CN117603338B

CN117603338B - A purification method for smegglutinin

Info

Publication number: CN117603338B
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: 2025-11-25
Anticipated expiration: 2043-05-19
Also published as: CN117603338A

Abstract

This invention belongs to the field of pharmaceutical technology and discloses a purification method for semagrapeptide. The invention purifies crude semagrapeptide using a two-step reversed-phase chromatography method, and finally, after lyophilization, obtains refined semagrapeptide with a purity ≥99.80% and a maximum single impurity ≤0.06%. The first step of purification using an acidic system containing acetate significantly reduces the content of isomer impurities; the second step, using an alkaline system containing phosphate, effectively removes or reduces acetylated impurities and some unknown precursor impurities, keeping the single impurity level controlled at ≤0.06%. The purification process of this invention eliminates the need for salt conversion or desalting, making it simpler to operate, reducing the production cycle, and facilitating industrial production.

Description

Semex lattice method for purifying lupeptide

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 mellitus can be divided into two points, insulin resistance and insulin secretion deficiency. 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 Nor and Norde company through a gene recombination technology, has 94 percent of homology with GLP-1, and is structurally modified compared with the GLP-1 analogue by 1) replacing Ala8 on a peptide chain with Aib8 to cover a hydrolysis site of dipeptidyl peptidase IV and prevent the degradation by enzyme, 2) attaching a modified fatty chain to Lys26, wherein the fatty chain comprises 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 as to reduce kidney clearance rate, and 3) replacing Lys34 with Arg34. Modification at 3 positions makes the half-life of semaglutin as long as 165h.

At present, the preparation method of the semaglutin mainly comprises the steps of firstly obtaining Arg34GLP-1 (9-37) peptide chain by a biological recombination method, then adding the non-natural amino acid dipeptide and a side chain by a chemical synthesis method, secondly, directly gradually adding amino acid by a chemical method to synthesize a straight peptide chain, and then adding the 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, a semaglutin sample with the purity of more than 99 percent and the maximum single impurity of less than 0.2 percent can be obtained through three times of reversed phase purification and one-time film evaporation, the purification steps are more, wherein the reversed phase purification needs to use a plurality of stationary phases and needs to newly input film evaporation equipment to increase the equipment cost, the eluting mobile phase in the patent CN 105777872B adopts two organic solvents of acetonitrile and isopropanol to increase the risk of organic solvent residue in the sample, the semaglutin sample with the purity of 99.38 percent and the single impurity of less than 0.15 percent is obtained through one-step salt conversion process after two-step reversed phase purification, the semaglutin sample with the purity of more than 99 percent is obtained through two-step reversed phase purification in the patent CN 111848777A after 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 longer, and the industrial scale-up 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 of purification, namely, an acid buffer solution containing acetate and an ion pairing agent and an organic solvent are used as mobile phases, and a reverse phase filler is used as a stationary phase;

(2) And the second step of purification, namely taking an alkaline buffer solution containing phosphate and an organic solvent as mobile phases and taking a reverse phase filler 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 liquid to prepare other buffers (such as formate buffer liquid), the buffer capacity is better, the buffer capacity can be kept when the buffer liquid is matched with strong acid TFA, and the buffer capacity of the system can be destroyed when the formate buffer liquid is matched with TFA.

As one 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% -1%, optionally 0.025% -0.1%, such as further optionally 0.05%, and 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, and is preferably adjusted with acetic acid, since it does not introduce new anions, does not destroy 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 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%, optionally 55%, and the termination gradient of the mobile phase RP-B is 70% -80%, optionally 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 an embodiment of the invention, the buffer salt concentration is 0.1-500mM, optionally 10-50mM, such as further optionally 20mM, the pH of the phosphate-containing alkaline buffer is >7.0, optionally 7.5-8.5, such as further optionally 8.0, and the pH is preferably adjusted with 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 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% and the termination gradient of the mobile phase RP-B is 41% -53%, such as 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 application, 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 particularly, such as an octaalkyl-bonded silica gel or an octadecyl-bonded silica gel, such as one or more types selected from Sepax BR-C18、Sepax GP-C18、Unisil 15-100 C18、Sepax Bio-C8(2)、BR-C18(2)、HPLCONE 8C18-100AA、HPLCONE 8C18K、HPLCONE 8C8K、YMC-C18 or YMC-C8, more particularly, such as one of the first purification steps, preferably BR-C18 (2) or YMC-C18 packing, and one of the second purification steps, preferably HPLCONE C18K or YMC-C18 packing.

