Smeglutide main chain precursor degradation impurity and preparation method thereof

Abstract

The invention provides a degradation impurity of a stemeglutide main chain precursor and a preparation method of the degradation impurity. Specifically, the invention also provides a quality control or purification method and a quality control kit of the main chain precursor of the semeglutide, and application, a preparation kit and a preparation device of degradation impurities of the main chain precursor of the semeglutide. According to the method disclosed by the invention, a large amount of pyroglutamic acid cyclization impurities originally existing in a small amount can be obtained by adopting a method which is simple to operate, the purity of the impurities is high, and the method can be used for quality control in the production process of the semeglutide and is beneficial to obtaining a high-purity final product.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and in particular to a semaglutide main chain precursor degradation impurity and a preparation method thereof. Background Art

[0002] Semaglutide is a glucose-lowering drug developed by Novo Nordisk. The product can significantly reduce the glycated hemoglobin (HbA1c) level and reduce weight in patients with type 2 diabetes, while greatly reducing the risk of hypoglycemia.

[0003] Semaglutide is obtained by modifying and transforming GLP-1 (7-37). The sequence of Semaglutide is:

[0004] His7-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26(AEEA-AEEA-γ-Glu-Octadeca-nedioic Acid)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37 (corresponding to SEQ ID NO: 1 in the sequence listing).

[0005] The sequence of the semaglutide backbone is:

[0006] His7-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37 (corresponding to SEQ ID NO: 2 in the sequence listing).

[0007] At present, there are two main methods for preparing the main chain of semaglutide: one is to directly synthesize the main chain of semaglutide using solid phase synthesis technology (CN103848910B, CN106478806A, etc.) or synthesize fragments first and then condense and connect them (CN106749613A, CN104356224A, etc.); the other is to first obtain the main chain without non-natural amino acid Aib by biological fermentation method, and then connect it with a short peptide containing Aib (CN110498849A, CN113801234A, etc.). The latter has the advantages of less racemic impurities, high yield and low cost, which is more conducive to industrial production.

[0008] Although there are many reports on the preparation method of semaglutide, there are few reports on related impurities and preparation methods in this field. Impurity research is an important part of drug quality research and one of the key factors in drug quality assurance. Impurities will affect the quality of the final product, so it is necessary to conduct detailed research on process impurities generated during the production process of semaglutide and degradation impurities generated during storage.

[0009] Since the N-terminus of the semaglutide main chain obtained by the biological fermentation method lacks the two amino acids His and Aib, the glutamic acid at the N-terminus is easily dehydrated to form pyroglutamic acid impurities during freeze-drying and storage, resulting in a decrease in the pure peptide content of the main chain, affecting subsequent modification and purification, and is not conducive to the synthesis of a high-purity final product.

[0010] Therefore, there is a need in the art to carry out targeted degradation and purification preparation of potential impurities in semaglutide for the stability study of the semaglutide backbone. Summary of the invention

[0011] The purpose of the present invention is to provide a semaglutide main chain precursor degradation impurity and a preparation method thereof.

[0012] Another object of the present invention is to provide a quality control or purification method for a semaglutide main chain precursor.

[0013] In a first aspect of the present invention, a quality control method for a semaglutide main chain precursor is provided, the quality control method comprising the steps of:

[0014] (a) providing a degradation impurity of a semaglutide backbone precursor and a semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample to be tested; and

[0015] (b) using the semaglutide main chain precursor degradation impurities as impurity reference substances, and determining the content of the semaglutide main chain precursor degradation impurities in the semaglutide main chain precursor sample to be tested by chromatography, thereby performing quality control of the semaglutide main chain precursor.

[0016] In another preferred embodiment, the degradation impurity of the semaglutide main chain precursor has a sequence as shown in SEQ ID NO:4.

[0017] In another preferred embodiment, the Lys26(Boc)-Arg34-GLP-1(9-37) sample is in a non-salt free state.

[0018] In another preferred embodiment, the Lys26(Boc)-Arg34-GLP-1(9-37) sample includes a suspension sample or a solid sample.

[0019] In another preferred embodiment, the solid sample comprises freeze-dried powder.

[0020] In another preferred embodiment, the suspension sample is a 10%-70% suspension, preferably a 30%-60% suspension, and more preferably a 50% suspension.

[0021] In another preferred embodiment, the chromatography is high performance liquid chromatography (HPLC).

[0022] In another preferred embodiment, the filler used in the chromatographic column used in the chromatography method is a reverse phase chromatography filler.

[0023] In another preferred embodiment, the filler type is selected from one of C18 and C8; preferably C18.

[0024] In another preferred example, the particle size of the filler is 5 μm.

[0025] In another preferred embodiment, the chromatography uses mobile phase A and mobile phase B for gradient elution.

[0026] In another preferred embodiment, the mobile phase A is diammonium hydrogen phosphate.

[0027] In another preferred embodiment, the mobile phase B is acetonitrile (ACN) or an acetonitrile (ACN) aqueous solution.

[0028] In another preferred embodiment, the concentration of the mobile phase A is 5-20 mM, preferably 10 mM.

[0029] In another preferred embodiment, the pH of the mobile phase A is 10.2.

[0030] In another preferred embodiment, the gradient elution program used in the chromatography is as follows:

[0031] (S1) When the elution start time is 0 min, the percentage content of mobile phase A is 75%, and the percentage content of mobile phase B is 25%;

[0032] (S2) When the elution start time is 2 min, the percentage content of mobile phase A is 75%, and the percentage content of mobile phase B is 25%;

[0033] (S3) When the elution start time is 21 min, the percentage content of mobile phase A is 35%, and the percentage content of mobile phase B is 65%;

[0034] (S4) when the elution start time is 21.5 min, the percentage content of mobile phase A is 0%, and the percentage content of mobile phase B is 100%;

[0035] (S5) when the elution start time is 25 min, the percentage content of mobile phase A is 0%, and the percentage content of mobile phase B is 100%;

[0036] (S6) when the elution start time is 25.1 min, the percentage content of mobile phase A is 75%, and the percentage content of mobile phase B is 25%; and

[0037] (S7) When the elution start time is 30 min, the percentage content of mobile phase A is 75%, and the percentage content of mobile phase B is 25%.

[0038] In another preferred embodiment, the chromatography method uses a detection wavelength of 214 nm, a flow rate of 1.0 mL / min, and a column temperature of 30-40° C. (preferably 35° C.).

[0039] In another preferred embodiment, the quality control method further comprises the steps of: preparing the impurity reference substance into standard solutions of different concentrations, and making a standard curve according to the impurity peak area after HPLC analysis.

[0040] In another preferred embodiment, the quality control method further includes the steps of: determining the concentration of the semaglutide main chain precursor in the Lys26(Boc)-Arg34-GLP-1(9-37) suspension, and performing HPLC analysis on the suspension, and calculating the content of semaglutide main chain precursor degradation impurities (target impurities) according to the impurity peak area and the standard curve; or, performing HPLC analysis on the Lys26(Boc)-Arg34-GLP-1(9-37) suspension with a known semaglutide main chain precursor concentration, and calculating the content of the target impurity according to the impurity peak area and the standard curve.

[0041] In another preferred embodiment, the quality control method further comprises the steps of: preparing the Lys26(Boc)-Arg34-GLP-1(9-37) solid sample (e.g., lyophilized powder) into a solution of known concentration, performing HPLC analysis, and calculating the content of the target impurity based on the impurity peak area and the standard curve.

[0042] In a second aspect of the present invention, a method for purifying a semaglutide main chain precursor is provided, the purification method comprising the steps of:

[0043] (a) providing a sample of degradation impurities of a semaglutide backbone precursor and a semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) to be purified; and

[0044] (b) using the semaglutide backbone precursor degradation impurities as impurity reference substances, using chromatography, and optimizing the chromatographic conditions and / or elution conditions to effectively separate the semaglutide backbone precursor degradation impurities in the semaglutide backbone precursor sample, thereby obtaining a purified semaglutide backbone precursor.

