Smeglutide main chain precursor enzymolysis impurity and preparation method thereof

Abstract

The invention provides a stemeglutide main chain precursor enzymolysis impurity and a preparation method thereof. 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 the enzymolysis impurity of the main chain precursor of the semeglutide. According to the method disclosed by the invention, a large amount of the stemeglutide main chain precursor enzymolysis impurities which only exist in a small amount after an enzyme digestion process originally can be obtained by adopting a method which is simple to operate, and the impurities are high in purity, can be used for quality control in a stemeglutide production process and are 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 enzymatic hydrolysis 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-nedioicAcid)-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. The impurity situation will affect the quality of the final product. Therefore, in this field, high-purity semaglutide main chain precursor impurities are needed as impurity reference substances for quality control of the semaglutide production process and to help obtain high-purity final products. Therefore, there is an urgent need in this field to develop a semaglutide main chain precursor enzymatic hydrolysis impurity and a preparation method thereof. Summary of the invention

[0009] The purpose of the present invention is to provide a semaglutide main chain precursor enzymatic hydrolysis impurity and a preparation method thereof.

[0010] Another object of the present invention is to provide a quality control or purification method for a semaglutide main chain precursor.

[0011] 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:

[0012] (a) providing a semaglutide main chain precursor enzymatic impurity and a semaglutide main chain precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample to be tested; and

[0013] (b) using the enzymatic impurities of the semaglutide main chain precursor as impurity reference substances, and determining the content of the enzymatic impurities of the semaglutide main chain precursor in the semaglutide main chain precursor sample to be tested by chromatography, thereby performing quality control of the semaglutide main chain precursor.

[0014] In another preferred embodiment, the enzymatic hydrolysis impurity of the semaglutide main chain precursor has a sequence as shown in SEQ ID NO:4.

[0015] In another preferred embodiment, the Lys26(Boc)-Arg34-GLP-1(9-37) sample is derived from the precursor fusion protease cleavage solution or the pure Lys26(Boc)-Arg34-GLP-1(9-37).

[0016] In another preferred embodiment, the structure of the precursor fusion protein is:

[0017] Leader peptide-DDDDK-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17

[0018] -Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26(Boc)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37.

[0019] In another preferred example, the precursor fusion protein includes the sequence shown in SEQ ID NO:6.

[0020] In another preferred embodiment, the concentration of Lys26(Boc)-Arg34-GLP-1(9-37) is 0.5-3 mg / mL, preferably 0.7-2 mg / mL.

[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, C8, PS / DVB, and PMMA; 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 precursor fusion protease digestion liquid, and performing HPLC analysis on the precursor fusion protease digestion liquid, and calculating the content of semaglutide main chain precursor enzymatic impurities (target impurities) according to the impurity peak area and the standard curve; or, performing HPLC analysis on the fusion protease digestion liquid 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 pure Lys26(Boc)-Arg34-GLP-1(9-37) 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 semaglutide backbone precursor enzymatic impurities and a semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) to be purified; and

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

[0045] In another preferred embodiment, the enzymatic hydrolysis 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 derived from the precursor fusion protease cleavage solution or the pure Lys26(Boc)-Arg34-GLP-1(9-37).

[0047] In another preferred embodiment, the structure of the precursor fusion protein is:

[0048] Leader peptide-DDDDK-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17

[0049] -Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26(Boc)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37.

[0050] In another preferred example, the precursor fusion protein includes the sequence shown in SEQ ID NO:6.

[0051] In another preferred embodiment, the concentration of Lys26(Boc)-Arg34-GLP-1(9-37) is 0.5-3 mg / mL, preferably 0.7-2 mg / mL.

[0052] In another preferred embodiment, the chromatography is high performance liquid chromatography (HPLC).

[0053] In another preferred embodiment, the filler used in the chromatographic column used in the chromatography method is a reverse phase chromatography filler.

[0054] In another preferred embodiment, the filler type is selected from one of C18, C8, PS / DVB, and PMMA; preferably C18.

[0055] In another preferred embodiment, the chromatography uses mobile phase A and mobile phase B for gradient elution.

[0056] In another preferred embodiment, the mobile phase A is diammonium hydrogen phosphate.

[0057] In another preferred embodiment, the mobile phase B is acetonitrile (ACN) or an acetonitrile (ACN) aqueous solution.

[0058] 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.

[0059] 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 enzymatic impurities in the semaglutide backbone precursor sample according to the impurity peak area and the standard curve.

[0060] 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 main chain precursor and the enzymatic impurities (impurity peak) of the semaglutide main chain precursor, thereby effectively separating the enzymatic impurities of the semaglutide main chain precursor.

[0061] In another preferred embodiment, the content of semaglutide main chain precursor enzymatic impurities in the semaglutide main chain precursor to be purified is ≥1%, preferably ≥1.5%, and more preferably ≥2%.

[0062] In another preferred embodiment, the content of semaglutide main chain precursor enzymatic impurities in the purified semaglutide main chain precursor is ≤0.8%, preferably ≤0.6%, and more preferably ≤0.5%.

[0063] In another preferred embodiment, the purified semaglutide backbone precursor is used to synthesize semaglutide (high purity).

[0064] In a third aspect of the present invention, a semaglutide main chain precursor enzymatic impurity is provided, wherein the semaglutide main chain precursor enzymatic impurity has a sequence as shown in SEQ ID NO:4.

[0065] In the fourth aspect of the present invention, there is provided a use of the enzymatic hydrolysis impurities of the semaglutide main chain precursor according to the third aspect of the present invention, for:

[0066] (Z1) as an impurity reference substance for quality control of the semaglutide production process (e.g., semaglutide backbone precursor); and / or

[0067] (Z2) was used as an impurity control substance for the purification of semaglutide backbone precursor.

[0068] 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.

[0069] 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.

[0070] In a fifth aspect of the present invention, a method for preparing enzymatic impurities of a semaglutide main chain precursor is provided, the preparation method comprising the steps of:

[0071] (a) providing a reaction system, the reaction system comprising: a semaglutide main chain precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample, and the reaction system undergoes a cleavage reaction under the conditions of pH 9 to 12 and enterokinase;

[0072] (b) allowing the cleavage reaction in step (a) to proceed for a period of time to obtain a cleaved solution; and

[0073] (c) purifying the enzymatically cleaved solution obtained in step (b) to obtain enzymatically cleaved impurities of the semaglutide main chain precursor.

