A method of purifying semaglutide

Abstract

The application belongs to the technical field of medicine, and discloses a purification method of semaglutide. The crude semaglutide peptide is purified by two-step reverse phase chromatography, and finally, the semaglutide fine peptide with purity ≥ 99.80% and maximum single impurity ≤ 0.06% is obtained by freeze-drying. The purification of the first step in the acid system containing acetate can greatly reduce the content of isomer impurities; the purification of the second step in the alkaline system containing phosphate can effectively remove or reduce acetylation impurities and part of unknown front impurities, so that the single impurity is controlled to be ≤ 0.06%. The purification process of the application does not need the step of salt conversion or desalination, is more convenient in operation, reduces the production cycle, and is beneficial to industrial production.

Description

Technical Field

[0001] This invention relates to a method for purifying polypeptides, specifically to a method for purifying crude smegglutinin peptide, belonging to the field of pharmaceutical technology. Background Technology

[0002] Diabetes mellitus is an endocrine disorder characterized by hyperglycemia, which can lead to kidney damage, retinopathy, and cardiovascular disease through various factors. Based on different pathogenesis mechanisms, diabetes is divided into type 1 and type 2 diabetes, with type 2 accounting for over 90% of all diabetes cases. The pathogenesis of type 2 diabetes can be divided into two aspects: insulin resistance and insulin secretion defects. Most cases of type 2 diabetes are hyperglycemic disorders arising from insulin resistance. As blood glucose levels increase, pancreatic β-cells produce glucotoxicity, which impairs glucose oxidation and glucose signaling, ultimately leading to insulin secretion defects and persistently elevated blood glucose levels. Therefore, current clinical treatment for type 2 diabetes primarily involves oral hypoglycemic agents or long-acting insulin injections. With the excellent efficacy of human glucagon-1 (GLP-1) and its analogues in treating type 2 diabetes, they are occupying an increasingly important position in the field of novel drugs for diabetes treatment. GLP-1 is a gastrointestinal hormone containing 37 amino acid residues. It can bind to the pancreatic GLP-1 receptor, activating it in a glucose concentration-dependent mode, thereby effectively stimulating pancreatic β-cells, promoting insulin secretion, restoring damaged pancreatic β-cell function, and inhibiting glucagon synthesis and release, ultimately lowering blood sugar. However, natural GLP-1 is rapidly hydrolyzed and inactivated by the plasma enzyme dipeptidyl peptidase IV (DPP-IV), with a half-life of about 2 minutes, thus increasing the difficulty of its clinical application. Therefore, modifying the structure of GLP-1 to retain the same pharmacological activity while prolonging its half-life is a key research focus for novel GLP-1 analogues.

[0003] Currently, GLP-1 analogs approved by the FDA for the treatment of type 2 diabetes include exenatide injection, liraglutide injection, semaglutide injection, and semaglutide tablets. Semaglutide is a GLP-1 analog produced by Novo Nordisk using recombinant gene technology. It shares 94% homology with GLP-1, but its structure has the following modifications: 1) Ala8 on the peptide chain is replaced with Aib8, which masks the dipeptidyl peptidase IV hydrolysis site, preventing enzymatic degradation; 2) A modified fatty chain is attached to Lys26, consisting of two 8-amino-3,6-dioxanoic acid structural units, one glutamate structure, and octadecanoic acid. The resulting long fatty chain can bind tightly to albumin, thereby reducing renal clearance; 3) Lys34 is replaced with Arg34. These three modifications extend the half-life of semaglutide to 165 hours.

[0004] Currently, there are two main categories of methods for preparing smegglutinin: 1. Obtaining the Arg34GLP-1(9-37) peptide chain through biological recombination, followed by chemical synthesis to add a dipeptide containing non-natural amino acids and a side chain; 2. Directly synthesizing a straight peptide chain by gradually adding amino acids through chemical methods, followed by the addition of side chains. Because chemical synthesis easily generates many impurities, such as side reaction impurities, isomer impurities, racemic impurities, and impurities caused by amino acid deletions or additions, the purification of crude smegglutinin is mostly performed using reversed-phase chromatography to effectively remove these impurities. Patent CN 110845602A requires three reverse-phase purification steps and one membrane evaporation step to obtain a smegglutinin sample with a purity greater than 99% and a maximum single impurity of less than 0.2%. This involves numerous purification steps, with reverse-phase purification requiring multiple stationary phases and necessitating the investment in new membrane evaporation equipment, increasing equipment costs. Patent CN 105777872B uses acetonitrile and isopropanol as the elution mobile phase, increasing the risk of residual organic solvents in the sample. After two reverse-phase purification steps, a salt conversion process is required to obtain a smegglutinin sample with a purity of 99.38% and a single impurity of less than 0.15%. Patent CN 111848777A involves two reverse-phase purification steps, followed by desalting, concentration, and lyophilization to obtain a smegglutinin sample with a purity greater than 99%. However, it does not mention the maximum single impurity level, and the desalting process uses an ultrafiltration membrane, resulting in a long operation time, which is not conducive to industrial-scale production.