[0045] In another preferred embodiment, the degradation impurity of the semaglutide main chain precursor has a sequence as shown in SEQ ID NO:4.

[0046] In another preferred embodiment, the Lys26(Boc)-Arg34-GLP-1(9-37) sample is in a non-salt free state.

[0047] In another preferred embodiment, the Lys26(Boc)-Arg34-GLP-1(9-37) sample includes a suspension sample or a solid sample.

[0048] In another preferred embodiment, the solid sample comprises freeze-dried powder.

[0049] In another preferred embodiment, the suspension sample is a 10%-70% suspension, preferably a 30%-60% suspension, and more preferably a 50% suspension.

[0050] In another preferred embodiment, the chromatography is high performance liquid chromatography (HPLC).

[0051] In another preferred embodiment, the filler used in the chromatographic column used in the chromatography method is a reverse phase chromatography filler.

[0052] In another preferred embodiment, the filler type is selected from one of C18 and C8; preferably C18.

[0053] In another preferred embodiment, the chromatography uses mobile phase A and mobile phase B for gradient elution.

[0054] In another preferred embodiment, the mobile phase A is diammonium hydrogen phosphate.

[0055] In another preferred embodiment, the mobile phase B is acetonitrile (ACN) or an acetonitrile (ACN) aqueous solution.

[0056] In another preferred embodiment, the purification method further comprises the steps of: preparing the impurity reference substance into standard solutions of different concentrations, and making a standard curve according to the impurity peak area after HPLC analysis.

[0057] In another preferred embodiment, the purification method further comprises the steps of: preparing the semaglutide backbone precursor sample to be purified into a solution of known concentration, performing HPLC analysis, and calculating the content of semaglutide backbone precursor degradation impurities in the semaglutide backbone precursor sample according to the impurity peak area and the standard curve.

[0058] In another preferred embodiment, the purification method further includes the step of optimizing the chromatographic conditions and / or elution conditions to improve the separation between the chromatographic peak (main peak) of the semaglutide backbone precursor and the degradation impurities (impurity peak) of the semaglutide backbone precursor, thereby effectively separating the degradation impurities of the semaglutide backbone precursor.

[0059] In another preferred embodiment, the content of degradation impurities of the semaglutide main chain precursor in the semaglutide main chain precursor to be purified is ≥2%, preferably ≥3%, and more preferably ≥5%.

[0060] In another preferred embodiment, the content of degradation impurities of the semaglutide backbone precursor in the purified semaglutide backbone precursor is ≤1%, preferably ≤0.8%, more preferably ≤0.5% (such as ≤0.45%).

[0061] In another preferred embodiment, the purified semaglutide backbone precursor is used to synthesize semaglutide (high purity).

[0062] In a third aspect of the present invention, a semaglutide backbone precursor degradation impurity is provided, wherein the semaglutide backbone precursor degradation impurity has a sequence as shown in SEQ ID NO:4.

[0063] In the fourth aspect of the present invention, there is provided a use of the semaglutide main chain precursor for degrading impurities according to the third aspect of the present invention, for:

[0064] (Z1) as an impurity reference substance for quality control or optimization of the semaglutide production process (e.g., semaglutide backbone precursor); and / or

[0065] (Z2) was used as an impurity control substance for the purification of semaglutide backbone precursor.

[0066] In another preferred embodiment, the use includes use in the quality control method as described in the first aspect of the present invention, or use in the purification method as described in the second aspect of the present invention.

[0067] In another preferred embodiment, the (Z1) includes: as an impurity reference substance, so as to perform quantitative analysis on impurities in the semaglutide main chain precursor.

[0068] In another preferred embodiment, the (Z1) comprises: optimizing the freeze-drying process of the semaglutide main chain precursor by quantitative analysis of impurities.

[0069] In a fifth aspect of the present invention, a method for preparing degradation impurities of a semaglutide main chain precursor is provided, the preparation method comprising the steps of:

[0070] (a) providing a semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample;

[0071] (b) heat treating the Lys26(Boc)-Arg34-GLP-1(9-37) sample at 55-85°C for a period of time; and

[0072] (c) purifying the product obtained in step (b) to obtain degradation impurities of semaglutide main chain precursor.

[0073] In another preferred embodiment, the heat treatment temperature in step (b) is 55°C, 65°C, 75°C or 85°C.

[0074] In another preferred embodiment, the heat treatment temperature in step (b) is 65°C or 75°C.

[0075] In another preferred embodiment, the heat treatment time in step (b) is 6-36 hours, preferably 12-24 hours, and more preferably 24 hours.

[0076] In another preferred embodiment, the Lys26(Boc)-Arg34-GLP-1(9-37) sample is in a non-salt free state.

[0077] In another preferred embodiment, the Lys26(Boc)-Arg34-GLP-1(9-37) sample includes a suspension sample or a solid sample.

[0078] In another preferred embodiment, the solid sample comprises freeze-dried powder.

[0079] In another preferred embodiment, the suspension sample is a 10%-70% suspension, preferably a 30%-60% suspension, and more preferably a 50% suspension.

[0080] In another preferred embodiment, the semaglutide main chain precursor Lys26(Boc)-Arg34-GLP-1(9-37) sample has a sequence as shown in SEQ ID NO: 3:

[0081] Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-A la24-Ala25-Lys26(Boc)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37(SEQ ID NO:3).

[0082] In another preferred embodiment, the degradation impurity of the semaglutide main chain precursor has a sequence as shown in SEQ ID NO: 4:

[0083] Pyr9 (pyroglutamate)

[0084] -Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala 24-Ala25-Lys26(Boc)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37(SEQ ID NO:4).

[0085] In another preferred embodiment, the purification method in step (c) is a chromatographic purification method.

[0086] In another preferred embodiment, the chromatographic purification method comprises the steps of:

[0087] (c1) The product obtained in step (b) is loaded onto a high performance liquid chromatography column, using phase A acidic aqueous solution and phase B organic solvent as mobile phases, performing gradient elution, and collecting the eluate.

[0088] In another preferred embodiment, the filler used in the HPLC preparative chromatography column is a reverse phase chromatography filler.

[0089] In another preferred embodiment, the filler type is selected from one of C18 and C8.

[0090] In another preferred embodiment, the particle size of the filler is 10-30 μm, preferably 10 μm.

[0091] In another preferred embodiment, the phase A acidic aqueous solution is selected from one of formic acid, acetic acid and trifluoroacetic acid.

[0092] In another preferred embodiment, the organic solvent of phase B is selected from one of methanol, ethanol and acetonitrile.

[0093] In another preferred embodiment, the organic solvent of phase B is an acidic organic solvent, preferably an organic solvent added with trifluoroacetic acid; more preferably acetonitrile added with 0.1% volume fraction of trifluoroacetic acid (0.1% trifluoroacetic acid / acetonitrile).

[0094] In another preferred embodiment, the proportion of the acid additive in the phase A acidic aqueous solution is 0.1%-0.5% by volume, preferably 0.1% by volume.

[0095] In another preferred embodiment, the acidic aqueous solution of phase A is 0.1% formic acid, and the organic solvent of phase B is acetonitrile.

[0096] In another preferred embodiment, the acidic aqueous solution of phase A is 0.1% trifluoroacetic acid, and the organic solvent of phase B is acetonitrile to which trifluoroacetic acid is added at a volume fraction of 0.1% (0.1% trifluoroacetic acid / acetonitrile).

[0097] In another preferred embodiment, the gradient elution procedure is as follows:

[0098] (S1) When the elution start time is 0 min, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 5-10%, and the percentage content of the organic solvent of phase B is 95-90%; preferably, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 10%, and the percentage content of the organic solvent of phase B is 90%;

[0099] (S2) when the elution start time is 10 min, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 65-58%, and the percentage content of the organic solvent of phase B is 35-42%; preferably, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 59%, and the percentage content of the organic solvent of phase B is 41%; and

[0100] (S3) When the elution start time is 70 min, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 45-38%, and the percentage content of the organic solvent of phase B is 55-62%; preferably, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 39%, and the percentage content of the organic solvent of phase B is 61%.

