Purification preparation method of GLP-1 precursor protein and main peptide chain intermediate

Abstract

The invention belongs to the technical field of medicine, and relates to a purification preparation method of protein and polypeptide, in particular to a purification preparation method of GLP-1 precursor protein and a main peptide chain intermediate. After fermentation is finished, fermentation liquor containing GLP-1 precursor protein is directly centrifuged and filtered to obtain a GLP-1 precursor protein solution, and an enzyme digestion solution is purified, subjected to acid precipitation and freeze-dried in a chromatography mode of combining reverse polymer filler and reverse silica gel chromatography filler to prepare the main peptide chain intermediate. The complex and tedious steps of ultrafiltration, high-pressure homogeneous crushing, inclusion body collection, inclusion body redenaturation and the like are innovatively removed, the process steps are simplified, the cost is saved, the high-purity and high-yield intermediate is obtained, and the method can be directly used for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a method for purifying and preparing precursor proteins and intermediates, specifically a method for purifying and preparing GLP-1 precursor protein and main peptide chain intermediates. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by hyperglycemia. In recent years, the global prevalence of diabetes has been increasing year by year. Currently, medications for treating type 2 diabetes include biguanides, meglitinides, and sulfonylureas. While these have some therapeutic effect, they also have significant side effects, easily causing hypoglycemia and gastrointestinal discomfort. Human glucagon-1 (GLP-1) and its analogues, as a novel treatment for type 2 diabetes, are highly favored due to their significant therapeutic effect, long duration of action, and fewer adverse side effects, especially semaglutide.

[0003] Smegglutide injection is a GLP-1 analog produced by Novo Nordisk using biosynthesis technology. Its peptide sequence is as follows: His7 Aib8 Glu9 Gly10 Thr11 Phe12 Thr13 Ser14 Asp15 Val16 Ser17 Ser18 Tyr19 Leu20 Glu21 ​​Gly22 Gln23 Ala24 Ala25 Lys26(AEEA AEEAγ GluOctadecanedioic) Glu27 Phe28 Ile29 Ala30 Trp31 Leu32 Val33 Arg34 Gly35 Arg36 Gly37 OH. Compared to human GLP-1, Ala8 is replaced with Aib8 (2-amino acid isobutyric acid), Lys34 is replaced with Arg34, and the lysine residue at position 26 of the peptide sequence is linked to a fatty acid acylated side chain (one 1,18-octadecanoic acid, one γ-glutamic acid group, and two 8-amino-3,6-dioxanoic acid groups), which greatly enhances the hydrophilicity of the entire peptide. It can not only bind tightly to albumin and resist dipeptidyl peptidase 4 (DPP 4) degradation, but also reduce renal excretion and significantly prolong the half-life. It is suitable for weekly subcutaneous injection to achieve a long-circulating effect. Therefore, smegglutide has a very wide market application.

[0004] Novo Nordisk's biosynthetic technology has solved the problem of exogenous protein degradation by the yeast's own proteases in the yeast expression system, enabling the biological preparation of smegglutinin fusion protein and intermediate peptide Arg34GLP 1 (9-37). However, this technology is quite challenging, and no specific preparation method has been reported. Currently, preparation using E. coli expression systems and chemical synthesis methods is common, but there are few reports on the purification process of smegglutinin intermediate peptide Arg34GLP 1 (9-37). In patent CN114057886A, the preparation of smegglutinin intermediate involves centrifugation, high-pressure homogenization to prepare inclusion bodies, inclusion body renaturation, filtration, fusion protein purification, desalting, enzymatic digestion, and reverse-phase chromatography. This process results in an inclusion body fusion protein content of approximately 31%, a purification purity of over 65%, a loading of 18 mg / mL, and a yield greater than 80%; after enzymatic digestion, reverse-phase chromatography yields a purity greater than 90% and a yield greater than 80%. Patent CN110498849A describes a process involving centrifugation, high-pressure homogenization to disrupt the bacterial cell count, centrifugation to collect inclusion bodies, inclusion body washing, renaturation, enzymatic digestion, and purification to obtain the intermediate Arg34GLP-1(9-37). This process uses ion exchange to purify the digested solution, achieving a peptide purity of over 89% and a yield greater than 82%. However, the type of ions used and the eluent are not disclosed. In patent CN111378027A, the intermediate preparation steps for smegglutinin include centrifugation to collect bacterial cells, high-pressure homogenization to disrupt the bacterial cells, centrifugation to collect inclusion bodies, washing inclusion bodies, renaturation, enzymatic digestion, ion exchange, and reversed-phase chromatography. The sample loading amount, ions, reversed-phase chromatography, and the specific type of eluent are not disclosed.