[0074] In another preferred embodiment, the Lys26(Boc)-Arg34-GLP-1(9-37) sample is derived from the precursor fusion protease cleavage solution or the pure Lys26(Boc)-Arg34-GLP-1(9-37).

[0075] In another preferred embodiment, the structure of the precursor fusion protein is:

[0076] Leader peptide-DDDDK-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17

[0077] -Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26(Boc)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37.

[0078] In another preferred example, the precursor fusion protein includes the sequence shown in SEQ ID NO:6.

[0079] In another preferred embodiment, the concentration of Lys26(Boc)-Arg34-GLP-1(9-37) is 0.5-3 mg / mL, preferably 0.7-2 mg / mL.

[0080] In another preferred embodiment, the concentration of enterokinase is 0.1-1.0 U / mg, preferably 0.5 U / mg.

[0081] In another preferred embodiment, the reaction system further contains a buffer substance.

[0082] In another preferred embodiment, the reaction system contains sodium chloride.

[0083] In another preferred embodiment, the sodium chloride concentration is 50-200 mM, preferably 150 mM.

[0084] In another preferred embodiment, the reaction system contains 20-50 mM Tris, preferably 50 mM Tris.

[0085] In another preferred embodiment, the cleavage reaction is carried out at room temperature.

[0086] In another preferred embodiment, NaOH solution is used in step (a) to make the reaction system reach pH 9-12.

[0087] 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:

[0088] 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).

[0089] In another preferred embodiment, the semaglutide main chain precursor enzymatic impurity has a sequence as shown in SEQ ID NO: 4:

[0090] Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly2 2-Gln23-Ala24-Ala25-Lys26(Boc)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34(SEQ ID NO:4).

[0091] In another preferred embodiment, the cleavage reaction in step (b) is carried out for 24 to 48 hours.

[0092] In another preferred embodiment, the purification method in step (c) is a chromatographic purification method.

[0093] In another preferred embodiment, the chromatographic purification method comprises the steps of:

[0094] (c1) The digested solution obtained in step (b) is loaded onto a high performance liquid chromatography column, and gradient elution is performed using a phase A buffer solution and a phase B organic solvent as mobile phases, and the eluate is collected.

[0095] In another preferred embodiment, the filler used in the HPLC preparative chromatography column is a reverse phase chromatography filler.

[0096] In another preferred embodiment, the filler type is selected from one of C18, C8, PS / DVB, and PMMA.

[0097] In another preferred embodiment, the particle size of the filler is 10-30 μm, preferably 10 μm.

[0098] In another preferred embodiment, the phase A buffer aqueous solution is selected from one of ammonium acetate, ammonium formate, ammonium bicarbonate, and ammonia solution.

[0099] In another preferred embodiment, the organic solvent of phase B is selected from one of methanol, ethanol, isopropanol and acetonitrile.

[0100] In another preferred embodiment, the buffered aqueous solution of phase A is an aqueous ammonia solution, and the organic solvent of phase B is acetonitrile.

[0101] In another preferred embodiment, the buffered aqueous solution of phase A is ammonium bicarbonate, and the organic solvent of phase B is acetonitrile.

[0102] In another preferred embodiment, the concentration of the phase A buffer solution is 20-50 mM, preferably 50 mM.

[0103] In another preferred embodiment, the pH of the phase A buffer solution is 8-11.

[0104] In another preferred embodiment, the buffer composition of the organic solvent of phase B is all volatile substances.

[0105] In another preferred embodiment, the preparation method does not include a step involving a desalting process.

[0106] In another preferred embodiment, the gradient elution procedure is as follows:

[0107] (S1) When the elution start time is 0 min, the percentage content of the phase A buffer solution in the mobile phase is 97-95%, and the percentage content of the phase B organic solvent is 3-5%; preferably, the percentage content of the phase A buffer solution in the mobile phase is 95%, and the percentage content of the phase B organic solvent is 5%;

[0108] (S2) when the elution start time is 10 min, the percentage content of the buffer solution of phase A in the mobile phase is 97-95%, and the percentage content of the organic solvent of phase B is 3-5%; preferably, the percentage content of the buffer solution of phase A in the mobile phase is 95%, and the percentage content of the organic solvent of phase B is 5%; and

[0109] (S3) When the elution start time is 40 min, the percentage content of the phase A buffer aqueous solution in the mobile phase is 85-80%, and the percentage content of the phase B organic solvent is 15-20%; preferably, the percentage content of the phase A buffer aqueous solution in the mobile phase is 82%, and the percentage content of the phase B organic solvent is 18%.

[0110] In another preferred embodiment, the preparation method further comprises the steps of:

[0111] (d) Lyophilization: The eluate obtained in step (c1) is diluted with water until the organic solvent in phase B is less than 5%, and lyophilized for 10 to 16 hours under the conditions of vacuum degree ≤ 10 mbar and baffle temperature control at 25° C., thereby obtaining a lyophilized preparation of the enzymatic hydrolysis impurities of the semaglutide main chain precursor.

[0112] In another preferred embodiment, the organic solvent of phase B in step (d) is acetonitrile.

[0113] In a sixth aspect of the present invention, a kit for preparing enzymatic impurities of a semaglutide main chain precursor is provided, the kit comprising:

[0114] (a) a first container and a sample of semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) located in the first container; and

[0115] (b) a second container and enterokinase or a buffered saline solution containing enterokinase located in the second container.

[0116] In another preferred embodiment, the enzymatic hydrolysis impurity of the semaglutide main chain precursor has a sequence as shown in SEQ ID NO:4.

[0117] In another preferred embodiment, the Lys26(Boc)-Arg34-GLP-1(9-37) sample is derived from the precursor fusion protease cleavage solution or the pure Lys26(Boc)-Arg34-GLP-1(9-37).

[0118] In another preferred embodiment, the concentration of Lys26(Boc)-Arg34-GLP-1(9-37) is 0.5-3 mg / mL, preferably 0.7-2 mg / mL.

[0119] In another preferred embodiment, the use concentration of enterokinase is 0.1-1.0 U / mg, preferably 0.5 U / mg.

[0120] In another preferred embodiment, the enterokinase is a dry powder preparation.

[0121] In another preferred embodiment, the pH of the buffered saline solution is 8-11.