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 crude peptide of the semaglutin 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 alkaline solution containing Tris has pH of 7.5 or more, such as 8.0-9.0, such as 8.5, and preferably has pH adjusted by hydrochloric acid.

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) Crude peptide pretreatment, namely dissolving the crude peptide of the semaglutin by using an alkaline solution containing Tris (Tris (hydroxymethyl aminomethane) and acetonitrile;

(2) The first step of purification, namely taking octadecylsilane chemically bonded silica filler as a stationary phase, taking buffer salt solution containing ammonium acetate-TFA and acetonitrile as a mobile phase RP-A1, taking acetonitrile solution as a mobile phase RP-B, carrying out linear gradient elution, and collecting main peak section components;

(3) And purifying by taking octadecylsilane chemically bonded silica filler as a stationary phase, taking buffer salt solution containing phosphate and acetonitrile as a mobile phase RP-A2 phase and acetonitrile solution as a mobile phase RP-B, performing linear gradient elution, and collecting main peak components.

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 is carried out by using the mobile phase RP-A1 and the mobile phase RP-B, a solvent for pretreatment of crude peptide, 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 the mobile phase system in the chromatographic column consistent with that of the sample, the acetonitrile with the concentration of 5-15% is used for balancing the chromatographic column to remove the salt introduced in the last step, and finally the mobile phase RP-A1 is used for balancing the chromatographic column.

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) According to the invention, the content of the isomer impurities can be greatly reduced by adopting an acid system containing acetate for purification in the first step (the relative retention time RRT is 0.954-RRT is 0.973 is less than or equal to 0.2%), so that the content of the isomer impurities is controlled to be less than or equal to 0.06% after the second step of purification, and the content of the isomer impurities is difficult to be controlled to be less than or equal to 0.2% by adopting other acid systems containing formate, so that the content of the isomer impurities is difficult to be controlled to be less than or equal to 0.06% after the second step of purification.

(3) In the second step, an alkaline system of phosphate is adopted for purification, so that acetylated impurities (RRT 1.107-RRT 1.218) and partial unknown pre-impurities (RRT 0.630-RRT 0.840) can be effectively removed or reduced, and 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, pH 2.5 was adjusted with acetic acid.

The RP-A2 mobile phase was formulated as 20mM dipotassium hydrogen phosphate, 10% acetonitrile, and pH was adjusted to 8.0 with phosphoric acid.

RP-A3 mobile phase formulation was 0.05% TFA,10% acetonitrile, 30mM ammonium acetate, and pH was adjusted to 2.5 with acetic acid.

The RP-A4 mobile phase was formulated as 20mM disodium hydrogen phosphate, 10% acetonitrile, and pH was adjusted to 8.0 with phosphoric acid.

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

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

RP-A7 mobile phase was formulated with 0.05% TFA,10% acetonitrile, 30mM ammonium acetate, and pH adjusted to 3.0 with acetic acid.

The RP-A8 mobile phase was formulated as 20mM disodium hydrogen phosphate, 10% acetonitrile, and pH was adjusted to 8.5 with phosphoric acid.

