of the isomer (the relative retention time RRT is 0.954-RRT is 0.973 is less than or equal to 0.2 percent), thereby controlling the content of the isomer after the second step of purification to be less than or equal to 0.06 percent; and the content of the isomer impurities is difficult to control to be less than or equal to 0.2 percent by using other acid systems containing formate, so that the content of the isomer impurities is difficult to control to be less than or equal to 0.06 percent after the second step of purification.

(3) The second step adopts alkaline system purification of phosphate to effectively remove or reduce acetylated impurities (RRT 1.107-RRT 1.218) and partial unknown pre-impurities (RRT 0.630-RRT 0.840), so that single impurities are controlled to be less than or equal to 0.06%.

(4) The second alkaline system of the invention uses a phosphate system, and because the semaglutin exists in the form of phosphate, the purification process of the invention does not need a salt conversion or desalination step, has simpler operation and shorter time period, and is suitable for industrialized scale-up production.

Drawings

FIG. 1 is a UPLC spectrum of a sample purified from semaglutin in example 2.

FIG. 2 is a UPLC profile of two samples of the semaglutin purification of example 2.

FIG. 3 is a UPLC spectrum of a sample purified from semaglutin in example 3.

FIG. 4 is a UPLC profile of two samples of the semaglutin purification of example 3.

FIG. 5 is a UPLC spectrum of a sample purified from semaglutin in example 4.

FIG. 6 is a UPLC profile of two samples of the semaglutin purification of example 4.

FIG. 7 is a UPLC spectrum of a sample purified from semaglutin in example 5.

FIG. 8 is a UPLC profile of two samples of the semaglutin purification of example 5.

FIG. 9 is a UPLC spectrum of two samples of the semaglutin purification of example 6.

Fig. 10 is a UPLC profile of a semaglutinin peptide sample from example 7.

FIG. 11 is a UPLC spectrum of a sample purified from semaglutin in comparative example 1.

FIG. 12 is a UPLC spectrum of a sample purified from semaglutin in comparative example 2.

Fig. 13 is a UPLC profile of two samples of semaglutin purification in comparative example 2.

Detailed Description

In order to make the technical features, objects and advantageous effects of the present invention more clearly understood, the technical solution of the present invention will be described in detail below with reference to the specific embodiments, but the protection content of the present invention is not limited to the following embodiments.

The raw materials, reagents and the like used in the following examples are all commercially available unless otherwise specified.

The intermediate polypeptide raw material Arg34GLP-1 (9-37) peptide chain used by the crude semaglutin peptide is prepared by Nanjing Hanchen medical science and technology limited company, wherein the preparation process of the crude semaglutin peptide can refer to the patent CN202210686496.7 of the invention declared by the company, and the preparation process of the intermediate polypeptide raw material Arg34GLP-1 (9-37) peptide chain can refer to the patent CN202111664146.2 of the invention declared by the company, and the whole content of the intermediate polypeptide raw material Arg34GLP-1 (9-37) peptide chain can be directly introduced into the invention.

The flow matching used in this example is as follows:

RP-A1 mobile phase formulation: 0.05% TFA,10% acetonitrile, 30mM sodium acetate, pH2.5 with acetic acid.

The formula of the RP-A2 mobile phase is as follows: 20mM dipotassium hydrogen phosphate, 10% acetonitrile, pH was adjusted to 8.0 with phosphoric acid.

The formula of the RP-A3 mobile phase is as follows: 0.05% TFA,10% acetonitrile, 30mM ammonium acetate, pH adjusted to 2.5 with acetic acid.

The formula of the RP-A4 mobile phase is as follows: 20mM disodium hydrogen phosphate, 10% acetonitrile, pH was adjusted to 8.0 with phosphoric acid.

RP-A5 mobile phase formulation: 0.10% TFA,10% acetonitrile, 30mM ammonium acetate, pH was adjusted to 2.5 with acetic acid.

The RP-A6 mobile phase formula is as follows: 50mM disodium hydrogen phosphate, 10% acetonitrile, pH was adjusted to 8.0 with phosphoric acid.

The formula of the RP-A7 mobile phase is as follows: 0.05% TFA,10% acetonitrile, 30mM ammonium acetate, pH 3.0 with acetic acid.

The formula of the RP-A8 mobile phase is as follows: 20mM disodium hydrogen phosphate, 10% acetonitrile, pH was adjusted to 8.5 with phosphoric acid.

The formula of the RP-B mobile phase is as follows: 60% acetonitrile.

Example 1:

pretreatment of crude semaglutin peptides

1g of crude semaglutin peptide (purity: about 74%) was weighed, and 50 times the mass of the solvents shown in Table 1 were added to perform pretreatment of the crude semaglutin peptide, respectively, and the results are shown in Table 1 below.