[0122] In another preferred embodiment, the buffered saline solution contains sodium chloride.

[0123] In another preferred embodiment, the sodium chloride concentration is 50-200 mM, preferably 150 mM.

[0124] In another preferred embodiment, the buffered saline solution contains 20-50 mM Tris, preferably 50 mM Tris.

[0125] In another preferred embodiment, the kit further comprises: (c) a third container and an alkaline medium in the third container.

[0126] In another preferred embodiment, the alkaline medium is a NaOH solution.

[0127] In another preferred embodiment, the kit further includes: (d) a fourth container and a purification reagent located in the fourth container.

[0128] In another preferred embodiment, the purification reagent comprises a chromatographic purification reagent.

[0129] In another preferred embodiment, the purification reagent includes a phase A acidic aqueous solution and / or a phase B organic solvent.

[0130] In another preferred embodiment, the phase A buffer aqueous solution is selected from one of ammonium acetate, ammonium formate, ammonium bicarbonate, and ammonia solution.

[0131] In another preferred embodiment, the organic solvent of phase B is selected from one of methanol, ethanol, isopropanol and acetonitrile.

[0132] In another preferred embodiment, the buffered aqueous solution of phase A is an aqueous ammonia solution, and the organic solvent of phase B is acetonitrile.

[0133] In another preferred embodiment, the buffered aqueous solution of phase A is ammonium bicarbonate, and the organic solvent of phase B is acetonitrile.

[0134] In another preferred embodiment, the concentration of the phase A buffer solution is 20-50 mM, preferably 50 mM.

[0135] In another preferred embodiment, the pH of the phase A buffer solution is 8-11.

[0136] In another preferred embodiment, the buffer composition of the organic solvent of phase B is all volatile substances.

[0137] In another preferred example, the first container, the second container, the third container and / or the fourth container may be the same container or different containers.

[0138] In another preferred embodiment, the kit further comprises a label and instructions.

[0139] 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.

[0140] In the seventh aspect of the present invention, a quality control kit for a semaglutide main chain precursor is provided, the quality control kit comprising: the semaglutide main chain precursor enzymatic hydrolysis impurities as described in the third aspect of the present invention.

[0141] In another preferred example, the quality control kit further includes a label and instructions.

[0142] In another preferred embodiment, the description indicates the quality control method as described in the first aspect of the present invention.

[0143] In an eighth aspect of the present invention, a device for preparing enzymatic impurities of a semaglutide main chain precursor is provided, the device comprising:

[0144] (M1) a reaction module, wherein the reaction module is configured to: provide a reaction system, wherein the reaction system comprises: a semaglutide main chain precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample, and wherein the reaction system undergoes a cleavage reaction under the conditions of pH 9 to 12 and enterokinase;

[0145] (M2) a time control module, wherein the time control module is configured to: allow the cleavage reaction in the reaction module to proceed for a period of time, thereby obtaining a post-enzyme cleavage solution; and

[0146] (M3) A purification module, wherein the purification module is configured to purify the solution after enzymatic cleavage to obtain enzymatic impurities of semaglutide main chain precursor.

[0147] In another preferred example, the time control module is configured to: allow the cleavage reaction in the reaction module to proceed for 24 to 48 hours.

[0148] In another preferred example, the (M3) purification module also includes a gradient elution submodule, which is configured to: use phase A buffer aqueous solution and phase B organic solvent as mobile phases to perform gradient elution on the post-enzyme cleavage solution, and collect the eluate.

[0149] In another preferred embodiment, the procedure of gradient elution in the gradient elution submodule is as follows:

[0150] (S1) When the elution start time is 0 min, the percentage content of the phase A buffer solution in the mobile phase is 97-95%, and the percentage content of the phase B organic solvent is 3-5%; preferably, the percentage content of the phase A buffer solution in the mobile phase is 95%, and the percentage content of the phase B organic solvent is 5%;

[0151] (S2) when the elution start time is 10 min, the percentage content of the buffer solution of phase A in the mobile phase is 97-95%, and the percentage content of the organic solvent of phase B is 3-5%; preferably, the percentage content of the buffer solution of phase A in the mobile phase is 95%, and the percentage content of the organic solvent of phase B is 5%; and

[0152] (S3) When the elution start time is 40 min, the percentage content of the phase A buffer aqueous solution in the mobile phase is 85-80%, and the percentage content of the phase B organic solvent is 15-20%; preferably, the percentage content of the phase A buffer aqueous solution in the mobile phase is 82%, and the percentage content of the phase B organic solvent is 18%.

[0153] 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 5%, and freeze-dry for 10 to 16 hours under the conditions of vacuum degree ≤ 10 mbar and baffle temperature control at 25°C, thereby obtaining a freeze-dried preparation of enzymatic impurities of the semaglutide main chain precursor.

[0154] In another preferred example, the device further comprises a (M5) control module, wherein the control module is configured to control the operation of the (M1) reaction module, the (M2) time control module and the (M3) purification module.

[0155] In another preferred example, the (M5) control module also controls the operation of the (M4) freeze-drying module.

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

[0157] (M1) reaction module, (M2) time control module and (M3) purification module are operated in sequence of (M1) → (M2) → (M3).

[0158] In another preferred embodiment, the (M5) control module controls the following modules in the device to operate in sequence:

[0159] (M1) reaction module, (M2) time control module, (M3) purification module and (M4) freeze-drying module are operated in the order of (M1) → (M2) → (M3) → (M4).

[0160] 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

[0161] Figure 1 The HPLC spectrum of the enzyme cleavage reaction solution under condition 8 in Table 3 is shown.

[0162] Figure 2 The HPLC spectrum of the impurity after purification is shown.

[0163] Figure 3 The mass spectrum of the impurity (SEQ ID NO: 4) is shown.

[0164] Figure 4 It shows the use of impurity controls to detect the content of impurities in the precursor fusion proteinase digestion solution.

[0165] Figure 5It shows the use of impurity control substances to detect the content of impurities in another precursor fusion proteinase digestion solution.