[0005] Given the problems with existing technologies, it is urgent to find a purification method that can reduce the complexity of the process, shorten the cycle, and facilitate industrial production, while ensuring the purity, yield, and maximum single impurity content of smegglutinin. Summary of the Invention

[0006] The purpose of this invention is to provide a purification method for smegglutinin. In this invention, crude smegglutinin is purified using a two-step reversed-phase chromatography method, and finally, after lyophilization, refined smegglutinin with a purity ≥99.80% and a single impurity ≤0.06% is obtained.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0008] A method for purifying smegglutinin, using a two-step reversed-phase high-performance liquid chromatography method, includes the following steps:

[0009] (1) First step purification: using an acidic buffer containing acetate and ion-pairing agents and an organic solvent as the mobile phase, and a reversed-phase packing as the stationary phase;

[0010] (2) Second step of purification: use phosphate-containing alkaline buffer and organic solvent as mobile phase and reversed phase packing as stationary phase.

[0011] In one 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, and potassium acetate-trifluoroacetic acid buffer.

[0012] The present invention uses acetate buffer, which has better buffering capacity than other buffers (such as formate buffer) and can maintain buffering capacity when used in combination with strong acid TFA; while when formate buffer is used in combination with TFA, TFA will destroy the buffering capacity of the system.

[0013] In one embodiment of the present invention, the mass concentration of TFA in the ammonium acetate-trifluoroacetic acid (TFA) buffer, sodium acetate-trifluoroacetic acid buffer, and potassium acetate-trifluoroacetic acid buffer is 0.001%-1%, optionally 0.025%-0.1%, and further optionally 0.05%; the concentration of acetate is 0.1-500 mM, optionally 10-50 mM, and further optionally 30 mM.

[0014] As one embodiment of the present invention, the acidic buffer solution containing acetate has a pH < 5.0, and can be selected as 2.0-3.0, or even 2.5; it is preferred to use acetic acid to adjust the pH, because it does not introduce new anions, does not destroy the buffering capacity of the system, and does not affect the concentration of the buffer solution.

[0015] In one embodiment of the present invention, in the first step of purification, a mixed solution of acidic buffer containing acetate and ion-pairing agent and acetonitrile is used as mobile phase RP-A1, and an aqueous solution containing acetonitrile is used as mobile phase RP-B for linear gradient elution. The initial gradient of mobile phase RP-B is 50%-60%, optionally 55%; the final gradient of mobile phase RP-B is 70%-80%, optionally 75%.

[0016] In one embodiment of the present invention, the phosphate-containing alkaline buffer solution is selected from any one of sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.

[0017] In one embodiment of the present invention, the concentration of the buffer salt is 0.1-500 mM, preferably 10-50 mM, and further preferably 20 mM; the pH of the phosphate-containing alkaline buffer solution is >7.0, preferably 7.5-8.5, and further preferably 8.0; it is preferable to use phosphate to adjust the pH, because it will not introduce new anions, will not destroy the buffering capacity of the system, and will not affect the concentration of the buffer solution.

[0018] In one embodiment of the present invention, in the second purification step, the mobile phase used is a mixed solution of phosphate-containing alkaline buffer and acetonitrile as mobile phase RP-A2, and an aqueous solution containing acetonitrile as mobile phase RP-B. The initial gradient of mobile phase RP-B is 30%-40%, optionally 38%; the final gradient of mobile phase RP-B is 41%-53%, optionally 48%.

[0019] In one embodiment of the present invention, the organic solvent used in the first step of purification and the second step of purification is an aqueous solution containing acetonitrile, such as an aqueous solution containing 10%-60% acetonitrile (mass concentration).

[0020] In one embodiment of the present invention, the reversed-phase packing material used in the first and second purification steps is selected from porous silica particles or a stationary phase made of silica gel. For example, the stationary phase used in this application can be made of porous silica particles having a straight-chain alkyl chain with chemical bonds of 4 to 18 carbon atoms. For example, it may contain a straight-chain alkyl chain with four (C4), eight (C8), twelve (C12), or eighteen (C18) carbon atoms, i.e., butyl, octyl, dodecyl, or octadecyl moieties. More specifically, such as octadecyl bonded silica gel or octadecyl bonded silica gel, such as one or more of 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 specifically, for the first step of purification, BR-C18(2) or YMC-C18 packing is preferred, and for the second step of purification, HPLCONE 8C18K or YMC-C18 packing is preferred.