[0101] In another preferred embodiment, the preparation method further comprises the steps of:

[0102] (d) Lyophilization: The eluate obtained in step (c1) is diluted with water until the organic solvent in phase B is less than 10%, and lyophilized for 10-16 hours under the conditions of vacuum degree ≤ 10 mbar and baffle temperature control at 25° C., thereby obtaining a lyophilized preparation of degradation impurities of the semaglutide main chain precursor.

[0103] In another preferred embodiment, the organic solvent of phase B in step (d) is acetonitrile.

[0104] In a sixth aspect of the present invention, a kit for preparing degradation impurities of semaglutide main chain precursor is provided, the kit comprising:

[0105] (a) a first container and a sample of semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) located in the first container; and

[0106] (b) a second container and a heat treatment device located in the second container.

[0107] In another preferred embodiment, the degradation impurity of the semaglutide main chain precursor has a sequence as shown in SEQ ID NO:4.

[0108] In another preferred embodiment, the Lys26(Boc)-Arg34-GLP-1(9-37) sample is in a non-salt free state.

[0109] In another preferred embodiment, the Lys26(Boc)-Arg34-GLP-1(9-37) sample includes a suspension sample or a solid sample (such as lyophilized powder).

[0110] In another preferred embodiment, the heat treatment device is configured to make the heating temperature 55-85°C, preferably 55°C, 65°C, 75°C or 85°C; more preferably 65°C or 75°C.

[0111] In another preferred embodiment, the heat treatment device is configured so that a single heating time is 6-36 hours, preferably 12-24 hours, and more preferably 24 hours.

[0112] In another preferred embodiment, the kit further comprises: (c) a third container and a purification reagent located in the third container.

[0113] In another preferred example, the first container, the second container and / or the third container may be the same container or different containers.

[0114] In another preferred embodiment, the purification reagent comprises a chromatographic purification reagent.

[0115] In another preferred embodiment, the purification reagent includes a phase A acidic aqueous solution and / or a phase B organic solvent.

[0116] In another preferred embodiment, the phase A acidic aqueous solution is selected from one of formic acid, acetic acid and trifluoroacetic acid.

[0117] In another preferred embodiment, the organic solvent of phase B is selected from one of methanol, ethanol and acetonitrile.

[0118] In another preferred embodiment, the organic solvent of phase B is an acidic organic solvent, preferably an organic solvent added with trifluoroacetic acid; more preferably acetonitrile added with 0.1% volume fraction of trifluoroacetic acid (0.1% trifluoroacetic acid / acetonitrile).

[0119] In another preferred embodiment, the kit further comprises a label and instructions.

[0120] In another preferred embodiment, the description indicates the preparation method as described in the fifth aspect of the present invention and / or the use as described in the fourth aspect of the present invention.

[0121] In the seventh aspect of the present invention, a quality control kit for a semaglutide backbone precursor is provided, the quality control kit comprising: the semaglutide backbone degradation impurities as described in the third aspect of the present invention.

[0122] In another preferred example, the quality control kit further includes a label and instructions.

[0123] In another preferred embodiment, the description indicates the quality control method as described in the first aspect of the present invention.

[0124] In an eighth aspect of the present invention, a method for optimizing the freeze-drying process of a semaglutide backbone precursor is provided, the optimization method comprising the steps of:

[0125] (a) providing a degradation impurity of a semaglutide main chain precursor and a lyophilized sample of a semaglutide main chain precursor;

[0126] (b) using the semaglutide main chain precursor degradation impurities as an impurity reference substance, and determining the content of the semaglutide main chain precursor degradation impurities in the semaglutide main chain precursor lyophilized sample by chromatography; and

[0127] (c) by adjusting the conditions of the freeze-drying process, the content of degradation impurities of the semaglutide main chain precursor in the freeze-dried sample of the semaglutide main chain precursor is reduced, thereby obtaining the optimized conditions of the freeze-drying process.

[0128] In another preferred embodiment, the conditions of the freeze-drying process include: temperature and / or freeze-drying time.

[0129] In another preferred embodiment, the degradation impurity of the semaglutide main chain precursor has a sequence as shown in SEQ ID NO:4.

[0130] In another preferred embodiment, the chromatography is high performance liquid chromatography (HPLC).

[0131] In another preferred embodiment, the filler used in the chromatographic column used in the chromatography method is a reverse phase chromatography filler.

[0132] In another preferred embodiment, the filler type is selected from one of C18 and C8; preferably C18.

[0133] In another preferred embodiment, the chromatography uses mobile phase A and mobile phase B for gradient elution.

[0134] In another preferred embodiment, the mobile phase A is diammonium hydrogen phosphate.

[0135] In another preferred embodiment, the mobile phase B is acetonitrile (ACN) or an acetonitrile (ACN) aqueous solution.

[0136] In another preferred embodiment, the optimization method further comprises the steps of: preparing the impurity reference substance into standard solutions of different concentrations, and making a standard curve according to the impurity peak area after HPLC analysis.

[0137] In another preferred embodiment, the optimization method further comprises the steps of: preparing the semaglutide backbone precursor freeze-dried sample into a solution of known concentration, performing HPLC analysis, and calculating the content of semaglutide backbone precursor degradation impurities in the semaglutide backbone precursor freeze-dried sample according to the impurity peak area and the standard curve.

[0138] In another preferred embodiment, the optimization method further comprises the step of: using the optimized conditions of the freeze-drying process to prepare an optimized process freeze-dried sample.

[0139] In another preferred embodiment, the content of degradation impurities of the semaglutide backbone precursor in the lyophilized sample of the semaglutide backbone precursor is ≥2%, preferably ≥3%, and more preferably ≥5%.

[0140] In another preferred embodiment, the content of degradation impurities of semaglutide main chain precursor in the optimized freeze-dried sample is ≤1%, preferably ≤0.8%, and more preferably ≤0.5% (such as ≤0.45%).

[0141] In another preferred embodiment, the optimized freeze-dried sample is used to synthesize semaglutide (high purity).

[0142] In a ninth aspect of the present invention, a device for preparing semaglutide main chain precursor degradation impurities is provided, the device comprising:

[0143] (M1) a sample loading module, wherein the sample loading module is configured to: provide a semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample;

[0144] (M2) a heat treatment module, wherein the heat treatment module is configured to: make the heat treatment temperature 55-85° C. and the single heating time 6-36 hours, thereby obtaining a heat-treated product; and

[0145] (M3) a purification module, wherein the purification module is configured to purify the product to obtain degradation impurities of the semaglutide main chain precursor.

[0146] In another preferred embodiment, the (M3) purification module further includes a gradient elution submodule, and the gradient elution submodule is configured to: perform gradient elution on the degradation product using phase A acidic aqueous solution and phase B organic solvent as mobile phases, and collect the eluate.

[0147] In another preferred embodiment, the procedure of gradient elution in the gradient elution submodule is as follows:

[0148] (S1) When the elution start time is 0 min, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 5-10%, and the percentage content of the organic solvent of phase B is 95-90%; preferably, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 10%, and the percentage content of the organic solvent of phase B is 90%;

[0149] (S2) when the elution start time is 10 min, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 65-58%, and the percentage content of the organic solvent of phase B is 35-42%; preferably, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 59%, and the percentage content of the organic solvent of phase B is 41%; and

[0150] (S3) When the elution start time is 70 min, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 45-38%, and the percentage content of the organic solvent of phase B is 55-62%; preferably, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 39%, and the percentage content of the organic solvent of phase B is 61%.