[0166] Figure 6 The figure shows the use of impurity reference substances to detect the content of impurities in the main chain precursor pure solution. DETAILED DESCRIPTION

[0167] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that the amount of impurities generated by enzymatic hydrolysis of the main chain precursor of semaglutide can be significantly increased by adjusting the enzyme cleavage conditions, and a high-purity impurity sample can be obtained by combining separation, purification and post-treatment. Through the simple method of the present invention, high-purity impurities can be obtained as impurity reference substances for quality control or purification of the semaglutide production process (especially the semaglutide main chain precursor), and it is helpful to synthesize 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] The terms "impurity", "target impurity" and "semaglutide main chain precursor enzymatic impurity" are used interchangeably and refer to the semaglutide main chain precursor enzymatic impurity as described in the third aspect of the present invention, which is obtained by the preparation method described in the fifth aspect of the present invention and has 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 semaglutide main chain precursor enzymatic impurity and a semaglutide main chain precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample to be tested; and

[0178] (b) using the enzymatic impurities of the semaglutide main chain precursor as impurity reference substances, and determining the content of the enzymatic impurities of the semaglutide main chain precursor 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 precursor fusion protease digestion liquid, the quality control method may further include the steps of: determining the concentration of the semaglutide backbone precursor in the precursor fusion protease digestion liquid, and performing HPLC analysis on the precursor fusion protease digestion liquid, and calculating the content of semaglutide backbone precursor enzymatic impurities (target impurities) according to the impurity peak area and the standard curve; or, performing HPLC analysis on the fusion protease digestion liquid 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 pure semaglutide main chain precursor (Lys26(Boc)-Arg34-GLP-1(9-37)), the quality control method may further include the steps of: preparing the pure Lys26(Boc)-Arg34-GLP-1(9-37) 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 semaglutide main chain precursor is also provided, and the quality control kit comprises: the semaglutide main chain precursor enzymatic hydrolysis impurities 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 semaglutide backbone precursor enzymatic impurities and a semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) to be purified; and

[0187] (b) using the enzymatic impurities of the semaglutide backbone precursor as an impurity reference substance, using chromatography, and optimizing the chromatographic conditions and / or elution conditions to effectively separate the enzymatic impurities of the semaglutide backbone precursor in the semaglutide backbone precursor sample, thereby obtaining a purified semaglutide backbone precursor.

[0188] Preferably, the purification 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.

[0189] 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 enzymatic impurities in the semaglutide backbone precursor sample according to the impurity peak area and the standard curve.

[0190] 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 main chain precursor and the enzymatic impurities (impurity peak) of the semaglutide main chain precursor, thereby effectively separating the enzymatic impurities of the semaglutide main chain precursor.

[0191] Typically, the content of semaglutide main chain precursor enzymatic impurities in the semaglutide main chain precursor to be purified is ≥1%, preferably ≥1.5%, more preferably ≥2%; the content of semaglutide main chain precursor enzymatic impurities in the purified semaglutide main chain precursor is ≤0.8%, preferably ≤0.6%, more preferably ≤0.5%.

[0192] Therefore, when the enzymatic hydrolysis impurities of the semaglutide main chain precursor of the present invention are used as impurity reference substances, on the one hand, the content of the target impurities in the semaglutide main chain precursor can be calculated by chromatography (impurity peak area and standard curve), thereby performing quality control of the semaglutide main chain precursor; on the other hand, the impurities can be effectively separated by optimizing the chromatographic conditions and / or elution conditions, thereby obtaining a purified semaglutide main chain precursor. In addition, the purified semaglutide main chain precursor can be used to synthesize high-purity semaglutide.

[0193] Impurities in the enzymatic degradation of semaglutide backbone precursor

[0194] The main chain precursor enzymatic impurities of semaglutide are mainly made of the main chain precursor Lys26(Boc)-Arg34-GLP-1(9-37) as raw materials. They are small amounts of impurities produced after the enzymatic cleavage process of the main chain precursor of semaglutide (the content is relatively low in conventional enzymatic cleavage processes, about 2%). The main difference between the main chain precursor enzymatic impurities of semaglutide and the main chain of semaglutide is that two and three amino acids are missing at the N-terminus and C-terminus, respectively. The specific sequence of the main chain precursor enzymatic impurities of semaglutide is as follows:

[0195] Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly2 2-Gln23-Ala24-Ala25-Lys26(Boc)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34(SEQ ID NO:4).

[0196] Using the preparation method described in the fifth aspect of the present invention, the inventors significantly increased the amount of impurities generated by adjusting the enzyme cleavage conditions, 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 purification 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 and the purification method described in the second aspect of the present invention.

[0197] Preparation method of enzymatic hydrolysis impurities of semaglutide main chain precursor

[0198] The present invention provides a method for preparing enzymatically hydrolyzed impurities of a semaglutide main chain precursor, the method comprising the steps of:

[0199] (a) providing a reaction system, the reaction system comprising: a semaglutide main chain precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample, and the reaction system undergoes a cleavage reaction under the conditions of pH 9 to 12 and enterokinase;

[0200] (b) allowing the cleavage reaction in step (a) to proceed for a period of time to obtain a cleaved solution; and

[0201] (c) purifying the enzymatically cleaved solution obtained in step (b) to obtain enzymatically cleaved impurities of the semaglutide main chain precursor.

[0202] The obtained semaglutide main chain precursor enzymatic impurity has the sequence shown in SEQ ID NO: 4:

[0203] Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly2 2-Gln23-Ala24-Ala25-Lys26(Boc)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34(SEQ ID NO:4).

[0204] When the purification method in step (c) is a chromatographic purification method, preferably the chromatographic purification method may include the steps of:

[0205] (c1) The digested solution obtained in step (b) is loaded onto a high performance liquid chromatography column, and gradient elution is performed using a phase A buffer solution and a phase B organic solvent as mobile phases, and the eluate is collected.

[0206] Furthermore, the preparation method of the present invention may also include a freeze-drying step:

[0207] (d) Lyophilization: The eluate obtained in step (c1) is diluted with water until the organic solvent in phase B is less than 5%, and lyophilized for 10 to 16 hours under the conditions of vacuum degree ≤ 10 mbar and baffle temperature control at 25° C., thereby obtaining a lyophilized preparation of the enzymatic hydrolysis impurities of the semaglutide main chain precursor.