[0021] In one embodiment of the present invention, before the first purification step, the crude semaglutide peptide is dissolved in a mixed solvent consisting of an alkaline solution containing Tris (tris(hydroxymethyl)aminomethane) and an organic solvent. The mass ratio of the mixed solvent to the crude semaglutide peptide is 30-70:1. After the dissolution step, a filtration operation can optionally be added to remove insoluble particles.

[0022] In one embodiment of the present invention, the crude smegglutide peptide is dissolved in an alkaline solution containing Tris (tris(hydroxymethyl)aminomethane) and an organic solvent before purification, more preferably in a mixed solution containing an aqueous solution of Tris and acetonitrile. The concentration of Tris is 0.1-1000 mM, preferably 10-100 mM, and further optionally 20-80 mM, 30-70 mM, or 40-60 mM. The mass concentration of acetonitrile is 5-20%, and further optionally 10%. The pH of the Tris-containing alkaline solution is ≥7.5, preferably 8.0-9.0, and further optionally 8.5; hydrochloric acid is preferably used to adjust the pH.

[0023] In one embodiment of the present invention, smegglutinin peptide is separated after purification, wherein the separation method is selected from one or a combination of lyophilization, antisolvent crystallization and isoelectric point precipitation.

[0024] As a more specific embodiment of the present invention, a purification method for smegglutinin, comprising crude peptide pretreatment and two-step reversed-phase high-performance liquid chromatography, includes the following steps:

[0025] (1) Crude peptide pretreatment: Smegglutinin crude peptide was dissolved in an alkaline solution containing Tris (tris(hydroxymethylaminomethane)) and acetonitrile;

[0026] (2) First step purification: Using octadecylsilane bonded silica gel packing as the stationary phase, a buffer salt solution containing ammonium acetate-TFA and acetonitrile as the mobile phase RP-A1, and an acetonitrile solution as the mobile phase RP-B, linear gradient elution was performed, and the main peak fraction was collected.

[0027] (3) Second step purification: Using octadecylsilane bonded silica gel as the stationary phase, a buffer salt solution containing phosphate and acetonitrile as the mobile phase RP-A2, and an acetonitrile solution as the mobile phase RP-B, linear gradient elution was performed, and the main peak components were collected.

[0028] As one embodiment of the present invention, the purified smegglutinin can be dried by freeze drying or spray drying.

[0029] In one embodiment of the present invention, before eluting with mobile phases RP-A1 and RP-B in the first step of purification, the column can be equilibrated with a solvent used during crude peptide pretreatment, such as a mixture of an alkaline solution containing Tris (tris(hydroxymethyl)aminomethane) and acetonitrile, to maintain the acidity or alkalinity of the mobile phase system in the column consistent with that of the sample; then, the column is equilibrated with acetonitrile at a concentration of 5-15% to remove the salts introduced in the previous step of column equilibration; finally, the column is equilibrated with mobile phase RP-A1.

[0030] As one embodiment of the present invention, before eluting with mobile phases RP-A2 and RP-B in the second purification step, the column can be equilibrated with mobile phase RP-A2.

[0031] In one embodiment of the present invention, the detection wavelength of the reversed-phase high-performance liquid chromatography method is 250-300 nm, preferably 280 nm.

[0032] In this invention, the solvent used for dissolution, the type and concentration of the elution mobile phase (buffer salt type, acid / base type), pH, and gradient elution were all determined through experimental screening. The optimal system was determined by comparing purity, yield, and the effect on impurity removal. The purification mobile phase system used in this invention has a better effect on impurity removal and yield than other systems in smegglutinin crude peptide.

[0033] The crude smegglutinin used in the purification method of this invention can be obtained through fermentation recombination and solid-phase synthesis (sequential coupling or fragment coupling).

[0034] The purification method for smegglutinin provided by this invention has the following advantages over existing technologies:

[0035] (1) The present invention completes the purification of crude smegglutinin peptide through two-step reversed-phase chromatography. The purity of the refined smegglutinin peptide obtained after freeze-drying is ≥99.80%, and the maximum single impurity content is ≤0.06%, which is a great improvement compared with the prior art.

[0036] (2) The first step of the present invention uses an acidic system containing acetate for purification, which can greatly reduce the content of isomer impurities (relative retention time RRT0.954~RRT0.973≤0.2%), so that the content can be controlled at ≤0.06% after the second step of purification; while using other acidic systems containing formate, it is difficult to control the content of isomer impurities at ≤0.2%, so that the content can be controlled at ≤0.06% after the second step of purification.

[0037] (3) The second step of purification using an alkaline phosphate system can effectively remove or reduce acetylated impurities (RRT1.107~RRT1.218) and some unknown precursor impurities (RRT0.630~RRT0.840), so that the single impurities are all controlled to ≤0.06%.