[0005] In existing technologies for the purification and preparation of smegglutinin intermediates, conventional production steps typically involve centrifugation to collect bacterial cells, high-pressure homogenization to break down bacterial cells, centrifugation to collect inclusion bodies, washing inclusion bodies, ultrafiltration, refolding, enzymatic digestion, filtration, chromatography, precipitation, and freeze-drying. The process is complex, the production cost is high, and the purity and yield of the intermediates obtained are not high, which is not conducive to large-scale commercial production.

[0006] Given the existing problems with the purification and preparation techniques for the semaglutide intermediate Arg34GLP-1(9-37), there is an urgent need to find a simpler, more efficient, and more suitable method for industrial-scale purification and preparation of the intermediate. Summary of the Invention

[0007] In view of this, one of the objectives of the present invention is to provide a method for purifying and preparing GLP-1 precursor protein and main peptide chain intermediate.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A method for purifying and preparing GLP-1 precursor protein and main peptide chain intermediate, characterized by comprising the following steps: (1) Centrifugation and filtration: Take the fermentation broth containing the target protein, centrifuge and filter it directly to obtain the GLP-1 precursor protein solution; (2) Enzyme digestion: Take the GLP-1 precursor protein solution obtained in step (1), dilute it, and digest it with recombinant enterokinase to obtain a mixed solution containing tag protein, linker, target intermediate and culture medium; (3) High pressure chromatography 1: Take the mixed solution obtained in step (2), use the reversed polymer packing as the stationary phase, and use mobile phase A and mobile phase B to perform gradient elution on the mixed solution. Collect the target intermediate solution after the first purification at 280 nm. (4) High-pressure chromatography 2: Take the target intermediate solution obtained in step (3), dilute it with water 3 to 5 times, use reverse silica gel chromatography packing as stationary phase, use mobile phase A and mobile phase B to perform gradient elution on the mixed solution, and collect the target intermediate solution after the second purification at 280 nm. (5) Acid precipitation: Take the target intermediate solution obtained in step (4), adjust the pH of the solution to acidic using a pH adjuster, let it stand at low temperature, discard the supernatant, add purified water, stir evenly, let it stand at low temperature, discard the supernatant, and collect the precipitate after centrifugation; (6) Freeze-drying: Resuspend the wet solid in step (5) and freeze-dry to obtain the purified GLP-1 main peptide chain intermediate.

[0009] When GLP-1 is semaglutide, the main peptide chain intermediate is Arg34GLP-1(9-37); when GLP-1 is liraglutide, the main peptide chain intermediate is Arg34GLP-1(7-37).

[0010] Preferably, in step (1), the solid content of the waste residue after centrifugation is ≥75%, and filtration is performed using a 0.45 μm hollow fiber membrane; Preferably, in step (2), the concentration of the diluted GLP-1 precursor protein solution is 1.7~17 mg / mL, the amount of recombinant enterokinase added is 6~16 U / g, the enzyme digestion temperature is 15~25℃, the enzyme digestion time is 5~15 h, and 1 mmol / L of calcium chloride is added during enzyme digestion. Preferably, the reverse polymer packing material in step (3) is one of polystyrene / divinylbenzene, polystyrene / polymethyl methacrylate, and polystyrene / divinylbenzene, and the reverse silica gel chromatography packing material in step (4) is one of C18, C4, and C8; Preferably, in steps (3) and (4), mobile phase A is one or more of phosphate buffer, citrate buffer, acetate buffer, tris(hydroxymethyl)aminomethane buffer, and glycine buffer, and the pH of mobile phase A is 7-8; mobile phase B is one or more of methanol, anhydrous ethanol, and acetonitrile. Preferably, in step (3), mobile phase A is 10-30 mmol / L phosphate buffer, mobile phase B is acetonitrile, the mobile phase velocity is 3.8 cm / min, and the injection volume of the mixed solution is Arg34GLP-1(9-37) or Arg34GLP-1(7-37) ≤24 g / L; the gradient elution program is as follows: (a) Over a 2-minute time period, the volume fraction of mobile phase B increased from 10% to 20%; (b) Maintain the volume fraction of mobile phase B at 20% for 18 min; (c) During the 3-minute time period, the volume fraction of mobile phase B increased from 20% to 35%; (d) During the 20 min time period, maintain the volume fraction of mobile phase B at 35%. Preferably, in step (4), mobile phase A is 3-8 mmol / L phosphate buffer, mobile phase B is acetonitrile, the mobile phase velocity is 3.8 cm / min, and the injection volume of the mixed solution is ≤16 g / L of Arg34GLP-1(9-37) or Arg34GLP-1(7-37); the gradient elution program is as follows: (a) Over a 3-minute time period, the volume fraction of mobile phase B increased from 10% to 29%; (b) During the 45 min time period, the volume fraction of mobile phase B increased from 29% to 35%.