[0208] In the above preparation method, typically:

[0209] For step (a): the Lys26(Boc)-Arg34-GLP-1(9-37) sample is derived from a precursor fusion protease digestion solution or a pure Lys26(Boc)-Arg34-GLP-1(9-37); for example, when a digestion solution or a pure solution is used, the concentration of the Lys26(Boc)-Arg34-GLP-1(9-37) is 0.5 to 3 mg / mL, preferably 0.7 to 2 mg / mL;

[0210] The semaglutide main chain precursor Lys26(Boc)-Arg34-GLP-1(9-37) sample has a sequence as shown in SEQ ID NO: 3:

[0211] 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);

[0212] The concentration of enterokinase is 0.1-1.0 U / mg, preferably 0.5 U / mg;

[0213] The reaction system may contain a buffer substance, such as sodium chloride, the concentration of which is 50-200 mM, preferably 150 mM; for example, the reaction system may also contain 20-50 mM Tris, preferably 50 mM Tris;

[0214] The cleavage reaction in step (a) is carried out at room temperature; NaOH solution is used to make the reaction system reach pH 9-12.

[0215] For step (b): it should be understood that the reaction time of the cleavage reaction in step (b) can be adjusted by those skilled in the art according to actual conditions and needs, and is not limited to the reaction time listed in the examples. For example, the reaction time can include but is not limited to 6-72 hours, and preferably the cleavage reaction in step (b) is carried out for 24 to 48 hours.

[0216] For step (c): the purification method is preferably a chromatographic purification method, which may include the steps of:

[0217] (c1) applying the digested solution obtained in step (b) to a high performance liquid chromatography column, using a phase A buffer solution and a phase B organic solvent as mobile phases, performing gradient elution, and collecting the eluate;

[0218] The filler used in the high performance liquid preparative chromatography column is a reverse phase chromatography filler; the filler type is selected from one of C18, C8, PS / DVB, and PMMA; the particle size of the filler is 10 to 30 μm, preferably 10 μm;

[0219] The phase A buffer solution is selected from one of ammonium acetate, ammonium formate, ammonium bicarbonate, and ammonia solution. Preferably, the concentration of the phase A buffer solution is 20-50 mM, preferably 50 mM; the pH of the phase A buffer solution is 8-11; the phase B organic solvent is selected from one of methanol, ethanol, isopropanol, and acetonitrile; preferably, the buffer composition of the phase B organic solvent is volatile substances, so the post-treatment can be exempted from the desalting process, that is, the method does not include a step involving the desalting process;

[0220] For example, the phase A buffer solution is an ammonia solution, and the phase B organic solvent is acetonitrile; or, the phase A buffer solution is ammonium bicarbonate, and the phase B organic solvent is acetonitrile;

[0221] The procedure of the gradient elution is as follows:

[0222] (S1) When the elution start time is 0 min, the percentage content of the phase A buffer solution in the mobile phase is 97-95%, and the percentage content of the phase B organic solvent is 3-5%; preferably, the percentage content of the phase A buffer solution in the mobile phase is 95%, and the percentage content of the phase B organic solvent is 5%;

[0223] (S2) when the elution start time is 10 min, the percentage content of the buffer solution of phase A in the mobile phase is 97-95%, and the percentage content of the organic solvent of phase B is 3-5%; preferably, the percentage content of the buffer solution of phase A in the mobile phase is 95%, and the percentage content of the organic solvent of phase B is 5%; and

[0224] (S3) When the elution start time is 40 min, the percentage content of the phase A buffer aqueous solution in the mobile phase is 85-80%, and the percentage content of the phase B organic solvent is 15-20%; preferably, the percentage content of the phase A buffer aqueous solution in the mobile phase is 82%, and the percentage content of the phase B organic solvent is 18%.

[0225] For step (d): the organic solvent of phase B is preferably acetonitrile.

[0226] 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.

[0227] The inventors unexpectedly discovered through experiments that in a high pH environment, enterokinase still has significant activity and can continue to cleave the main chain precursor of semaglutide Lys26 (Boc) -Arg34-GLP-1 (9-37) to generate a large number of SEQ ID NO: 4 impurities. Those skilled in the art know that it is also possible to directly express the target impurities by fermentation through molecular construction, but the operation is cumbersome, the cycle is long, and the cost is high, so it does not have the advantages of the method of the present invention. In addition, impurity degradation can also be carried out by other enzymes, such as trypsin, clostripain, etc., but their selectivity is poor and not as good as the enterokinase used in the method of the present invention.

[0228] Kit for the preparation of enzymatic impurities of semaglutide main chain precursor

[0229] The present invention provides a kit for preparing enzymatic hydrolysis impurities of a semaglutide main chain precursor, the kit comprising:

[0230] (a) a first container and a sample of semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) located in the first container; and

[0231] (b) a second container and enterokinase or a buffered saline solution containing enterokinase located in the second container.

[0232] Preferably, the Lys26(Boc)-Arg34-GLP-1(9-37) sample is derived from a precursor fusion protease digestion solution or a pure Lys26(Boc)-Arg34-GLP-1(9-37); for example, when a digestion solution or a pure solution is used, the concentration of the Lys26(Boc)-Arg34-GLP-1(9-37) can be 0.5 to 3 mg / mL, preferably 0.7 to 2 mg / mL;

[0233] The enterokinase in the kit can be a dry powder preparation or a solution, such as a buffered saline solution containing enterokinase; the concentration of the enterokinase can be a higher concentration that needs to be diluted before use, or it can be directly prepared as a working concentration for convenience of use. The working concentration of the enterokinase can be 0.1-1.0 U / mg, preferably 0.5 U / mg;

[0234] The pH of the buffered saline solution may be 8-11; it may contain sodium chloride, and the concentration of sodium chloride is 50-200 mM, preferably 150 mM; preferably, the buffered saline solution contains 20-50 mM Tris, preferably 50 mM Tris.

[0235] The kit of the present invention may optionally further include: (c) a third container and an alkaline medium in the third container; the alkaline medium is preferably a NaOH solution.

[0236] The kit of the present invention may further optionally include: (d) a fourth container and a purification reagent located in the fourth container.

[0237] 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.

[0238] Preferably, the A phase buffer aqueous solution is selected from one of ammonium acetate, ammonium formate, ammonium bicarbonate, and ammonia solution. Preferably, the concentration of the A phase buffer aqueous solution is 20-50 mM, preferably 50 mM; the pH of the A phase buffer aqueous solution is 8-11; the B phase organic solvent is selected from one of methanol, ethanol, isopropanol and acetonitrile; preferably, the buffer composition of the B phase organic solvent is volatile substances, so the post-treatment can avoid the desalting process;

[0239] For example, the phase A buffer aqueous solution is an ammonia solution, and the phase B organic solvent is acetonitrile; or, the phase A buffer aqueous solution is ammonium bicarbonate, and the phase B organic solvent is acetonitrile.