[0038] (4) The alkaline system used in the second step of the present invention is a phosphate system. Since smegglutinin exists in the form of phosphate, the purification process of the present invention does not require the step of salt conversion or desalting, making the operation simpler and the time cycle shorter, which is suitable for industrial scale-up production. Attached Figure Description

[0039] Figure 1This is the UPLC spectrum of the purified smegglutinin sample from Example 2.

[0040] Figure 2 These are the UPLC spectra of the two purified smegglutinin samples from Example 2.

[0041] Figure 3 This is the UPLC spectrum of the purified smegglutinin sample from Example 3.

[0042] Figure 4 These are the UPLC spectra of the two purified smegglutinin samples from Example 3.

[0043] Figure 5 This is the UPLC spectrum of the purified smegglutinin sample from Example 4.

[0044] Figure 6 These are the UPLC spectra of the two purified samples of smegglutinin from Example 4.

[0045] Figure 7 This is the UPLC spectrum of the purified smegglutinin sample from Example 5.

[0046] Figure 8 These are the UPLC spectra of the two purified samples of smegglutinin from Example 5.

[0047] Figure 9 These are the UPLC spectra of the two purified samples of smegglutinin from Example 6.

[0048] Figure 10 This is the UPLC spectrum of the smegglutinin peptide sample from Example 7.

[0049] Figure 11 This is the UPLC spectrum of the purified smegglutinin sample from Comparative Example 1.

[0050] Figure 12 This is the UPLC spectrum of the purified smegglutinin sample from Comparative Example 2.

[0051] Figure 13 This is the UPLC spectrum of two purified samples of smegglutinin in Comparative Example 2. Detailed Implementation

[0052] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail with reference to specific embodiments. However, the scope of protection of the present invention is not limited to the following embodiments.

[0053] Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available.

[0054] Both the crude semaglutide peptide and the intermediate polypeptide raw material Arg34GLP-1(9-37) peptide chain used in the crude semaglutide peptide are manufactured in-house by Nanjing Hanxin Pharmaceutical Technology Co., Ltd. The preparation process of the crude semaglutide peptide can be referred to the invention patent CN202210686496.7 that has been filed by our company, and the preparation process of the intermediate polypeptide raw material Arg34GLP-1(9-37) peptide chain can be referred to the invention patent CN202111664146.2 that has been filed by our company and has been published. All of their contents can be directly incorporated into this invention.

[0055] The mobile phase formulation used in this embodiment is as follows:

[0056] RP-A1 mobile phase formulation: 0.05% TFA, 10% acetonitrile, 30mM sodium acetate, pH adjusted to 2.5 with acetic acid.

[0057] The RP-A2 mobile phase formulation is: 20 mM dipotassium hydrogen phosphate, 10% acetonitrile, and pH adjusted to 8.0 with phosphoric acid.

[0058] The RP-A3 mobile phase formulation is: 0.05% TFA, 10% acetonitrile, 30mM ammonium acetate, with the pH adjusted to 2.5 using acetic acid.

[0059] The RP-A4 mobile phase formulation is: 20 mM disodium hydrogen phosphate, 10% acetonitrile, and pH adjusted to 8.0 with phosphoric acid.

[0060] RP-A5 mobile phase formulation: 0.10% TFA, 10% acetonitrile, 30mM ammonium acetate, pH adjusted to 2.5 with acetic acid.

[0061] The RP-A6 mobile phase formulation is: 50 mM disodium hydrogen phosphate, 10% acetonitrile, and pH adjusted to 8.0 with phosphoric acid.

[0062] The RP-A7 mobile phase formulation is: 0.05% TFA, 10% acetonitrile, 30mM ammonium acetate, with the pH adjusted to 3.0 using acetic acid.

[0063] The RP-A8 mobile phase formulation is: 20 mM disodium hydrogen phosphate, 10% acetonitrile, and pH adjusted to 8.5 with phosphoric acid.

[0064] The RP-B mobile phase formulation is: 60% acetonitrile.

[0065] Example 1:

[0066] Pretreatment of crude smegglutinin

[0067] Weigh 1g of crude smegglutinin peptide (purity approximately 74%) and add 50 times its mass of the solvent shown in Table 1 to pretreat the crude smegglutinin peptide. The results are shown in Table 1 below.

[0068] Table 1. Effects of different solvents on the solubility of crude semaglutide.

[0069]

[0070] The results showed that the crude smegglutinin peptide is difficult to dissolve due to the presence of side chain groups in its structure. It is insoluble in water, dissolves slowly in 10% acetonitrile, and forms a gel in a mixture of 1% acetic acid and 10% acetonitrile. In a mixed solvent of 50 mM sodium sulfate solution and 10% acetonitrile, it first dissolves and then precipitates as a flocculent precipitate. This invention uses a 50 mM Tris + 10% acetonitrile (pH 8.5) solution, which can improve the solubility of smegglutinin. It can rapidly dissolve the crude peptide sample within 10 minutes, which is faster than the dissolution rate of 50 mM ammonium carbonate solution. The resulting solution is clear and transparent, and remains stable after standing without reprecipitation, facilitating sample loading, avoiding column clogging, and extending column life.