TABLE 1 Effect of different solvents on the dissolution of crude Semiglutide peptides

The result shows that the crude peptide of the semaglutin is relatively insoluble in water and slowly soluble in 10% acetonitrile due to the side chain groups in the structure, and is gelatinous in a mixed system of 1% acetic acid and 10% acetonitrile, and flocculent precipitate can be firstly dissolved and then separated out in a mixed solvent of 50mM sodium sulfate solution and 10% acetonitrile. According to the invention, 50mM Tris+10% acetonitrile (pH 8.5) solution is adopted, so that the solubility of the semaglutinin can be improved, a crude peptide sample can be rapidly dissolved in 10min, the dissolution speed is higher than that of 50mM ammonium carbonate solution, the formed solution is clear and transparent, and is stable and can not be re-separated after being placed, the sample loading is facilitated, the chromatographic column blockage is avoided, and the service life of the chromatographic column is prolonged.

Example 2: purification of crude peptide of semaglutin

Sample treatment: 1g of crude peptide of semaglutin was weighed, and a 50-fold mass of a solution containing 50mM Tris and 10% acetonitrile (pH 8.5) was added to dissolve the crude peptide sample for purification.

First purification: taking the dissolved crude peptide of the semaglutin as a sample, taking octadecylsilane chemically bonded silica filler of BR-C18 (2) type as a stationary phase, wherein the flow rate is 200cm/hr, and the detection wavelength is 280nm. The column was equilibrated with a solution containing 50mM Tris and 10% acetonitrile for 2 CV; loading according to the loading amount of 10g/L resin of total protein of the loaded sample; after loading, the column was equilibrated with 10% acetonitrile for 2 CVs; then using RP-A1 mobile phase equilibrium chromatographic column for 3 CV; and finally, performing linear gradient elution (RP-B is from 55 to 75 percent and eluted for 90 minutes) by using RP-A1 and RP-B, and obtaining a collected main peak section which is the purified sample of the semaglutin. By UPLC detection, the purity of a purified sample is 99.46% (the impurity content of the front impurity with the peak time of 10.415min, namely rrt=0.744 is 0.15%, the impurity content of the front impurity with the peak time of 11.666min, namely rrt=0.833 is 0.15%, the impurity content of the isomer with the peak time of 13.560min, namely rrt=0.969 is 0.08%, the impurity content is far less than 0.2%, the impurity content of the acetylated impurity with the peak time of 15.788min, namely rrt=1.128 is 0.11%, and the maximum single impurity is 0.15%) and the purification yield is 61.2%. The UPLC spectrum is shown in FIG. 1.

Sample treatment: the thus obtained semaglutin purified sample was diluted with one volume of purified water, and the pH was adjusted to 8.0 with 10% aqueous ammonia, and was allowed to purify.

Second purification: taking the diluted semaglutin purified sample as a sample loading sample, taking octadecylsilane chemically bonded silica filler of HPLCONE 8C18K type as a stationary phase, wherein the flow rate is 200cm/hr, and the detection wavelength is 280nm. 2 CVs in the column were equilibrated with RP-A2 mobile phase; loading according to the loading amount of 10g/L resin of total protein of the loaded sample; after loading, the column was equilibrated with RP-A2 mobile phase for 3 CVs; and finally, performing linear gradient elution by using RP-A2 and RP-B (the RP-B is eluted for 45min from 38% -48%), and collecting a main peak section to obtain the semaglutin purified two samples. Through UPLC detection, the purity of the purified two samples is 99.82 percent (the maximum single impurity is an isomer impurity with the peak time of 12.908min, namely RRT=0.964 impurity content is 0.06 percent, the peak time is 14.828min, namely RRT=1.107 impurity content is 0.03 percent, which is obviously reduced by one order of magnitude compared with the purified one sample, the pre-impurity content is obviously reduced), and the purification yield is 76.8 percent. The UPLC spectrum is shown in FIG. 2.