[0151] In another preferred embodiment, the device also includes a (M4) freeze-drying module, which is configured to: dilute the eluate obtained in the gradient elution submodule with water until the organic solvent of phase B is less than 10%, and freeze-dry for 10-16 hours under the conditions of vacuum degree ≤10mbar and baffle temperature control of 25°C, thereby obtaining a freeze-dried preparation of degradation impurities of the semaglutide main chain precursor.

[0152] In another preferred example, the device further comprises a control module (M5), which is configured to control the operation of the sample loading module (M1), the heat treatment module (M2) and the purification module (M3).

[0153] In another preferred example, the (M5) control module also controls the operation of the (M4) freeze-drying module.

[0154] In another preferred embodiment, the (M5) control module controls the following modules in the device to operate in sequence:

[0155] (M1) sample loading module, (M2) heat treatment module, (M3) purification module, that is, they are operated in the order of (M1) → (M2) → (M3).

[0156] In another preferred embodiment, the (M5) control module controls the following modules in the device to operate in sequence:

[0157] (M1) sample loading module, (M2) heat treatment module, (M3) purification module and (M4) freeze drying module are operated in the order of (M1) → (M2) → (M3) → (M4).

[0158] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0159] Figure 1 The HPLC spectrum of the precursor before freeze-drying is shown, wherein the main peak appearing at about 15-16 min is the target product semaglutide main chain precursor (SEQ ID NO: 3).

[0160] Figure 2 The HPLC spectrum of the precursor after freeze-drying is shown, wherein the impurity peak appearing at about 16-17 min is the degradation impurity of the semaglutide main chain precursor (SEQ ID NO: 4).

[0161] Figure 3 The HPLC spectrum of the degradation product under condition 5 in Table 1 is shown.

[0162] Figure 4 The HPLC spectrum of the impurities after purification in Example 2 is shown.

[0163] Figure 5 The HPLC spectrum of the impurities after purification in Example 3 is shown.

[0164] Figure 6 The mass spectrum of the impurity (SEQ ID NO: 4) is shown.

[0165] Figure 7 The figure shows the use of impurity reference substances to detect the content of impurities in the main chain precursor lyophilized powder solution.

[0166] Figure 8 The HPLC spectrum after freeze-drying process optimization is shown, in which the content of the impurity (SEQ ID NO: 4) is significantly reduced. DETAILED DESCRIPTION

[0167] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that the solid or suspension of the semaglutide main chain precursor can be degraded by heating and other methods, which can greatly increase the generation of pyroglutamic acid cyclized impurities (semaglutide main chain precursor degradation impurities, having the sequence shown in SEQ ID NO: 4) that originally only existed in a small amount in the semaglutide main chain precursor. Combined with separation, purification and post-treatment, a high-purity impurity sample can be obtained. Through the simple method of the present invention, high-purity impurities can be obtained as impurity reference substances, which are used for quality control or optimization of the semaglutide production process (especially the semaglutide main chain precursor), and are helpful for synthesizing high-purity final product semaglutide. The present invention was completed on this basis.

[0168] the term

[0169] In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning given below.

[0170] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0171] As used herein, the terms "impurity", "target impurity", "degradation impurity", and "semaglutide backbone precursor degradation impurity" are used interchangeably and refer to the semaglutide backbone precursor degradation impurity as described in the third aspect of the present invention, obtained by the preparation method described in the fifth aspect of the present invention, having a sequence as shown in SEQ ID NO:4.

[0172] Semaglutide

[0173] Semaglutide was developed by Novo Nordisk, CAS No. 910463-68-2, and is a human glucagon-like peptide-1 (GLP-1) analog, with the sequence: His7-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26(AEEA-AEEA-γ-Glu-Octadeca-nedioicAcid)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37 (corresponding to SEQ ID NO: 1). The sequence homology with human natural GLP-1 is 97%.

[0174] Semaglutide is a hypoglycemic drug that can significantly reduce the level of glycated hemoglobin (HbA1c) and reduce body weight in patients with type 2 diabetes, while greatly reducing the risk of hypoglycemia. Semeglutide is obtained by modifying and transforming GLP-1 (7-37). Compared with Liraglutide, Semeglutide has a longer fatty chain and increased hydrophobicity, but Semeglutide has been modified with short-chain PEG, and its hydrophilicity has been greatly enhanced. After PEG modification, it can not only bind tightly to albumin and mask the DPP-4 enzyme hydrolysis site, but also reduce renal excretion, prolong the biological half-life, and achieve a long circulation effect. It can significantly reduce fasting or postprandial blood sugar in patients with type 2 diabetes to regulate blood sugar levels in the body, while reducing the patient's weight and reducing the risk of death in patients with cardiovascular disease.

[0175] Quality control method and quality control kit for semaglutide main chain precursor

[0176] In the present invention, a quality control method for a semaglutide main chain precursor is provided, and the quality control method comprises the steps of:

[0177] (a) providing a degradation impurity of a semaglutide backbone precursor and a semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample to be tested; and

[0178] (b) using the semaglutide main chain precursor degradation impurities as impurity reference substances, and determining the content of the semaglutide main chain precursor degradation impurities in the semaglutide main chain precursor sample to be tested by chromatography, thereby performing quality control of the semaglutide main chain precursor.

[0179] Preferably, the quality control method may further include the steps of: preparing the impurity reference substance into standard solutions of different concentrations, and making a standard curve according to the impurity peak area after HPLC analysis.

[0180] When the semaglutide backbone precursor sample to be tested is derived from / is a Lys26(Boc)-Arg34-GLP-1(9-37) suspension, the quality control method may further include the steps of: determining the concentration of the semaglutide backbone precursor in the Lys26(Boc)-Arg34-GLP-1(9-37) suspension, and performing HPLC analysis on the suspension, and calculating the content of semaglutide backbone precursor degradation impurities (target impurities) according to the impurity peak area and the standard curve; or, performing HPLC analysis on the Lys26(Boc)-Arg34-GLP-1(9-37) suspension with a known semaglutide backbone precursor concentration, and calculating the content of the target impurity according to the impurity peak area and the standard curve.

[0181] When the semaglutide main chain precursor sample to be tested is derived from / is a Lys26(Boc)-Arg34-GLP-1(9-37) solid sample (e.g., lyophilized powder), the quality control method may further include the steps of: preparing the Lys26(Boc)-Arg34-GLP-1(9-37) solid sample (e.g., lyophilized powder) into a solution of known concentration, performing HPLC analysis, and calculating the content of the target impurity based on the impurity peak area and the standard curve.

[0182] In the present invention, a quality control kit for a semaglutide main chain precursor is also provided, and the quality control kit comprises: the degradation impurities of the semaglutide main chain precursor as described in the third aspect of the present invention.

[0183] Preferably, the quality control kit further comprises a label and instructions, and the instructions may indicate the quality control method as described in the first aspect of the present invention.

[0184] Purification method of semaglutide main chain precursor

[0185] In the present invention, a method for purifying a semaglutide main chain precursor is provided, and the purification method comprises the steps of:

[0186] (a) providing a sample of degradation impurities of a semaglutide backbone precursor and a semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) to be purified; and

[0187] (b) using the semaglutide main chain precursor degradation impurities as impurity reference substances, using chromatography, and optimizing the chromatographic conditions and / or elution conditions to effectively separate the semaglutide main chain precursor degradation impurities in the semaglutide main chain precursor sample, thereby obtaining a purified semaglutide main chain precursor. Preferably, the purification method may further include the steps of preparing the impurity reference substances into standard solutions of different concentrations, and making a standard curve according to the impurity peak area after HPLC analysis.

[0188] The purification method may further include the steps of preparing the semaglutide backbone precursor sample to be purified into a solution of known concentration, performing HPLC analysis, and calculating the content of semaglutide backbone precursor degradation impurities in the semaglutide backbone precursor sample according to the impurity peak area and the standard curve.