[0240] The first container, the second container, the third container and / or the fourth container mentioned above may be the same container or different containers.

[0241] 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.

[0242] Preparation device for enzymatic hydrolysis impurities of semaglutide main chain precursor

[0243] The present invention provides a device for preparing semaglutide main chain precursor enzymatic hydrolysis impurities, the device comprising:

[0244] (M1) a reaction module, wherein the reaction module is configured to: provide a reaction system, wherein the reaction system comprises: a semaglutide main chain precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample, and wherein the reaction system undergoes a cleavage reaction under the conditions of pH 9 to 12 and enterokinase;

[0245] (M2) a time control module, wherein the time control module is configured to: allow the cleavage reaction in the reaction module to proceed for a period of time, thereby obtaining a post-enzyme cleavage solution; and

[0246] (M3) A purification module, wherein the purification module is configured to purify the solution after enzymatic cleavage to obtain enzymatic impurities of semaglutide main chain precursor.

[0247] Typically, the (M2) time control module is configured to allow the cleavage reaction in the reaction module to proceed for 24 to 48 hours.

[0248] The (M3) purification module also includes a gradient elution submodule, which is configured to: use the A phase buffer solution and the B phase organic solvent as mobile phases to perform gradient elution on the post-enzyme cleavage solution, and collect the eluate.

[0249] The procedure of gradient elution in the gradient elution submodule is as follows:

[0250] (S1) When the elution start time is 0 min, the percentage content of the phase A buffer solution in the mobile phase is 97-95%, and the percentage content of the phase B organic solvent is 3-5%; preferably, the percentage content of the phase A buffer solution in the mobile phase is 95%, and the percentage content of the phase B organic solvent is 5%;

[0251] (S2) when the elution start time is 10 min, the percentage content of the buffer solution of phase A in the mobile phase is 97-95%, and the percentage content of the organic solvent of phase B is 3-5%; preferably, the percentage content of the buffer solution of phase A in the mobile phase is 95%, and the percentage content of the organic solvent of phase B is 5%; and

[0252] (S3) When the elution start time is 40 min, the percentage content of the phase A buffer aqueous solution in the mobile phase is 85-80%, and the percentage content of the phase B organic solvent is 15-20%; preferably, the percentage content of the phase A buffer aqueous solution in the mobile phase is 82%, and the percentage content of the phase B organic solvent is 18%.

[0253] 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 5%, and freeze-dry for 10 to 16 hours under the conditions of vacuum degree ≤10mbar and baffle temperature control at 25°C, thereby obtaining a freeze-dried preparation of enzymatic impurities of the semaglutide main chain precursor.

[0254] The device further comprises a control module (M5), which is configured to: control the operation of the reaction module (M1), the time control module (M2) and the purification module (M3), for example, the control module (M5) controls the following modules in the device to operate in sequence:

[0255] (M1) reaction module, (M2) time control module and (M3) purification module, that is, they are operated in the order of (M1) → (M2) → (M3);

[0256] 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:

[0257] (M1) reaction module, (M2) time control module, (M3) purification module and (M4) freeze-drying module are operated in the order of (M1) → (M2) → (M3) → (M4).

[0258] Main advantages of the present invention

[0259] 1. For the fusion protein digestion solution containing the main chain precursor Lys26(Boc)-Arg34-GLP-1(9-37) of semaglutide or the digestion solution containing the pure product of Lys26(Boc)-Arg34-GLP-1(9-37), the present invention unexpectedly found that the degradation of the main peak (semaglutide main chain precursor Lys26(Boc)-Arg34-GLP-1(9-37)) was accelerated and the content of the target impurity was significantly increased by simply increasing the pH of the above solution. In particular, the main peak pure product obtained after the normal process purification is further digested with enterokinase (high pH), which can obtain the target impurity more specifically and is more conducive to separation and purification. In addition, the digestion site in the above digestion process is not the conventional site DDDDK (SEQ ID NO: 5) of enterokinase.

[0260] 2. When the solution after enzyme cleavage is subsequently purified, the present invention only needs to use a simple chromatographic purification method or other simple purification methods, and the mobile phase uses volatile components, the post-treatment is simple, and the desalting process can be avoided.

[0261] 3. Direct fermentation expression of target impurities by molecular construction is also possible, but the operation is cumbersome, the cycle is long, and the cost is high, so it does not have the advantages of the method of the present invention. In addition, impurities can also be degraded by other enzymes, such as trypsin, clostripain, etc., but their selectivity is poor, not as good as the enterokinase used in the method of the present invention.

[0262] 4. The high-purity impurity prepared in the present invention can be used as an impurity reference substance for quality control of the semaglutide production process (eg, semaglutide main chain precursor-related process), and can also be used for the purification of semaglutide main chain precursor.

[0263] For example, when the enzymatic hydrolysis impurities of the semaglutide main chain precursor of the present invention are used as impurity reference substances, on the one hand, the content of the target impurities in the semaglutide main chain precursor can be calculated by chromatography (impurity peak area and standard curve), thereby performing quality control of the semaglutide main chain precursor; on the other hand, the impurities can be effectively separated by optimizing the chromatographic conditions and / or elution conditions, 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.

[0264] 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.

[0265] 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.

[0266] Sequence Listing

[0267]

[0268]

[0269] General Methods

[0270] The pure Lys26(Boc)-Arg34-GLP-1(9-37) (semaglutide main chain precursor) and the precursor fusion protease cleavage solution (which can be cleaved by enterokinase to obtain semaglutide main chain precursor) used in the present invention were prepared by the methods described in Examples 7 and 6 of Patent CN113801233A.

[0271] Example 1: Discovery of impurities in enzymatic degradation of semaglutide backbone precursor

[0272] The semaglutide main chain precursor enzymatic impurity (SEQ ID NO: 4) of the present invention is unexpectedly discovered when the precursor fusion protein is cleaved by enterokinase (the conventional cleavage site of enterokinase is DDDDK) to prepare the semaglutide main chain precursor (SEQ ID NO: 3).