Example 3: purification of crude peptide of semaglutin

First purification: taking the dissolved crude peptide of the semaglutin as a sample, taking octadecylsilane chemically bonded silica filler of BR-C18 (2) type as a stationary phase, wherein the flow rate is 200cm/hr, and the detection wavelength is 280nm. The column was equilibrated with a solution containing 50mM Tris and 10% acetonitrile for 2 CV; loading according to the loading amount of 10g/L resin of total protein of the loaded sample; after loading, the column was equilibrated with 10% acetonitrile for 2 CVs; then using RP-A3 mobile phase equilibrium chromatographic column for 3 CV; and finally, performing linear gradient elution (RP-B is from 55 to 75 percent and eluted for 90 minutes) by using RP-A3 and RP-B, and obtaining a collected main peak section which is the purified sample of the semaglutin. By UPLC detection, the purity of a purified sample is 99.25% (the impurity content of the front impurity with the peak time of 10.506min, namely RRT=0.739 is 0.20 percent, the impurity content of the isomer with the peak time of 13.801min, namely RRT=0.971 is 0.06 percent, the impurity content is far less than 0.2 percent, the impurity content of the acetylated impurity with the peak time of 15.987min, namely RRT=1.125 is 0.03 percent, and the maximum single impurity content is 0.20 percent), and the purification yield is 63.7 percent. The UPLC spectrum is shown in FIG. 3.

Second purification: taking the diluted semaglutin purified sample as a sample loading sample, taking octadecylsilane chemically bonded silica filler of HPLCONE 8C18K type as a stationary phase, wherein the flow rate is 200cm/hr, and the detection wavelength is 280nm. 2 CVs in the column were equilibrated with RP-A4 mobile phase; loading according to the loading amount of 10g/L resin of total protein of the loaded sample; after loading, the column was equilibrated with RP-A4 mobile phase for 3 CVs; and finally, performing linear gradient elution by using RP-A4 and RP-B (the RP-B is eluted for 45min from 38% -48%), and collecting a main peak section to obtain the semaglutin purified two samples. The purity of the purified two samples was 99.84% (the maximum single impurity was the isomer impurity with peak time 13.998min, i.e. rrt=0.967 impurity content was 0.04%; where the acetylated impurity had been completely removed; the pre-impurity content was significantly reduced) and the purification yield was 75.6%. The UPLC spectrum is shown in FIG. 4.

Example 4: purification of crude peptide of semaglutin

First purification: taking the dissolved crude peptide of the semaglutin as a sample, taking octadecylsilane chemically bonded silica filler of BR-C18 (2) type as a stationary phase, wherein the flow rate is 200cm/hr, and the detection wavelength is 280nm. The column was equilibrated with a solution containing 50mM Tris and 10% acetonitrile for 2 CV; loading according to the loading amount of 10g/L resin of total protein of the loaded sample; after loading, the column was equilibrated with 10% acetonitrile for 2 CVs; then using RP-A5 mobile phase equilibrium chromatographic column for 3 CV; and finally, performing linear gradient elution (RP-B is from 55 to 75 percent and eluted for 90 minutes) by using RP-A5 and RP-B, and obtaining a collected main peak section which is the purified sample of the semaglutin. The UPLC test shows that the purity of a purified sample is 99.20% (the impurity content of the impurity with the peak time of 10.494min, namely rrt=0.737 is 0.12%, the impurity content of the impurity with the peak time of 11.772min, namely rrt=0.826 is 0.19%, the impurity content of the isomer with the peak time of 13.717min, namely rrt=0.963 is 0.08%, the impurity content of the impurity is far less than 0.2%, the impurity content of the acetylated impurity with the peak time of 17.347min, namely rrt=1.218 is 0.14%, and the maximum impurity content of the impurity is 0.19%) and the purification yield is 64.1%. The UPLC spectrum is shown in FIG. 5.

Second purification: taking the diluted semaglutin purified sample as a sample loading sample, taking octadecylsilane chemically bonded silica filler of HPLCONE 8C18K type as a stationary phase, wherein the flow rate is 200cm/hr, and the detection wavelength is 280nm. 2 CVs in the column were equilibrated with RP-A6 mobile phase; loading according to the loading amount of 10g/L resin of total protein of the loaded sample; after loading, the column was equilibrated with RP-A6 mobile phase for 3 CVs; and finally, performing linear gradient elution by using RP-A6 and RP-B (the RP-B is eluted for 45min from 38% -48%), and collecting a main peak section to obtain the semaglutin purified two samples. The purity of the purified two samples was 99.86% (the maximum single impurity was the isomer impurity with peak time 13.940min, i.e. rrt=0.965 impurity content 0.05%, wherein the acetylated impurity had been completely removed, the pre-impurity content was significantly reduced) and the purification yield was 76.2% by UPLC detection. The UPLC spectrum is shown in FIG. 6.