The RP-B mobile phase formula is 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 Semiglutide peptide

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

And (3) the first purification, namely 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 method comprises the steps of balancing 2 CVs of a chromatographic column by using a solution containing 50mM Tris and 10% acetonitrile, loading according to the loading amount of 10g/L resin of total protein of a loaded sample, balancing 2 CVs of the chromatographic column by using 10% acetonitrile after loading, balancing 3 CVs of the chromatographic column by using RP-A1 mobile phase, and finally carrying out linear gradient elution (55% -75% of RP-B and 90min of elution) by using RP-A1 and RP-B, wherein the collected main peak section is the semaglutinin purified sample. 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 is 0.15%, the impurity content of the front impurity with the peak time of 11.666min is 0.15%, the impurity content of the isomer with the peak time of 13.560min is 0.833, the impurity content of the isomer with the peak time of 13.560min is 0.08%, the impurity content of the impurity of RRT=0.969 is far less than 0.2%, the impurity content of the acetylated impurity with the peak time of 15.788min is 0.11%, the impurity content of RRT=1.128 is 0.15% of the maximum single impurity), and the purification yield is 61.2%. The UPLC spectrum is shown in FIG. 1.

Sample treatment, namely adding one volume of purified water into the obtained semaglutin purified sample to dilute, and adjusting the pH value to 8.0 by using 10% ammonia water to be purified.

And purifying for the second time, namely taking a diluted semaglutin purified sample as a sample loading sample, taking HPLCONE C18K octadecylsilane chemically bonded silica filler as a stationary phase, wherein the flow rate is 200cm/hr, and the detection wavelength is 280nm. Loading the sample according to the loading amount of 10g/L resin of total protein of the sample by using 2 CV of RP-A2 mobile phase equilibrium chromatographic column, loading the sample by using 3 CV of RP-A2 mobile phase equilibrium chromatographic column, and finally performing linear gradient elution (from 38% -48% of RP-B for 45 min) by using RP-A2 and RP-B, wherein the collected main peak section is the two samples of the semaglutinin purification. Through UPLC detection, the purity of the purified two samples is 99.82 percent (the maximum single impurity is isomer impurity with peak time of 12.908min, namely RRT=0.964 impurity content is 0.06 percent, the peak time is acetylated impurity with 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 Semiglutide peptide

And (3) the first purification, namely 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 method comprises the steps of balancing 2 CVs of a chromatographic column by using a solution containing 50mM Tris and 10% acetonitrile, loading according to the loading amount of 10g/L resin of total protein of a loaded sample, balancing 2 CVs of the chromatographic column by using 10% acetonitrile after loading, balancing 3 CVs of the chromatographic column by using RP-A3 mobile phase, and finally carrying out linear gradient elution (55% -75% of RP-B and 90min of elution) by using RP-A3 and RP-B, wherein the collected main peak section is the semaglutinin purified sample. By UPLC detection, the purity of a purified sample is 99.25% (the impurity content of front impurity with peak time of 10.506min, namely RRT=0.739 is 0.20%, the impurity content of isomer with peak time of 13.801min, namely RRT=0.971 is 0.06%, the impurity content is far less than 0.2%, the impurity content of acetylated impurity with peak time of 15.987min, namely RRT=1.125 is 0.03%, and the maximum single impurity is 0.20%), and the purification yield is 63.7%. The UPLC spectrum is shown in FIG. 3.

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

EXAMPLE 4 purification of crude Semiglutide peptide

And (3) the first purification, namely 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 method comprises the steps of balancing 2 CVs of a chromatographic column by using a solution containing 50mM Tris and 10% acetonitrile, loading according to the loading amount of 10g/L resin of total protein of a loaded sample, balancing 2 CVs of the chromatographic column by using 10% acetonitrile after loading, balancing 3 CVs of the chromatographic column by using RP-A5 mobile phase, and finally carrying out linear gradient elution (55% -75% of RP-B and 90min of elution) by using RP-A5 and RP-B, wherein the collected main peak section is the semaglutinin purified sample. The UPLC test shows that the purity of the purified sample is 99.20% (the impurity content of the impurity with the peak time of 10.494min is 0.12%, the impurity content of the impurity with the peak time of 11.772min is 0.826, the impurity content of the impurity with the peak time of 13.717min is 0.19%, the impurity content of the isomer with the peak time of 13.717min is 0.08%, the impurity content of the impurity with the peak time of 17.347min is far less than 0.2%, the impurity content of the acetylated impurity with the peak time of 17.347min is 0.14%, the impurity content of the impurity with the peak time of RRT=1.218 is 0.19%, and the maximum single impurity content is 0.19%) and the purification yield is 64.1%. The UPLC spectrum is shown in FIG. 5.