[0273] The structure of the precursor fusion protein is: leader peptide-conventional cleavage site of enterokinase-Lys26 (Boc)-Arg34-GLP-1 (9-37), that is:

[0274] Leader peptide-DDDDK-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26(Boc)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37 (corresponding to SEQ ID NO: 6)

[0275] Enterokinase should have performed enzymatic cleavage at the C-terminus of the conventional enzymatic cleavage site "DDDDK" to obtain SEQ ID NO: 3 (i.e., Lys26(Boc)-Arg34-GLP-1(9-37)). However, it was unexpectedly found that enterokinase also performed enzymatic cleavage at the C-terminus of Arg34, thereby producing an impurity SEQ ID NO: 4 (i.e., Lys26(Boc)-Arg34-GLP-1(9-34)) with 3 amino acids missing from the C-terminus (i.e., -Gly35-Arg36-Gly37). This impurity is often present during the enzymatic cleavage process and is difficult to completely remove.

[0276] Example 2: Preparation method of target impurities in precursor fusion proteinase cleavage solution

[0277] (1) Enzymatic degradation

[0278] Take 500mL of the precursor fusion protein (i.e., the precursor fusion protein in Example 1) digestion solution, wherein the concentration of Lys26(Boc)-Arg34-GLP-1(9-37) is 0.7mg / mL (containing 0.5U / mg of initial enterokinase, 50mM Tris pH 8.0, and 150mM sodium chloride), add 1M NaOH to adjust to different pH values ​​(e.g., pH 9.0, 10.0, and 11.0), stir at room temperature, and monitor the degradation of the main peak semaglutide main chain precursor Lys26(Boc)-Arg34-GLP-1(9-37) (corresponding to SEQ ID NO: 3) and the generation of the target impurity Lys26(Boc)-Arg34-GLP-1(9-34) (corresponding to SEQ ID NO: 4) in the solution. The results are shown in Table 1.

[0279] Table 1 Precursor fusion protease digestion results

[0280]

[0281] According to Table 1, as the pH value was increased from 8.0 to 9.0, 10.0, 11.0, and 12.0, it was unexpectedly found that the degradation of the main peak was accelerated and the content of the target impurity increased.

[0282] When pH=9.0, the target impurity content percentage reaches about 1.8-2.0 times the target impurity content percentage when pH=8.0.

[0283] When pH=10.0, the target impurity content percentage reaches about 2.6-2.9 times the target impurity content percentage when pH=8.0.

[0284] When pH=11.0, the target impurity content percentage reaches about 3.6 times the target impurity content percentage when pH=8.0.

[0285] When pH=12.0, the target impurity content percentage reaches about 2.1-2.9 times the target impurity content percentage when pH=8.0.

[0286] Although the target impurity content percentage at pH = 12.0 is lower than that at pH = 11.0, it is still significantly higher than the target impurity content percentage at pH = 8.0.

[0287] (2) Sample purification

[0288] The chromatographic column used was UniPS (10 μm, 10*250mm), 0.1% ammonia water as phase A, acetonitrile as phase B. The degradation solution of condition 8 in Table 1 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 98.5%.

[0289] Table 2 Elution gradient during purification

[0290] Time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 10 95 5 40 82 18

[0291] like Figure 1 As shown, the HPLC spectrum of the enzyme cleavage reaction solution under condition 8 in Table 3 is shown.

[0292] like Figure 2 As shown, the HPLC spectrum of the impurities after purification is shown.

[0293] like Figure 3 As shown, a mass spectrum of an impurity (SEQ ID NO: 4) is shown.

[0294] (3) Freeze-drying

[0295] The eluate from step (2) was diluted with water to a concentration of less than 5% acetonitrile, 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 98.4%.

[0296] Example 3: Preparation method of target impurities in pure Lys26(Boc)-Arg34-GLP-1(9-37)

[0297] (1) Enzymatic degradation

[0298] 200 mg of pure Lys26(Boc)-Arg34-GLP-1(9-37) was dissolved in 100 mL of solution (containing enterokinase 0.5 U / mg, 50 mM Tris pH 8.0, sodium chloride 150 mM), and 1 M NaOH or 1 M HCL was added to adjust to different pH values ​​(e.g. pH 7.0, 8.0, 9.0, 10.0, 11.0), stirred at room temperature, and the degradation of the main peak in the solution and the generation of the target impurities were monitored. The results are shown in Table 3.

[0299] Table 3 Lys26(Boc)-Arg34-GLP-1(9-37) pure product enzymatic degradation results

[0300]

[0301]

[0302] According to Table 3, as the pH value was increased from 7.0, 8.0 to 9.0, 10.0, 11.0, and 12.0, it was unexpectedly found that the degradation of the main peak was significantly accelerated and the content of the target impurity was significantly increased.

[0303] When pH=9.0, the target impurity content percentage reaches about 21.4-24.8 times the target impurity content percentage when pH=7.0.

[0304] When pH=10.0, the target impurity content percentage reaches about 42.1-51.3 times the target impurity content percentage when pH=7.0.

[0305] When pH=11.0, the target impurity content percentage reaches about 50.8-63.2 times the target impurity content percentage when pH=7.0.

[0306] When pH=12.0, the target impurity content percentage reaches about 40.0-54.4 times the target impurity content percentage when pH=7.0.

[0307] Although the target impurity content percentage at pH = 12.0 is lower than that at pH = 11.0, it is still significantly higher than the target impurity content percentage at pH = 7.0 and 8.0.

[0308] (2) Sample purification

[0309] The chromatographic column was C8 (10 μm, 10*250mm), 50mM ammonium bicarbonate (pH 8.0) as phase A, acetonitrile as phase B. The degradation solution of condition 10 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 95.8%, and the impurity purity was 98.2%.

[0310] (3) Freeze-drying

[0311] The eluate from step (2) was decompressed to remove acetonitrile, 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 98.2%.

[0312] Example 4: Enzymatic impurities in the main chain precursor of semaglutide in the quality control or purification of the main chain precursor of semaglutide Use in law

[0313] By preparing the impurity reference substance, it is convenient to carry out quantitative analysis of impurities in the semaglutide main chain precursor or the precursor fusion protease cleavage solution containing the main chain precursor in the process, such as the following examples.