Example 5: purification of crude peptide of semaglutin

First purification: taking the dissolved crude peptide of the semaglutin as a sample, taking octadecylsilane chemically bonded silica filler of BR-C18 (2) type as a stationary phase, wherein the flow rate is 200cm/hr, and the detection wavelength is 280nm. The column was equilibrated with a solution containing 50mM Tris and 10% acetonitrile for 2 CV; loading according to the loading amount of 10g/L resin of total protein of the loaded sample; after loading, the column was equilibrated with 10% acetonitrile for 2 CVs; then using RP-A7 mobile phase equilibrium chromatographic column for 3 CV; and finally, performing linear gradient elution (RP-B is from 55 to 75 percent and eluted for 90 minutes) by using RP-A7 and RP-B, and obtaining a collected main peak section which is the purified sample of the semaglutin. The UPLC test shows that the purity of the purified sample is 99.00% (the impurity content of the impurity with the peak time of 10.514min, namely RRT= 0.743 is 0.12%, the maximum impurity is the isomer impurity with the peak time of 13.775min, namely RRT=0.973, the impurity content is 0.14% and less than 0.2%) and the purification yield is 63.7%. The UPLC spectrum is shown in FIG. 7.

Second purification: taking the diluted semaglutin purified sample as a sample loading sample, taking octadecylsilane chemically bonded silica filler of HPLCONE 8C18K type as a stationary phase, wherein the flow rate is 200cm/hr, and the detection wavelength is 280nm. 2 CVs in the column were equilibrated with RP-A8 mobile phase; loading according to the loading amount of 10g/L resin of total protein of the loaded sample; after loading, the column was equilibrated with RP-A8 mobile phase for 3 CVs; and finally, performing linear gradient elution by using RP-A8 and RP-B (the RP-B is eluted for 45min from 38% -48%), and collecting a main peak section to obtain the semaglutin purified two samples. Through UPLC detection, the purity of the purified two samples is 99.82 percent (the maximum single impurity is the isomer impurity with peak-off time of 12.286min, namely RRT=0.966 impurity content is 0.05 percent, the pre-impurity content is obviously reduced), and the purification yield is 74.4 percent. The UPLC spectrum is shown in FIG. 8.

Example 6: purification of crude peptide of semaglutin

The YMC-C18 filler was used in place of the octadecyl silane chemically bonded silica filler of BR-C18 (2) type used in the first purification in example 2 and the octadecyl silane chemically bonded silica filler of HPLCONE 8C18K type used in the second purification, and the other conditions and operation steps were the same as in example 2, and the purity of the sample after the second purification was 99.82% (the impurity content of the largest single impurity was 0.04%, the pre-impurity content was significantly reduced), and the purification yield was 75.1%. The UPLC spectrum is shown in FIG. 9.

Example 7: spin steaming and freeze-drying of two purified samples of semaglutin

And (3) placing the second sample of the semaglutin purification obtained in the example 2 into a rotary evaporator to remove acetonitrile, then placing the second sample into a minus 80 ℃ for prefreezing, and then placing the second sample into a freeze dryer for freeze drying after prefreezing, wherein the finally obtained freeze-dried sample is the semaglutin refined peptide sample. Through UPLC detection, the purity of the semaglutin refined peptide is 99.86 percent (the maximum single impurity is 0.04 percent), the UPLC spectrum is shown as figure 10, and the result shows that the product is very stable under the conditions of rotary evaporation and freeze drying, and the purity and the impurity content are hardly affected.

Comparative example 1

Referring to example 2, 30mM ammonium acetate was replaced with 30mM ammonium formate in mobile phase RP-A1, and the other conditions and steps were the same as in the first purification of example 2, and the effect of buffer salts in mobile phase on the purity, impurities, yield, etc. of the semaglutin was examined. As a result, as shown in fig. 11, the purity of the purified sample was 98.62%, and the impurity content of the isomer having a peak time of 12.956min, i.e., rrt=0.963 impurity content was 0.28%; the acetylated impurity with the peak time of 15.017min, namely RRT=1.117, has the impurity content of 0.29 percent and the maximum single impurity content of 0.44 percent, which shows that the ammonium acetate buffer salt has better purification effect on the crude peptide of the semaglutin than the ammonium formate buffer salt, and can effectively reduce the impurity content of the impurity which is difficult to remove, especially the impurity content of the isomer with the relative retention time of RRT of 0.954-RRT of 0.973, so that the impurity content is stably controlled to be less than or equal to 0.06 percent after the second purification step.