[0071] Example 2: Purification of crude smegglutinin peptide

[0072] Sample preparation: Weigh 1g of crude smegglutinin peptide and add 50 times its weight of a solution containing 50mM Tris and 10% acetonitrile (pH 8.5) to dissolve the crude peptide sample for purification.

[0073] First purification: The dissolved crude smegglutinin was used as the sample, and BR-C18(2) octadecylsilane-bonded silica gel was used as the stationary phase. The flow rate was 200 cm / hr, and the detection wavelength was 280 nm. The column was equilibrated for 2 CVs with a solution containing 50 mM Tris and 10% acetonitrile. The sample was loaded with a total protein content of 10 g / L resin. After loading, the column was equilibrated for 2 CVs with 10% acetonitrile. Then, the column was equilibrated for 3 CVs with RP-A1 mobile phase. Finally, linear gradient elution was performed with RP-A1 and RP-B (RP-B from 55% to 75%, elution for 90 min). The collected main peak segment was the purified smegglutinin sample. UPLC analysis showed that the purity of the purified sample was 99.46% (the impurity at peak time 10.415 min, i.e., RRT = 0.744, impurity content 0.15%; the impurity at peak time 11.666 min, i.e., RRT = 0.833, impurity content 0.15%; the isomer impurity at peak time 13.560 min, i.e., RRT = 0.969, impurity content 0.08%, much less than 0.2%; the acetylated impurity at peak time 15.788 min, i.e., RRT = 1.128, impurity content 0.11%; the maximum single impurity was 0.15%), and the purification yield was 61.2%. The UPLC chromatogram is shown below. Figure 1 As shown.

[0074] Sample preparation: Dilute the obtained smegglutinin purified sample with an equal volume of purified water, adjust the pH to 8.0 with 10% ammonia, and wait for purification.

[0075] Second purification: The diluted semaglutide purified sample I was used as the loading sample, with HPLCONE 8C18K octadecylsilane-bonded silica gel as the stationary phase, a flow rate of 200 cm / hr, and a detection wavelength of 280 nm. The column was equilibrated with RP-A2 mobile phase for 2 CVs; the sample was loaded with a total protein content of 10 g / L resin; after loading, the column was equilibrated with RP-A2 mobile phase for 3 CVs; finally, linear gradient elution was performed with RP-A2 and RP-B (RP-B from 38% to 48%, elution for 45 min), and the collected main peak segment was the semaglutide purified sample II. UPLC analysis showed that the purity of purified sample 2 was 99.82% (the largest single impurity was an isomer with a peak time of 12.908 min, i.e., RRT = 0.964, with a content of 0.06%; the acetylated impurity with a peak time of 14.828 min, i.e., RRT = 1.107, with a content of 0.03%, significantly lower than that of purified sample 1 by an order of magnitude; the content of precursor impurities was significantly reduced), and the purification yield was 76.8%. The UPLC chromatogram is shown below. Figure 2 As shown.

[0076] Example 3: Purification of crude smegglutinin peptide

[0077] Sample preparation: Weigh 1g of crude smegglutinin peptide and add 50 times its weight of a solution containing 50mM Tris and 10% acetonitrile (pH 8.5) to dissolve the crude peptide sample for purification.

[0078] First purification: The dissolved crude smegglutinin was used as the sample, and BR-C18(2) octadecylsilane-bonded silica gel was used as the stationary phase. The flow rate was 200 cm / hr, and the detection wavelength was 280 nm. The column was equilibrated for 2 CVs with a solution containing 50 mM Tris and 10% acetonitrile. The sample was loaded with a total protein content of 10 g / L resin. After loading, the column was equilibrated for 2 CVs with 10% acetonitrile. Then, the column was equilibrated for 3 CVs with RP-A3 mobile phase. Finally, linear gradient elution was performed with RP-A3 and RP-B (RP-B from 55% to 75%, elution for 90 min). The collected main peak segment was the purified smegglutinin sample. UPLC analysis showed that the purity of the purified sample was 99.25% (the impurity at peak time 10.506 min, i.e., RRT = 0.739, with a content of 0.20%; the isomer impurity at peak time 13.801 min, i.e., RRT = 0.971, with a content of 0.06%, much less than 0.2%; the acetylated impurity at peak time 15.987 min, i.e., RRT = 1.125, with a content of 0.03%; the maximum single impurity was 0.20%), and the purification yield was 63.7%. The UPLC chromatogram is shown below. Figure 3 As shown.

[0079] Sample preparation: Dilute the obtained smegglutinin purified sample with an equal volume of purified water, adjust the pH to 8.0 with 10% ammonia, and wait for purification.