[0314] 4.1 Quantitative analysis of impurities in precursor fusion proteinase digestion solution

[0315] First, a high-purity semaglutide main chain precursor enzymatic hydrolysis impurity is obtained by the preparation method of the present invention. Subsequently, the precursor fusion protease cleavage solution is analyzed by HPLC method, and the target impurity is used as a control, and the position of the impurity in the HPLC spectrum can be conveniently determined according to the control.

[0316] 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 fusion protease cleavage solution with the main chain precursor concentration determined is subjected to HPLC analysis, and the content of the target impurity can be calculated according to the impurity peak area and the standard curve.

[0317] Thus, quantitative analysis of target impurities and main chain precursors in the precursor fusion protein digestion solution is achieved. In addition, the above method can also be used to monitor the impurity content during the digestion process.

[0318] like Figure 4 As shown, the content of impurities in the precursor fusion proteinase digestion solution was detected using an impurity control substance.

[0319] like Figure 5 As shown, the impurity content in another precursor fusion proteinase digestion solution was detected using an impurity control substance.

[0320] Therefore, for the precursor fusion protein digestion solution, the use of semaglutide main chain precursor enzymatic hydrolysis impurity reference material can not only realize the quantitative analysis of the impurity content in the enzymatic digestion solution, but also realize the dynamic monitoring of the impurity content during the enzymatic digestion process.

[0321] 4.2 Quantitative analysis of impurities in the pure product of the main chain precursor Lys26(Boc)-Arg34-GLP-1(9-37)

[0322] Similarly, the pure product of the main chain precursor Lys26(Boc)-Arg34-GLP-1(9-37) was prepared into a solution and subjected to HPLC analysis. 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.

[0323] In addition, 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; the main chain precursor is then prepared into a solution of known concentration and subjected to HPLC analysis, and the content of the target impurity can be calculated according to the impurity peak area and the standard curve.

[0324] like Figure 6 As shown, the content of impurities in the main chain precursor pure solution is detected using an impurity reference substance.

[0325] Therefore, for the pure product of the main chain precursor or its solution, the quantification of the impurity content therein can also be achieved by using the semaglutide main chain precursor enzymatic hydrolysis impurity reference substance.

[0326] The above HPLC analysis method is as follows:

[0327] Chromatographic column: C18 (4.6*250mm, 5μm)

[0328] Mobile phase A: 10 mM diammonium phosphate pH 10.2

[0329] Mobile phase B: 80% ACN in water

[0330] Detection wavelength: 214nm

[0331] Flow rate: 1.0mL / min

[0332] Column temperature: 35°C

[0333] The elution gradient is shown in Table 4:

[0334] Table 4 Elution gradient table during HPLC analysis

[0335] 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

[0336] Therefore, the enzymatic impurities of the semaglutide main chain precursor can be used as impurity reference substances for quality control of the semaglutide main chain precursor.

[0337] Furthermore, by optimizing the chromatographic conditions and / or elution conditions, the enzymatic 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 enzymatic impurities (impurity peak) of the semaglutide backbone precursor can be improved), thereby obtaining a purified semaglutide backbone precursor.

[0338] The purified semaglutide main chain precursor thus obtained can be used to synthesize high-purity semaglutide. Therefore, the enzymatic impurities of the semaglutide main chain precursor are used as impurity reference substances, which are of great significance for the quality control and process optimization of the semaglutide production process.

[0339] 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 enzymatic impurity and a semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample to be tested, wherein the semaglutide backbone precursor enzymatic impurity has a sequence as shown in SEQ ID NO: 4; and (b) using the enzymatic impurities of the semaglutide main chain precursor as impurity reference substances, and determining the content of the enzymatic impurities of the semaglutide main chain precursor 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 enzymatic impurity and a semaglutide backbone precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample to be purified, wherein the semaglutide backbone precursor enzymatic impurity has a sequence as shown in SEQ ID NO: 4; and (b) using the enzymatic impurities of the semaglutide backbone precursor as an impurity reference substance, using chromatography, and optimizing the chromatographic conditions and / or elution conditions to effectively separate the enzymatic impurities of the semaglutide backbone precursor in the semaglutide backbone precursor sample, thereby obtaining a purified semaglutide backbone precursor.

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

4.

4. A use of enzymatic hydrolysis impurities of semaglutide main chain precursor according to claim 3, characterized in that: Used for: (Z1) as an impurity reference substance for quality control 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 enzymatically hydrolyzed impurities of a semaglutide main chain precursor, characterized in that: The preparation method comprises the steps of: (a) providing a reaction system, the reaction system comprising: a semaglutide main chain precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample, and the reaction system undergoes a cleavage reaction under the conditions of pH 9 to 12 and enterokinase; (b) allowing the cleavage reaction in step (a) to proceed for a period of time to obtain a cleaved solution; and (c) purifying the enzymatically cleaved solution obtained in step (b) to obtain semaglutide main chain precursor enzymatic impurities, wherein the semaglutide main chain precursor enzymatic impurities have a sequence as shown in SEQ ID NO:

4.

6. The preparation method according to claim 5, characterized in that: The Lys26(Boc)-Arg34-GLP-1(9-37) sample is derived from the precursor fusion protease digestion solution or the pure Lys26(Boc)-Arg34-GLP-1(9-37).

7. A kit for preparing the enzymatic hydrolysis impurities of the semaglutide main chain precursor 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 enterokinase or a buffered saline solution containing enterokinase located in the second container.

8. The kit according to claim 7, characterized in that The kit further comprises: (c) a third container and an alkaline medium in the third container.

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

10. A device for preparing the enzymatic hydrolysis impurities of the semaglutide main chain precursor as claimed in claim 3, characterized in that: The device comprises: (M1) a reaction module, wherein the reaction module is configured to: provide a reaction system, wherein the reaction system comprises: a semaglutide main chain precursor (Lys26(Boc)-Arg34-GLP-1(9-37)) sample, and wherein the reaction system undergoes a cleavage reaction under the conditions of pH 9 to 12 and enterokinase; (M2) a time control module, wherein the time control module is configured to: allow the cleavage reaction in the reaction module to proceed for a period of time, thereby obtaining a post-enzyme cleavage solution; and (M3) A purification module, wherein the purification module is configured to purify the solution after enzymatic cleavage to obtain enzymatic impurities of the semaglutide main chain precursor.

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