Comparative example 2

First purification: taking the dissolved crude peptide of the semaglutin as a sample, taking octadecylsilane chemically bonded silica filler of BR-C18 (2) type as a stationary phase, wherein the flow rate is 200cm/hr, and the detection wavelength is 280nm. The column was equilibrated with a solution containing 50mM Tris and 10% acetonitrile for 2 CV; loading according to the loading amount of 10g/L resin of total protein of the loaded sample; after loading, the column was equilibrated with 10% acetonitrile for 2 CVs; and then mobile phase RP-A:0.2% phosphoric acid +10% acetonitrile, 10% ammonia water to adjust ph2.5, equilibrate column 3 CVs; and finally, carrying out linear gradient elution (RP-B is eluted for 90min from 55% -75%) by using a mobile phase RP-A and a mobile phase RP-B (60% acetonitrile), and obtaining a collected main peak section which is the purified sample of the semaglutin. The purity of a purified sample was 98.19% (the maximum single impurity was an isomer impurity with a peak time of 14.021min, i.e., rrt=0.954 impurity content of 0.63%, and the peak time was 16.334min, i.e., rrt=1.112 impurity content of 0.19%) as determined by UPLC detection, and the purification yield was 76.8%. The UPLC profile is shown in fig. 12.

Second purification: taking the diluted semaglutin purified sample as a sample loading sample, taking octadecylsilane chemically bonded silica filler of HPLCONE 8C18K type as a stationary phase, wherein the flow rate is 200cm/hr, and the detection wavelength is 280nm. With mobile phase C:20mM ammonium acetate+10% acetonitrile, pH was adjusted to 8.0 with aqueous ammonia, and the column was equilibrated for 2 CV; loading according to the loading amount of 10g/Lresin of total protein of the loaded sample; after loading, the column was equilibrated for 3 CVs with mobile phase C; and finally, carrying out linear gradient elution (RP-B is eluted for 45min from 38% -48%) by using a mobile phase C and a mobile phase RP-B (60% acetonitrile), and obtaining a collected main peak section which is the second sample of the semaglutin purification. Through UPLC detection, the purity of the purified two samples is 98.96 percent (the impurity content of the front impurity with the peak time of 10.705min is 0.28 percent, namely RRT=0.733, the impurity content of the front impurity is higher, the impurity content of the isomer with the peak time of 13.997min is 0.19 percent, the impurity content of the acetylated impurity with the peak time of 16.342min is 0.16 percent, the impurity content of the acetylated impurity is not obviously changed from that of the purified one sample), the maximum single impurity content is 0.28 percent, and the purification yield is 75.4 percent. The UPLC spectrum is shown in FIG. 13. The purity of the semaglutin sample obtained by the purification method in the comparative example is obviously lower than that of the semaglutin sample obtained in the example 2, and the isomer, the acetylated impurity and the unknown pre-impurity are obviously increased.

Claims

1. A purification method of semaglutin is characterized by purifying by a two-step reversed-phase high performance liquid chromatography method, which is characterized in that: the method comprises the following steps:

2. The purification method according to claim 1, wherein: the acid buffer solution containing acetate and ion pairing agent is selected from any one of ammonium acetate-trifluoroacetic acid buffer solution, sodium acetate-trifluoroacetic acid buffer solution and potassium acetate-trifluoroacetic acid buffer solution.

3. The purification method according to claim 2, characterized in that: the mass concentration of the trifluoroacetic acid is 0.001% -1%; the acetate concentration is 0.1-500mM.

4. The purification method according to claim 2, characterized in that: the pH value of the acid buffer solution containing acetate is less than 5.0.

5. The purification method according to claim 1, wherein: in the mobile phase used in the first purification step, a mixed solution of an acid buffer solution containing acetate and an ion pairing agent and acetonitrile is taken as a mobile phase RP-A1, and an aqueous solution containing acetonitrile is taken as a mobile phase RP-B for linear gradient elution.

6. The purification method according to claim 1, wherein: the alkaline buffer solution containing phosphate is selected from any one of sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate.

7. The purification method according to claim 6, wherein: the concentration of phosphate in the alkaline buffer solution is 0.1-500mM; the pH of the alkaline buffer solution containing phosphate is more than 7.0.

8. The purification method according to claim 7, wherein: in the second step of purification, the mixed solution of alkaline buffer solution containing phosphate and acetonitrile is used as mobile phase RP-A2, and the aqueous solution containing acetonitrile is used as mobile phase RP-B.

9. The purification method according to claim 1, wherein: the reversed phase filler used in the first purification and the second purification is selected from a stationary phase made of porous silica particles or silica gel.

10. The purification method according to claim 1, wherein: the crude peptide of semaglutin is dissolved by using a mixed solvent composed of an alkaline solution containing tris and an organic solvent before the first purification step.

11. The purification method according to any one of claims 1 to 10, characterized in that: purification by crude peptide pretreatment and two-step reverse phase high performance liquid chromatography comprising the steps of:

(1) Pretreatment of crude peptide: dissolving the crude peptide of the semaglutin by using an alkaline solution containing tris and acetonitrile;