[0080] Second purification: The diluted semaglutide purified sample 1 was used as the loading sample, and HPLCONE 8C18K octadecylsilane-bonded silica gel was used as the stationary phase. The flow rate was 200 cm / hr, and the detection wavelength was 280 nm. The column was equilibrated with RP-A4 mobile phase for 2 CVs. The sample was loaded with a total protein loading of 10 g / L resin. After loading, the column was equilibrated with RP-A4 mobile phase for 3 CVs. Finally, linear gradient elution was performed using RP-A4 and RP-B (RP-B from 38% to 48%, elution for 45 min). The collected main peak was the semaglutide purified sample 2. UPLC analysis showed that the purity of the purified sample 2 was 99.84% (the largest single impurity was an isomer impurity with a peak time of 13.998 min, i.e., RRT = 0.967, impurity content was 0.04%; acetylated impurities were completely removed; the content of precursor impurities was significantly reduced), and the purification yield was 75.6%. UPLC spectra as follows Figure 4 As shown.

[0081] Example 4: Purification of crude smegglutinin peptide

[0082] Sample preparation: Weigh 1g of crude smegglutinin peptide and add 50 times its weight of a solution containing 50mM Tris and 10% acetonitrile (pH 8.5) to dissolve the crude peptide sample for purification.

[0083] First purification: The dissolved crude smegglutinin was used as the sample, and BR-C18(2) octadecylsilane-bonded silica gel was used as the stationary phase. The flow rate was 200 cm / hr, and the detection wavelength was 280 nm. The column was equilibrated for 2 CVs with a solution containing 50 mM Tris and 10% acetonitrile. The sample was loaded with a total protein content of 10 g / L resin. After loading, the column was equilibrated for 2 CVs with 10% acetonitrile. Then, the column was equilibrated for 3 CVs with RP-A5 mobile phase. Finally, linear gradient elution was performed with RP-A5 and RP-B (RP-B from 55% to 75%, elution for 90 min). The collected main peak segment was the purified smegglutinin sample. UPLC analysis showed that the purity of the purified sample was 99.20% (the impurity at peak time 10.494 min, i.e., RRT = 0.737, had a content of 0.12%; the impurity at peak time 11.772 min, i.e., RRT = 0.826, had a content of 0.19%; the isomer impurity at peak time 13.717 min, i.e., RRT = 0.963, had a content of 0.08%, much less than 0.2%; the acetylated impurity at peak time 17.347 min, i.e., RRT = 1.218, had a content of 0.14%; the largest single impurity was 0.19%), and the purification yield was 64.1%. The UPLC chromatogram is shown below. Figure 5 As shown.

[0084] Sample preparation: Dilute the obtained smegglutinin purified sample with an equal volume of purified water, adjust the pH to 8.0 with 10% ammonia, and wait for purification.

[0085] Second purification: The diluted semaglutide purified sample 1 was used as the loading sample, with HPLCONE 8C18K octadecylsilane-bonded silica gel as the stationary phase, a flow rate of 200 cm / hr, and a detection wavelength of 280 nm. The column was equilibrated with RP-A6 mobile phase for 2 CVs; the sample was loaded with a total protein loading of 10 g / L resin; after loading, the column was equilibrated with RP-A6 mobile phase for 3 CVs; finally, linear gradient elution was performed with RP-A6 and RP-B (RP-B from 38% to 48%, elution for 45 min), and the collected main peak was the semaglutide purified sample 2. UPLC analysis showed that the purity of the purified sample 2 was 99.86% (the largest single impurity was an isomer impurity with a peak time of 13.940 min, i.e., RRT = 0.965, impurity content was 0.05%; acetylated impurities were completely removed; the content of precursor impurities was significantly reduced), and the purification yield was 76.2%. UPLC spectra as follows Figure 6 As shown.

[0086] Example 5: Purification of crude smegglutinin peptide

[0087] Sample preparation: Weigh 1g of crude smegglutinin peptide and add 50 times its weight of a solution containing 50mM Tris and 10% acetonitrile (pH 8.5) to dissolve the crude peptide sample for purification.

[0088] First purification: The dissolved crude smegglutinin was used as the sample, and BR-C18(2) octadecylsilane-bonded silica gel was used as the stationary phase. The flow rate was 200 cm / hr, and the detection wavelength was 280 nm. The column was equilibrated for 2 CVs with a solution containing 50 mM Tris and 10% acetonitrile. The sample was loaded with a total protein content of 10 g / L resin. After loading, the column was equilibrated for 2 CVs with 10% acetonitrile. Then, the column was equilibrated for 3 CVs with RP-A7 mobile phase. Finally, linear gradient elution was performed with RP-A7 and RP-B (RP-B from 55% to 75%, elution for 90 min). The collected main peak segment was the purified smegglutinin sample. UPLC analysis showed that the purity of the purified sample was 99.00% (the impurity with a peak time of 10.514 min, i.e., RRT = 0.743, with a content of 0.12%; the largest single impurity was the isomer with a peak time of 13.775 min, i.e., RRT = 0.973, with a content of 0.14%, less than 0.2%), and the purification yield was 63.7%. The UPLC chromatogram is shown below. Figure 7 As shown.