[0189] The purification method may further include the step of optimizing the chromatographic conditions and / or elution conditions to improve the separation between the chromatographic peak (main peak) of the semaglutide backbone precursor and the semaglutide backbone precursor degradation impurities (impurity peaks), thereby effectively separating the semaglutide backbone precursor degradation impurities.

[0190] Typically, the content of semaglutide main chain precursor degradation impurities in the semaglutide main chain precursor to be purified is ≥2%, preferably ≥3%, more preferably ≥5%; the content of semaglutide main chain precursor degradation impurities in the purified semaglutide main chain precursor is ≤1%, preferably ≤0.8%, more preferably ≤0.5% (e.g. ≤0.45%).

[0191] In addition, the present invention also provides a method for optimizing the freeze-drying process of the main chain precursor of semaglutide, and the optimization method comprises the steps of:

[0192] (a) providing a degradation impurity of a semaglutide main chain precursor and a lyophilized sample of a semaglutide main chain precursor;

[0193] (b) using the semaglutide main chain precursor degradation impurities as an impurity reference substance, and determining the content of the semaglutide main chain precursor degradation impurities in the semaglutide main chain precursor lyophilized sample by chromatography; and

[0194] (c) by adjusting the conditions of the freeze-drying process, the content of degradation impurities of the semaglutide main chain precursor in the freeze-dried sample of the semaglutide main chain precursor is reduced, thereby obtaining the optimized conditions of the freeze-drying process.

[0195] Preferably, the conditions of the freeze-drying process include: temperature and / or freeze-drying time.

[0196] By optimizing the freeze-drying process, the content of degradation impurities of the semaglutide main chain precursor in the freeze-dried sample can be reduced, thereby achieving purification of the freeze-dried sample.

[0197] Therefore, when the degradation impurities of the semaglutide main chain precursor of the present invention are used as impurity reference substances, on the one hand, the impurities can be effectively separated by optimizing the chromatographic conditions and / or elution conditions, thereby obtaining a purified semaglutide main chain precursor; on the other hand, the generation amount of impurities and main chain precursors can be changed by optimizing the freeze-drying process (such as the temperature and / or freeze-drying time during freeze-drying), thereby obtaining a purified semaglutide main chain precursor. The purified semaglutide main chain precursor can be used to synthesize high-purity semaglutide.

[0198] Degradation impurities of semaglutide backbone precursor

[0199] The degradation impurity of the semaglutide main chain precursor is an obvious impurity that was unexpectedly found to be present after freeze-drying of the semaglutide main chain precursor (SEQ ID NO: 3) during the preparation of the semaglutide main chain precursor. This impurity often exists during the freeze-drying process, and its peak time is very close to that of the main chain precursor, so it is difficult to completely remove the impurity.

[0200] Compared with the semaglutide main chain precursor (SEQ ID NO: 3), the impurity is a product SEQ ID NO: 4 in which the N-terminal glutamic acid of SEQ ID NO: 3 is dehydrated to form pyroglutamic acid.

[0201] The difference between the degradation impurities of the semaglutide backbone precursor and the semaglutide backbone is that two amino acids are missing at the N-terminus, and the residual glutamic acid at the N-terminus is dehydrated to undergo pyroglutamic acid cyclization. The specific sequence of the degradation impurities of the semaglutide backbone precursor is as follows:

[0202] Pyr9 (pyroglutamate)

[0203] -Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala 24-Ala25-Lys26(Boc)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37(SEQ ID NO:4).

[0204] Using the preparation method described in the fifth aspect of the present invention, the inventors significantly increased the amount of impurities generated by heating and other methods, and performed separation, purification and post-treatment to obtain a high-purity impurity sample. The method of the present invention is simple to operate, and the obtained impurities are of high purity, so the high-purity impurities can be used as impurity reference substances for quality control or optimization of the semaglutide production process (especially the semaglutide main chain precursor-related process), and contribute to the synthesis of high-purity final products, such as the quality control method described in the first aspect of the present invention, the purification method described in the second aspect of the present invention, and the optimization method described in the eighth aspect of the present invention.

[0205] Preparation method of degradation impurities of semaglutide main chain precursor

[0206] The present invention provides a method for preparing a semaglutide main chain precursor degradation impurity, the method comprising the steps of:

[0207] (a) providing a semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample;

[0208] (b) heat treating the Lys26(Boc)-Arg34-GLP-1(9-37) sample at 55-85°C for a period of time; and

[0209] (c) purifying the product obtained in step (b) to obtain degradation impurities of semaglutide main chain precursor.

[0210] The prepared semaglutide main chain precursor degradation impurity has the sequence shown in SEQ ID NO: 4:

[0211] Pyr9 (pyroglutamate)

[0212] -Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala 24-Ala25-Lys26(Boc)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37(SEQ ID NO:4).

[0213] When the purification method in step (c) is a chromatographic purification method, preferably the chromatographic purification method may include the steps of:

[0214] (c1) The product obtained in step (b) is loaded onto a high performance liquid chromatography column, using phase A acidic aqueous solution and phase B organic solvent as mobile phases, performing gradient elution, and collecting the eluate.

[0215] Furthermore, the preparation method of the present invention may also include a freeze-drying step:

[0216] (d) Lyophilization: The eluate obtained in step (c1) is diluted with water until the organic solvent in phase B is less than 10%, and lyophilized for 10-16 hours under the conditions of vacuum degree ≤ 10 mbar and baffle temperature control at 25° C., thereby obtaining a lyophilized preparation of degradation impurities of the semaglutide main chain precursor.

[0217] In the above preparation method, typically:

[0218] For step (a): the Lys26(Boc)-Arg34-GLP-1(9-37) sample is in a non-salt free state; it may include a suspension sample or a solid sample, for example, the suspension sample is a 10%-70% suspension, preferably a 30%-60% suspension, and more preferably a 50% suspension;

[0219] The semaglutide main chain precursor Lys26(Boc)-Arg34-GLP-1(9-37) sample has a sequence as shown in SEQ ID NO: 3:

[0220] Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-A la24-Ala25-Lys26(Boc)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37(SEQ ID NO:3).

[0221] For step (b): the heat treatment temperature is 55-85°C, preferably 55°C, 65°C, 75°C or 85°C; more preferably 65°C or 75°C; the heat treatment time is 6-36 hours, preferably 12-24 hours, more preferably 24 hours.

[0222] For step (c): the purification method is preferably a chromatographic purification method, which may include the steps of:

[0223] (c1) applying the product obtained in step (b) to a high performance liquid chromatography column, using phase A acidic aqueous solution and phase B organic solvent as mobile phases, performing gradient elution, and collecting the eluate;

[0224] The filler used in the high performance liquid phase preparative chromatography column is a reverse phase chromatography filler; the filler type is selected from one of C18 and C8; the particle size of the filler is 10 to 30 μm, preferably 10 μm;

[0225] The phase A acidic aqueous solution is selected from one of formic acid, acetic acid, and trifluoroacetic acid. Preferably, the proportion of the acid additive in the phase A acidic aqueous solution is 0.1%-0.5% by volume, preferably 0.1% by volume; the phase B organic solvent is selected from one of methanol, ethanol, and acetonitrile, or the phase B organic solvent is an acidic organic solvent, preferably an organic solvent added with trifluoroacetic acid; more preferably, acetonitrile added with 0.1% by volume of trifluoroacetic acid (0.1% trifluoroacetic acid / acetonitrile);

[0226] For example, the acidic aqueous solution of phase A is 0.1% formic acid, and the organic solvent of phase B is acetonitrile; or, the acidic aqueous solution of phase A is 0.1% trifluoroacetic acid, and the organic solvent of phase B is acetonitrile to which trifluoroacetic acid is added at a volume fraction of 0.1% (0.1% trifluoroacetic acid / acetonitrile);

[0227] The procedure of the gradient elution is as follows:

[0228] (S1) When the elution start time is 0 min, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 5-10%, and the percentage content of the organic solvent of phase B is 95-90%; preferably, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 10%, and the percentage content of the organic solvent of phase B is 90%;

[0229] (S2) when the elution start time is 10 min, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 65-58%, and the percentage content of the organic solvent of phase B is 35-42%; preferably, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 59%, and the percentage content of the organic solvent of phase B is 41%; and

[0230] (S3) When the elution start time is 70 min, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 45-38%, and the percentage content of the organic solvent of phase B is 55-62%; preferably, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 39%, and the percentage content of the organic solvent of phase B is 61%.