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

EXAMPLE 5 purification of crude Semiglutide peptide

And (3) the first purification, namely 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 method comprises the steps of balancing 2 CVs of a chromatographic column by using a solution containing 50mM Tris and 10% acetonitrile, loading according to the loading amount of 10g/L resin of total protein of a loaded sample, balancing 2 CVs of the chromatographic column by using 10% acetonitrile after loading, balancing 3 CVs of the chromatographic column by using RP-A7 mobile phase, and finally carrying out linear gradient elution (55% -75% of RP-B and 90min of elution) by using RP-A7 and RP-B, wherein the collected main peak section is the semaglutinin purified sample. The UPLC test shows that the purity of the purified sample is 99.00% (the impurity content of rrt= 0.743 is 0.12% before the peak time is 10.514min, and the maximum impurity is the isomer impurity with the peak time of 13.775min, the impurity content of rrt=0.973 is 0.14% and less than 0.2%) and the purification yield is 63.7%. The UPLC spectrum is shown in FIG. 7.

And purifying for the second time, namely taking a diluted semaglutin purified sample as a sample loading sample, taking HPLCONE C18K octadecylsilane chemically bonded silica filler as a stationary phase, wherein the flow rate is 200cm/hr, and the detection wavelength is 280nm. Loading the sample according to the loading amount of 10g/L resin of total protein of the sample by using 2 CV of RP-A8 mobile phase equilibrium chromatographic column, loading the sample by using 3 CV of RP-A8 mobile phase equilibrium chromatographic column, and finally performing linear gradient elution (from 38% -48% of RP-B for 45 min) by using RP-A8 and RP-B, wherein the collected main peak section is the two samples of the semaglutinin purification. Through UPLC detection, the purity of the purified two samples is 99.82 percent (the maximum single impurity is 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 Semiglutide peptide

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 HPLCONE C18K type used in the second purification, and the other conditions and operation steps were the same as in example 2, except that the purity of the sample after the second purification was 99.82% (the maximum impurity content of single impurity was 0.04%, the previous 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 lyophilization of two samples of semaglutin purification

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 final 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 shown in FIG. 11, the purity of the purified sample is 98.62%, the impurity content of the isomer with the peak time of 12.956min, namely RRT=0.963 impurity content is 0.28%, the impurity content of the acetylated impurity with the peak time of 15.017min, namely RRT=1.117 impurity content is 0.29%, and the maximum single impurity is 0.44%, 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 the impurity content of the isomer with the impurity which is difficult to remove, particularly the impurity content of the isomer with the relative retention time of RRT 0.954-RRT 0.973, can be effectively reduced, so that the impurity content after the second purification step is stably controlled to be less than or equal to 0.06%.

Comparative example 2

And (3) the first purification, namely 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 method comprises the steps of balancing 2 CVs of a chromatographic column by using a solution containing 50mM Tris and 10% acetonitrile, loading according to the loading amount of 10g/L resin of total protein of a loaded sample, balancing 2 CVs of the chromatographic column by using 10% acetonitrile after loading, adjusting the pH value to 2.5 by using mobile phase RP-A, 0.2% phosphoric acid and 10% acetonitrile and 10% ammonia water, balancing 3 CVs of the chromatographic column, and finally performing linear gradient elution (the RP-B is eluted for 90min from 55% -75% by using mobile phase RP-A and mobile phase RP-B (60% acetonitrile)), wherein the collected main peak section is the semaglutinin purified sample. The purity of a purified sample was 98.19% (the maximum single impurity was isomer impurity with peak time 14.021min, i.e., rrt=0.954 impurity content was 0.63%, and the peak time 16.334min was acetylated impurity, i.e., rrt=1.112 impurity content was 0.19%) by UPLC detection, and the purification yield was 76.8%. The UPLC profile is shown in fig. 12.