[0089] Sample preparation: Dilute the obtained smegglutinin purified sample with an equal volume of purified water, adjust the pH to 8.0 with 10% ammonia, and wait for purification.

[0090] Second purification: The diluted semaglutide purified sample 1 was used as the loading sample. An HPLCONE 8C18K octadecylsilane-bonded silica gel packing material was used as the stationary phase at a flow rate of 200 cm / hr and a detection wavelength of 280 nm. The column was equilibrated with RP-A8 mobile phase for 2 CVs. The sample was loaded with a total protein loading of 10 g / L resin. After loading, the column was equilibrated with RP-A8 mobile phase for 3 CVs. Finally, a linear gradient elution was performed using RP-A8 and RP-B (RP-B from 38% to 48%, elution for 45 min). The collected main peak was the semaglutide purified sample 2. UPLC analysis showed that the purity of the purified sample 2 was 99.82% (the largest single impurity was an isomer impurity with a peak elution time of 12.286 min, i.e., RRT = 0.966, impurity content was 0.05%; the impurity content was significantly reduced), and the purification yield was 74.4%. The UPLC chromatogram is shown below. Figure 8 As shown.

[0091] Example 6: Purification of crude smegglutinin peptide

[0092] The BR-C18(2) octadecylsilane-bonded silica gel packing used in the first purification of Example 2 and the HPLCONE 8C18K octadecylsilane-bonded silica gel packing used in the second purification were replaced with YMC-C18 packing material. All other conditions and operating procedures were the same as in Example 2. After the second purification, the purity of the sample was 99.82% (the maximum single impurity content was 0.04%; the impurity content was significantly reduced), and the purification yield was 75.1%. The UPLC chromatogram is shown below. Figure 9 As shown.

[0093] Example 7: Rotary evaporation and lyophilization of two samples purified from smegglutinin

[0094] The purified semaglutide sample obtained in Example 2 was placed in a rotary evaporator to remove acetonitrile, then pre-frozen at -80°C. After pre-freezing, it was lyophilized in a freeze dryer. The resulting lyophilized sample was the semaglutide peptide sample. UPLC analysis showed that the purity of the semaglutide peptide was 99.86% (maximum single impurity 0.04%). The UPLC chromatogram is shown below. Figure 10 As shown, the results indicate that the product is very stable under rotary evaporation and freeze-drying conditions, and its purity and impurity content are hardly affected.

[0095] Comparative Example 1

[0096] Referring to Example 2, 30 mM ammonium formate was used instead of 30 mM ammonium acetate in the mobile phase RP-A1. All other conditions and procedures were the same as in the first purification in Example 2. The effect of the buffer salt in the mobile phase on the purity, impurities, and yield of smegglutinin was investigated. The results are as follows: Figure 11As shown, the purity of the purified sample was 98.62%. The isomer impurity with a peak elution time of 12.956 min (RRT = 0.963) had a content of 0.28%; the acetylated impurity with a peak elution time of 15.017 min (RRT = 1.117) had a content of 0.29%, and the maximum single impurity was 0.44%. This indicates that ammonium acetate buffer is superior to ammonium formate buffer in purifying the crude smegglutinin peptide, effectively reducing the content of difficult-to-remove impurities, especially isomer impurities with relative retention times of RRT 0.954–RRT 0.973, thus ensuring that the content of this impurity is stably controlled at ≤0.06% after the second purification step.

[0097] Comparative Example 2

[0098] Sample preparation: Weigh 1g of crude smegglutinin peptide and add 50 times its weight of a solution containing 50mM Tris and 10% acetonitrile (pH 8.5) to dissolve the crude peptide sample for purification.

[0099] First purification: The dissolved crude smegglutinin was used as the sample, and BR-C18(2) octadecylsilane-bonded silica gel was used as the stationary phase. The flow rate was 200 cm / hr, and the detection wavelength was 280 nm. The column was equilibrated for 2 CVs with a solution containing 50 mM Tris and 10% acetonitrile. The sample was loaded with a total protein content of 10 g / L resin. After loading, the column was equilibrated for 2 CVs with 10% acetonitrile. The column was then equilibrated for 3 CVs with mobile phase RP-A: 0.2% phosphoric acid + 10% acetonitrile and 10% ammonia to adjust the pH to 2.5. Finally, linear gradient elution was performed with mobile phases RP-A and RP-B (60% acetonitrile) (RP-B from 55% to 75%, elution for 90 min). The collected main peak was the purified smegglutinin sample. UPLC analysis showed that the purity of the purified sample was 98.19% (the largest single impurity was an isomer with a peak time of 14.021 min, i.e., RRT = 0.954, and a content of 0.63%; and an acetylated impurity with a peak time of 16.334 min, i.e., RRT = 1.112, and a content of 0.19%), with a purification yield of 76.8%. The UPLC chromatogram is shown below. Figure 12 As shown.