[0231] For step (d): the organic solvent of phase B is preferably acetonitrile.

[0232] It should be understood that for those skilled in the art, a variety of purification methods well known in the art can be used for step (c) in the preparation method of the present invention, and are not limited to the chromatographic purification method. The chromatographic purification method used in the embodiment of the present invention is only used as an example for illustration, and is not intended to limit the scope of the present invention. Similarly, the preferred features in the above steps are also only used as examples for illustration, and are not intended to limit the scope of the present invention.

[0233] In addition, although the degradation impurities of the semaglutide main chain precursor may be prepared by methods other than the preparation method of the present invention, the content of the impurities prepared by other methods is lower and the non-target impurities contained in the sample are uncontrollable, so the preparation method of the present invention has advantages.

[0234] Kit for the preparation of degradation impurities of semaglutide main chain precursor

[0235] The present invention provides a kit for preparing a semaglutide main chain precursor degradation impurity, the kit comprising:

[0236] (a) a first container and a sample of semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) located in the first container; and

[0237] (b) a second container and a heating device located within the second container.

[0238] Preferably, the Lys26(Boc)-Arg34-GLP-1(9-37) sample is in a non-salt free state; it can be a suspension sample or a solid sample; the heating device is configured to make the heating temperature 55-85°C, preferably 55°C, 65°C, 75°C or 85°C; more preferably 65°C or 75°C; the heating device is configured to make the single heating time 6-36 hours, preferably 12-24 hours, more preferably 24 hours.

[0239] The kit of the present invention may optionally further include: (c) a third container and a purification reagent located in the third container.

[0240] The purification reagent may include a chromatography purification reagent, for example, including a phase A acidic aqueous solution and / or a phase B organic solvent.

[0241] Preferably, the phase A acidic aqueous solution is selected from one of formic acid, acetic acid, and trifluoroacetic acid; the phase B organic solvent is selected from one of methanol, ethanol, and acetonitrile, or the phase B organic solvent is an acidic organic solvent, preferably an organic solvent added with trifluoroacetic acid; more preferably, acetonitrile added with 0.1% volume fraction of trifluoroacetic acid (0.1% trifluoroacetic acid / acetonitrile).

[0242] The first container, the second container and / or the third container mentioned above may be the same container or different containers.

[0243] The kit of the present invention may optionally further comprise a label and instructions, wherein the instructions may preferably indicate the preparation method as described in the fifth aspect of the present invention and / or the use as described in the fourth aspect of the present invention.

[0244] Preparation device for degradation impurities of semaglutide main chain precursor

[0245] The present invention provides a device for preparing a semaglutide main chain precursor and degrading impurities, the device comprising:

[0246] (M1) a sample loading module, wherein the sample loading module is configured to: provide a semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample;

[0247] (M2) a heat treatment module, wherein the heat treatment module is configured to: make the heat treatment temperature 55-85° C. and the single heating time 6-36 hours, thereby obtaining a heat-treated product; and

[0248] (M3) a purification module, wherein the purification module is configured to purify the product to obtain degradation impurities of the semaglutide main chain precursor.

[0249] Typically, the (M3) purification module further includes a gradient elution submodule, which is configured to: perform gradient elution on the degradation product using phase A acidic aqueous solution and phase B organic solvent as mobile phases, and collect the eluate.

[0250] The procedure of gradient elution in the gradient elution submodule is as follows:

[0251] (S1) When the elution start time is 0 min, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 5-10%, and the percentage content of the organic solvent of phase B is 95-90%; preferably, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 10%, and the percentage content of the organic solvent of phase B is 90%;

[0252] (S2) when the elution start time is 10 min, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 65-58%, and the percentage content of the organic solvent of phase B is 35-42%; preferably, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 59%, and the percentage content of the organic solvent of phase B is 41%; and

[0253] (S3) When the elution start time is 70 min, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 45-38%, and the percentage content of the organic solvent of phase B is 55-62%; preferably, the percentage content of the acidic aqueous solution of phase A in the mobile phase is 39%, and the percentage content of the organic solvent of phase B is 61%.

[0254] Preferably, the device also includes a (M4) freeze-drying module, which is configured to: dilute the eluate obtained in the gradient elution submodule with water until the organic solvent of phase B is less than 10%, and freeze-dry for 10-16 hours under the conditions of vacuum degree ≤10mbar and baffle temperature control of 25°C, thereby obtaining a freeze-dried preparation of degradation impurities of the semaglutide main chain precursor.

[0255] The device further comprises a control module (M5), which is configured to: control the operation of the sample loading module (M1), the heating degradation module (M2) and the purification module (M3), for example, the control module (M5) controls the following modules in the device to operate in sequence:

[0256] (M1) sample loading module, (M2) heating degradation module, (M3) purification module, that is, they are operated in the order of (M1) → (M2) → (M3);

[0257] Alternatively, when the device further includes a freeze-drying module (M4), the control module (M5) further controls the operation of the freeze-drying module (M4), for example, the control module (M5) controls the following modules in the device to operate in sequence:

[0258] (M1) sample loading module, (M2) heating degradation module, (M3) purification module and (M4) freeze drying module are operated in the order of (M1) → (M2) → (M3) → (M4).

[0259] Main advantages of the present invention

[0260] 1. The present invention unexpectedly found that the degradation of the semaglutide main chain precursor was not obvious in the solution state, but it degraded faster in the high concentration suspension state or even in the solid state, and high temperature significantly promoted the degradation, and significantly increased the yield of the target impurity of the present invention. After the solid pure product was heated at 65°C for 24 hours, more than 75% of the main peak (semaglutide main chain precursor) was converted into SEQ ID NO: 4 impurity.

[0261] 2. When the heated degradation products are subsequently purified, the present invention only needs to adopt a simple chromatographic purification method or other simple purification methods to obtain a high-purity impurity sample.

[0262] 3. Compared with the impurity samples prepared by the method of the present invention, the impurity content obtained by other degradation methods is lower, and the non-target impurities contained in the samples are uncontrollable.

[0263] 4. The high-purity impurities prepared in the present invention can be used as impurity reference substances for quality control of the semaglutide production process (e.g., semaglutide main chain precursor-related processes), and can also be used for purification of semaglutide main chain precursors and optimization of main chain precursor freeze-drying processes.

[0264] For example, when the degradation impurities of the semaglutide main chain precursor of the present invention are used as impurity reference substances, on the one hand, the impurities can be effectively separated by optimizing the chromatographic conditions and / or elution conditions, thereby obtaining a purified semaglutide main chain precursor; on the other hand, the generation amount of impurities and main chain precursors can be changed by optimizing the freeze-drying process (such as the temperature and / or freeze-drying time during freeze-drying), thereby obtaining a purified semaglutide main chain precursor. In addition, the purified semaglutide main chain precursor can be further used to synthesize high-purity semaglutide.

[0265] Therefore, the impurities and preparation methods and preparation kits of the present invention are of great significance to multiple links in the semaglutide synthesis process.

[0266] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0267] Sequence Listing

[0268]

[0269]

[0270] General Methods

[0271] The pure Lys26(Boc)-Arg34-GLP-1(9-37) (semaglutide main chain precursor) used in the present invention is prepared by the method described in Example 7 of patent CN113801233A.