And purifying for the second time, namely taking a diluted semaglutin purified sample as a sample loading sample, taking HPLCONE C18K octadecylsilane chemically bonded silica filler as a stationary phase, wherein the flow rate is 200cm/hr, and the detection wavelength is 280nm. Regulating pH to 8.0 with mobile phase C, ammonium acetate and 10% acetonitrile, balancing chromatographic column for 2 CV, loading according to loading amount of total protein 10g/Lresin, balancing chromatographic column for 3 CV with mobile phase C, and linear gradient eluting with mobile phase C and mobile phase RP-B (60% acetonitrile) (RP-B is from 38% -48%, eluting for 45 min), collecting main peak to obtain semaglutinin purified two samples. Through UPLC detection, the purity of the purified two samples is 98.96 percent (the impurity content before the peak time is 10.705min, namely RRT=0.733 is 0.28 percent, the impurity content before the peak time is higher, the impurity content after the peak time is 13.997min is 0.19 percent, the impurity content after the peak time is 16.342min, namely RRT= 1.119 is 0.16 percent, the impurity content after the impurity is acetylated 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. The purification method of the semaglutin is characterized by comprising the following steps of:

(1) Before the first purification step, a mixed solvent consisting of alkaline solution containing Tris and organic solvent is used for dissolving the crude peptide of the semaglutin, wherein the mixed solvent is a mixed solution containing Tris aqueous solution and acetonitrile;

(2) The first step of purification, namely, an acid buffer solution containing acetate and an ion pairing agent and an organic solvent are taken as mobile phases, a reversed-phase filler is taken as a stationary phase, and a mixed solution containing 30 mM sodium acetate or ammonium acetate, 0.05% -0.10% of an ion pairing agent trifluoroacetic acid and 10% of acetonitrile with the pH value of 2.5-3.0 is taken as a mobile phase A phase, and an aqueous solution containing 60% of acetonitrile is taken as a mobile phase B phase, so that linear gradient elution is carried out;

(3) The second step of purification, namely taking an alkaline buffer solution containing phosphate and an organic solvent as a mobile phase and a reversed-phase filler as a stationary phase, wherein the mobile phase takes a mixed solution containing 20-50 mM of dipotassium hydrogen phosphate or disodium hydrogen phosphate and 10% of acetonitrile with the pH value of 8.0-8.5 as a mobile phase A phase and takes an aqueous solution containing 60% of acetonitrile as a mobile phase B phase.

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

3. The method according to claim 1, wherein the crude peptide is purified by pretreatment and two-step reverse-phase high performance liquid chromatography, comprising the steps of:

(1) Crude peptide pretreatment, namely dissolving the crude peptide of the semaglutin by using an alkaline solution containing tris and acetonitrile;

(2) The first step of purification, namely, octadecylsilane chemically bonded silica filler is used as a stationary phase, buffer salt solution with pH of 2.5-3.0 and containing 30 mM sodium acetate or ammonium acetate, 0.05% -0.10% TFA and 10% acetonitrile is used as a mobile phase A phase, 60% acetonitrile solution is used as a mobile phase B phase, linear gradient elution is carried out, and main peak section components are collected;

(3) And the second step of purification, namely taking octadecylsilane chemically bonded silica filler as a stationary phase, taking buffer salt solution containing 20-50 mM dipotassium hydrogen phosphate or disodium hydrogen phosphate and 10% acetonitrile with the pH value of 8.0-8.5 as a mobile phase A phase, taking 60% acetonitrile solution as a mobile phase B phase, carrying out linear gradient elution, and collecting main peak section components.