[0100] Sample preparation: Dilute the obtained smegglutinin purified sample with an equal volume of purified water, adjust the pH to 8.0 with 10% ammonia, and wait for purification.

[0101] Second purification: The diluted semaglutide purified sample 1 was used as the loading sample. An HPLCONE 8C18K octadecylsilane-bonded silica gel packing material was used as the stationary phase at a flow rate of 200 cm / hr and a detection wavelength of 280 nm. The column was equilibrated with mobile phase C: 20 mM ammonium acetate + 10% acetonitrile, with the pH adjusted to 8.0 by ammonia. The column was equilibrated for 2 CVs. The sample was loaded with a total protein content of 10 g / L resin. After loading, the column was equilibrated with mobile phase C for 3 CVs. Finally, a linear gradient elution was performed using mobile phase C and mobile phase RP-B (60% acetonitrile) (RP-B from 38% to 48%, elution for 45 min). The collected peak segment was the semaglutide purified sample 2. UPLC analysis showed that the purity of purified sample 2 was 98.96% (the impurity at peak time 10.705 min, i.e., RRT = 0.733, had a content of 0.28%, which was relatively high; the isomer impurity at peak time 13.997 min, i.e., RRT = 0.959, had a content of 0.19%; and the acetylated impurity at peak time 16.342 min, i.e., RRT = 1.119, had a content of 0.16%, with no significant change in acetylated impurity content compared to purified sample 1), with a maximum single impurity content of 0.28% and a purification yield of 75.4%. The UPLC chromatogram is shown below. Figure 13 As shown, the purity of the semaglutide sample obtained by the purification method in this comparative example is significantly lower than that in Example 2, with a significant increase in isomers, acetylated impurities, and unknown precursors.

Claims

1. A method for purifying smegglutinin, comprising a two-step reversed-phase high-performance liquid chromatography method, characterized in that: Includes the following steps: (1) Before the first step of purification, the crude smegglutinin peptide is dissolved in a mixed solvent consisting of an alkaline solution containing tris(hydroxymethyl)aminomethane and an organic solvent; the mixed solvent is a mixed solution containing Tris aqueous solution and acetonitrile. (2) First step purification: using an acidic buffer solution containing acetate and an ion-pairing agent and an organic solvent as the mobile phase, and a reversed-phase packing as the stationary phase; in the mobile phase, a mixed solution of an acidic buffer solution containing 30 mM sodium acetate or ammonium acetate and 0.05%-0.10% trifluoroacetic acid as the ion-pairing agent and 10% acetonitrile with a pH of 2.5-3.0 is used as mobile phase A, and an aqueous solution containing 60% acetonitrile is used as mobile phase B, and linear gradient elution is performed; (3) Second step purification: use an alkaline buffer containing phosphate and an organic solvent as the mobile phase and a reversed-phase packing as the stationary phase; in the mobile phase, the mixed solution of an alkaline buffer containing 20-50 mM dipotassium hydrogen phosphate or disodium hydrogen phosphate and 10% acetonitrile with a pH of 8.0-8.5 is the mobile phase A, and the aqueous solution containing 60% acetonitrile is the mobile phase B.

2. The purification method according to claim 1, characterized in that: The reversed-phase packing material used in the first and second purification steps is selected from porous silica particles or silica gel as the stationary phase.

3. The purification method according to claim 1, characterized in that: Purification via crude peptide pretreatment and two-step reversed-phase high-performance liquid chromatography includes the following steps: (1) Crude peptide pretreatment: Smegglutinin crude peptide was dissolved in an alkaline solution containing tris(hydroxymethyl)aminomethane and acetonitrile; (2) First step purification: using octadecylsilane bonded silica gel as the stationary phase, a buffer salt solution with pH 2.5-3.0 containing 30 mM sodium acetate or ammonium acetate, 0.05%-0.10% TFA and 10% acetonitrile as the mobile phase A, and a 60% acetonitrile solution as the mobile phase B, linear gradient elution was performed, and the main peak fraction was collected; (3) Second step purification: Using octadecylsilane bonded silica gel as the stationary phase, a buffer salt solution containing 20-50 mM dipotassium hydrogen phosphate or disodium hydrogen phosphate and 10% acetonitrile with a pH of 8.0-8.5 is used as mobile phase A, and 60% acetonitrile solution is used as mobile phase B. Linear gradient elution is performed, and the main peak component is collected.

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