[0272] Example 1: Discovery of degradation impurities of semaglutide backbone precursor

[0273] The semaglutide main chain precursor degradation impurity (SEQ ID NO: 4) described in the present invention is an obvious impurity unexpectedly found in the preparation of the semaglutide main chain precursor (SEQ ID NO: 3) after freeze-drying. After research, it was determined that compared with the target product semaglutide main chain precursor (SEQ ID NO: 3), the impurity is the product SEQ ID NO: 4 in which the N-terminal glutamic acid of SEQ ID NO: 3 is dehydrated to form pyroglutamic acid.

[0274] like Figure 1As shown, the HPLC spectrum of the precursor before lyophilization is shown, wherein the main peak appearing at about 15-16 min is the target product semaglutide main chain precursor (SEQ ID NO: 3).

[0275] like Figure 2 As shown, the HPLC spectrum of the precursor after freeze-drying is shown, wherein the impurity peak appearing at about 16-17 min is the degradation impurity of the semaglutide main chain precursor (SEQ ID NO: 4).

[0276] From the above results, it can be seen that this impurity often exists in the freeze-drying process, and its peak time is very close to that of the target product, and this impurity is difficult to completely remove.

[0277] Example 2: Preparation of semaglutide (suspension) degradation impurities

[0278] (1) Degradation

[0279] 200 mg of Lys26(Boc)-Arg34-GLP-1(9-37) solid pure product was added with pure water to prepare a 50% suspension, which was heated and degraded at 35°C, 45°C, 55°C, 65°C, 75°C, and 85°C for 12-36 hours, and samples were taken for HPLC analysis. The results are shown in Table 1.

[0280] Table 1 Degradation results of pure semaglutide main chain precursor

[0281]

[0282] According to Table 1, it can be seen that as the temperature increases from 35°C to 45°C, 55°C, 65°C, 75°C, and 85°C (with the same heating time), it is unexpectedly found that the degradation of the main peak accelerates.

[0283] When the temperature is 85°C (Condition 8 in Table 1), the main peak content percentage is reduced to about 45.7% of the main peak content percentage when the temperature is 35°C (Condition 1 in Table 1).

[0284] As the temperature increases from 35°C to 45°C, 55°C, 65°C, and 75°C, the content of the target impurity gradually increases. However, when the temperature continues to increase to 85°C (Condition 8 in Table 1), the content of the target impurity is lower than that at 75°C (Condition 7 in Table 1), but is still higher than the target impurity content at 65°C, 55°C, 45°C, and 35°C.

[0285] When the temperature is 75° C. (Condition 7 in Table 1), the target impurity content percentage reaches about 9.6 times the target impurity content percentage when the temperature is 35° C. (Condition 1 in Table 1).

[0286] Moreover, a proper increase in degradation time will also accelerate the degradation of the main peak and increase the content of the target impurity. For example, at the same temperature (such as 65°C), when the degradation time is 24h, the target impurity content percentage reaches about 4.4 times the target impurity content percentage when the degradation time is 12h; the main peak content percentage is reduced to about 40.3% of the main peak content percentage when the degradation time is 12h.

[0287] However, when the degradation time is too long, although the main peak will continue to degrade, the target impurity content percentage will decrease. For example, at the same temperature (such as 65°C), when the degradation time is 36h (Table 1 Condition 6), the target impurity content percentage is 44.23%, which is lower than the target impurity content percentage of 55.01% when the degradation time is 24h (Table 1 Condition 5), but still higher than the target impurity content percentage of 12.52% when the degradation time is 12h (Table 1 Condition 4).

[0288] (2) Sample purification

[0289] The chromatographic column was C8 (10 μm, 10*250mm), 0.1% formic acid as phase A, acetonitrile as phase B. The degradation product under condition 5 in Table 1 (its HPLC spectrum is as shown in Figure 3 The column was loaded with the elution program shown in Table 2, and the eluate was collected at a wavelength of 210 nm. The purification yield of the target impurity in the eluate was 82.6%, and the impurity purity was 94.8% (the HPLC spectrum of the purified impurity was as shown in Figure 4 shown).

[0290] Table 2 Elution gradient table during purification

[0291]

[0292] (3) Freeze-drying

[0293] The eluate from step (2) was diluted with water until the acetonitrile content was less than 10%, and freeze-dried for 16 h at a vacuum degree of ≤10 mbar and a baffle temperature of 25°C to obtain a freeze-dried target impurity with a purity of 95.9%.

[0294] Example 3: Preparation of semaglutide (solid) degradation impurities

[0295] (1) Degradation

[0296] 200 mg of Lys26(Boc)-Arg34-GLP-1(9-37) solid pure product was directly heated and degraded at 35°C, 45°C, 55°C, 65°C, 75°C, and 85°C for 12-36 hours, and samples were taken for HPLC analysis. The results are shown in Table 3.

[0297] Table 3 Degradation results of pure semaglutide main chain precursor

[0298]

[0299] According to Table 3, it can be seen that as the temperature increases from 35°C to 45°C, 55°C, 65°C, 75°C, and 85°C (same heating time), it is unexpectedly found that the degradation of the main peak accelerates.

[0300] When the temperature is 85°C (Condition 8 in Table 3), the main peak content percentage is reduced to about 38.5% of the main peak content percentage when the temperature is 35°C (Condition 1 in Table 3).

[0301] As the temperature increases from 35°C to 45°C, 55°C, 65°C, and 75°C, the content of target impurities gradually increases. However, when the temperature continues to increase to 85°C (Condition 8 in Table 3), the content of target impurities is lower than that at 75°C (Condition 7 in Table 3), but is still close to the target impurity content at 65°C and 55°C (Conditions 4 and 3 in Table 3), and is higher than the target impurity content at 45°C and 35°C.

[0302] When the temperature is 75° C. (Condition 7 in Table 3), the target impurity content percentage reaches about 9.6 times the target impurity content percentage when the temperature is 35° C. (Condition 1 in Table 3).

[0303] Moreover, a proper increase in degradation time will also accelerate the degradation of the main peak and increase the content of the target impurity. For example, at the same temperature (such as 65°C), when the degradation time is 24h, the target impurity content percentage reaches about 1.7 times the target impurity content percentage when the degradation time is 12h; the main peak content percentage is reduced to about 26.2% of the main peak content percentage when the degradation time is 12h.

[0304] However, when the degradation time is too long, although the main peak will continue to degrade, the target impurity content percentage will decrease. For example, at the same temperature (such as 65°C), when the degradation time is 36h (Table 3 Condition 6), the target impurity content percentage is 73.56%, which is lower than the target impurity content percentage of 76.86% when the degradation time is 24h (Table 3 Condition 5), but still higher than the target impurity content percentage of 44.58% when the degradation time is 12h (Table 3 Condition 4).

[0305] (2) Sample purification

[0306] The chromatographic column was C8 (10 μm, 10*250mm), 0.1% trifluoroacetic acid as phase A, 0.1% trifluoroacetic acid / acetonitrile as phase B. The degradation solution of condition 5 in Table 3 was loaded onto the column, the elution program in Table 2 was run, and the eluate was collected at a wavelength of 210nm. The purification yield of the target impurity in the eluate was 85.3%, and the impurity purity was 98.9% (the HPLC spectrum of the purified impurity is shown in Figure 5 shown).

[0307] (3) Freeze-drying

[0308] The eluate from step (2) was diluted with water until the acetonitrile content was less than 10%, and freeze-dried for 16 h at a vacuum degree of ≤10 mbar and a baffle temperature of 25°C to obtain a freeze-dried target impurity with a purity of 99.02%.

[0309] Example 4: Quality control or purification method of degradation impurities of semaglutide main chain precursor Application in freeze-drying process optimization

[0310] By preparing the impurity reference substance, the impurity quantitative analysis of the semaglutide main chain precursor in the process can be conveniently performed, such as the following examples.

[0311] Impurity Quantification of Lyophilized Main Chain Precursor Powder

[0312] First, the mass spectrum of the degradation impurity of the main chain precursor of semaglutide (SEQ ID NO: 4) with high purity is obtained by the preparation method of the present invention. Figure 6 ). Subsequently, the solution prepared from the main chain precursor lyophilized powder was analyzed by HPLC method, and the target impurity was used as a control, and the position of the impurity in the HPLC spectrum could be conveniently determined based on the control.

[0313] Secondly, the prepared impurity reference substance is prepared into standard solutions of different concentrations, and a standard curve is made according to the peak area after HPLC analysis; then the main chain precursor is prepared into a solution of known concentration, HPLC analysis is performed, and the content of the target impurity is calculated according to the impurity peak area and the standard curve.

[0314] like Figure 7 As shown, the content of impurities in the main chain precursor lyophilized powder solution was detected using an impurity reference substance, and the content was 3.55%.

[0315] The above HPLC analysis method is as follows:

[0316] Chromatographic column: C18 (4.6*250mm, 5μm)

[0317] Mobile phase A: 10 mM diammonium phosphate pH 10.2

[0318] Mobile phase B: 80% ACN in water

[0319] Detection wavelength: 214nm

[0320] Flow rate: 1.0mL / min

[0321] Column temperature: 35°C

[0322] The elution gradient is shown in Table 4:

[0323] Table 4 Elution gradient table during HPLC analysis

[0324] Time (min) Mobile phase A (%) Mobile phase B (%) 0 75 25 2 75 25 21 35 65 21.5 0 100 25 0 100 25.1 75 25 30 75 25

[0325] Therefore, degradation impurities of the semaglutide backbone precursor can be used as impurity reference substances for quality control of the semaglutide backbone precursor.

[0326] Furthermore, by optimizing the chromatographic conditions and / or elution conditions, the degradation impurities of the semaglutide backbone precursor in the semaglutide backbone precursor sample can be effectively separated (for example, the separation between the chromatographic peak (main peak) of the semaglutide backbone precursor and the degradation impurities (impurity peak) of the semaglutide backbone precursor can be improved), thereby obtaining a purified semaglutide backbone precursor.

[0327] In addition, the inventors studied the degradation law of the impurities, combined with the quantitative analysis of impurities using impurity reference substances, and after multiple attempts and quantitative tests, found that temperature and freeze-drying time have a significant effect on controlling the impurity content in freeze-dried powder using the control variable method. Figure 8 The inventors finally reduced the content of this impurity in the freeze-dried powder from about 3.55% to less than 0.5% by optimizing the freeze-drying process of the main chain precursor.

[0328] Therefore, by preparing the impurity and using it as an impurity standard to achieve quantitative analysis of impurities, it can further help optimize the freeze-drying process of the main chain precursor and have guiding significance for the freeze-drying process.

[0329] The purified semaglutide main chain precursor (including lyophilized powder) thus obtained can be used to synthesize high-purity semaglutide. Therefore, the degradation impurities of the semaglutide main chain precursor are used as impurity reference substances, which are of great significance to the quality control and process optimization of the semaglutide production process.

[0330] Comparative Example

[0331] 200 mg of Lys26(Boc)-Arg34-GLP-1(9-37) solid pure product was added with pure water to prepare suspensions of different concentrations, which were heated and degraded at 75°C for 12 hours, and samples were taken for HPLC analysis. The results are shown in Table 5.

[0332] Table 5 Degradation results of pure semaglutide main chain precursor

[0333] NO. Concentration mg / mL. state Main peak% Target impurity% 1 0.5 Solution 97.47 0 2 2 Solution 96.87 0 3 10 Suspension 90.01 5.42 4 30 Suspension 70.53 20.32 5 70 Suspension 61.12 30.05

[0334] According to Table 5, when the pure solid product of Lys26(Boc)-Arg34-GLP-1(9-37) is prepared as a solution, the target impurity cannot be obtained; when the pure solid product of Lys26(Boc)-Arg34-GLP-1(9-37) is prepared as a 10% suspension, the target impurity can be generated but the content is low; when the pure solid product of Lys26(Boc)-Arg34-GLP-1(9-37) is prepared as a 30% or 70% suspension, the content of the target impurity is greatly increased.

[0335] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A quality control method for a semaglutide main chain precursor, characterized in that: The quality control method comprises the steps of: (a) providing a semaglutide backbone precursor degradation impurity and a semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample to be tested, wherein the semaglutide backbone precursor degradation impurity has a sequence as shown in SEQ ID NO: 4; and (b) using the semaglutide main chain precursor degradation impurities as impurity reference substances, and determining the content of the semaglutide main chain precursor degradation impurities in the semaglutide main chain precursor sample to be tested by chromatography, thereby performing quality control of the semaglutide main chain precursor.

2. A method for purifying a semaglutide main chain precursor, characterized in that: The purification method comprises the steps of: (a) providing a semaglutide backbone precursor degradation impurity and a semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample to be purified, wherein the semaglutide backbone precursor degradation impurity has a sequence as shown in SEQ ID NO: 4; and (b) using the semaglutide backbone precursor degradation impurities as impurity reference substances, using chromatography, and optimizing the chromatographic conditions and / or elution conditions to effectively separate the semaglutide backbone precursor degradation impurities in the semaglutide backbone precursor sample, thereby obtaining a purified semaglutide backbone precursor.

3. A degradation impurity of a semaglutide main chain precursor, characterized in that: The semaglutide main chain precursor degradation impurity has a sequence as shown in SEQ ID NO:

4.

4. A use of the semaglutide main chain precursor for degrading impurities according to claim 3, characterized in that: Used for: (Z1) as an impurity reference substance for quality control or optimization of the semaglutide production process (e.g., semaglutide backbone precursor); and / or (Z2) was used as an impurity control substance for the purification of semaglutide backbone precursor.

5. A method for preparing semaglutide main chain precursor degradation impurities, characterized in that: The preparation method comprises the steps of: (a) providing a semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample; (b) heat treating the Lys26(Boc)-Arg34-GLP-1(9-37) sample at 55-85° C. for a period of time; and (c) purifying the product obtained in step (b) to obtain semaglutide backbone precursor degradation impurities, wherein the semaglutide backbone precursor degradation impurities have a sequence as shown in SEQ ID NO:

4.

6. The preparation method according to claim 5, characterized in that: The heating temperature in step (b) is 55°C, 65°C, 75°C or 85°C; preferably 65°C or 75°C.

7. A kit for preparing the semaglutide main chain precursor degradation impurity as claimed in claim 3, characterized in that: The kit comprises: (a) a first container and a sample of semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) located in the first container; and (b) a second container and a heat treatment device located in the second container.

8. A quality control kit for semaglutide main chain precursor, characterized in that: The quality control kit comprises: the degradation impurities of the semaglutide main chain precursor as described in claim 3.

9. A method for optimizing the freeze-drying process of a semaglutide main chain precursor, characterized in that: The optimization method comprises the steps of: (a) providing a semaglutide backbone precursor degradation impurity and a semaglutide backbone precursor lyophilized sample, wherein the semaglutide backbone precursor degradation impurity has a sequence as shown in SEQ ID NO: 4; (b) using the semaglutide main chain precursor degradation impurities as an impurity reference substance, and determining the content of the semaglutide main chain precursor degradation impurities in the semaglutide main chain precursor lyophilized sample by chromatography; and (c) by adjusting the conditions of the freeze-drying process, the content of degradation impurities of the semaglutide main chain precursor in the freeze-dried sample of the semaglutide main chain precursor is reduced, thereby obtaining the optimized conditions of the freeze-drying process.

10. A device for preparing the semaglutide main chain precursor degradation impurity as claimed in claim 3, characterized in that: The device comprises: (M1) a sample loading module, wherein the sample loading module is configured to: provide a semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample; (M2) a heat treatment module, wherein the heat treatment module is configured to: make the heat treatment temperature 55-85° C. and the single heating time 6-36 hours, thereby obtaining a heat-treated product; and (M3) a purification module, wherein the purification module is configured to purify the product to obtain degradation impurities of the semaglutide main chain precursor.

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