[0011] Preferably, the pH adjuster in step (5) is one of glacial acetic acid, hydrochloric acid, phosphoric acid, and citric acid, and the solution pH is 4.5~5.5; Preferably, in step (5), the two low-temperature standing times are 1~6 h, the two volumes of supernatant discarded are 60%~80% of the original solution volume, the ratio of the volume of purified water added to the volume of the remaining supernatant is 1:1, the centrifugation speed is 5000~6000 rpm, and the centrifugation time is 3~5 min. Preferably, in step (6), the mass-volume ratio of Arg34GLP-1(9-37) or Arg34GLP-1(7-37) after resuspending in purified water is 4%~8%.

[0012] The present invention also provides Arg34GLP-1(9-37) or Arg34GLP-1(7-37) as the main peptide chain intermediate obtained by the above purification and preparation method.

[0013] The beneficial effects of this invention are as follows: (1) The purification and preparation method proposed in this invention directly centrifuges and filters the GLP-1 precursor protein solution after fermentation, and then prepares the main peptide chain intermediate Arg34GLP-1(9-37) or Arg34GLP-1(7-37) after enzymatic digestion, two-step chromatography, acid precipitation and freeze drying. It innovatively removes the complicated and cumbersome steps such as ultrafiltration, high pressure homogenization, collection of inclusion bodies and metamorphic inclusion bodies. By simplifying the process steps, it not only saves costs, but also obtains high-purity and high-yield intermediates that can be directly used for industrial production.

[0014] (2) This invention innovatively utilizes a chromatography method combining reverse polymer packing and reverse silica gel chromatography packing to prepare high-purity intermediates. Because the reverse polymer packing is stable and can be cleaned and restored using acids and alkalis, it eliminates the need for pretreatment of soluble components in the fermentation broth by ultrafiltration, precipitation, centrifugation, filtration, refolding, or other methods. Furthermore, its packing has a high sample loading capacity. Therefore, after enzymatic digestion, the reverse polymer packing is first used to pre-purify the complex system solution containing culture medium, inorganic salts, tag proteins, linkers, enterokinase, etc., and then the reverse silica gel chromatography packing is used for further purification, increasing the purity of the intermediate to over 95%.

[0015] In summary, this invention proposes a purification and preparation method for GLP-1 precursor protein and main peptide chain intermediates. The purification process is simple to operate, requires little equipment investment, has a high yield, and produces intermediate peptides with high purity, making it easy to scale up industrially.

[0016] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0017] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1 HPLC chromatogram of GLP-1 precursor protein digestion solution; Figure 2 The image shows the purification process of the GLP-1 precursor protease digestion solution using reverse polymer packing. Figure 3The HPLC chromatogram of the GLP-1 precursor protease digestion solution after purification with reverse polymer packing material; Figure 4 The image shows the purification process of GLP-1 precursor protein digestion solution using C18 packing material. Figure 5 The HPLC chromatogram of the GLP-1 precursor protease digestion solution after purification with C18 packing material; Figure 6 The HPLC chromatogram of Arg34GLP-1(9-37), the intermediate of the main peptide chain after acid precipitation. Detailed Implementation

[0018] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0019] Example 1 Purification and preparation of a GLP-1 precursor protein and main peptide intermediate Arg34GLP-1(9-37) The specific experimental steps are as follows: (1) Centrifugation and filtration: After fermentation, the sample was centrifuged using a disc centrifuge. During the centrifugation process, parameters such as the sample injection speed, rotation speed, and slag discharge time were adjusted to ensure that the solid content of the waste residue was ≥75%. The supernatant was filtered through a 0.45 μm hollow fiber membrane. The resulting GLP-1 precursor protein solution was directly introduced into the next step.

[0020] Experimental results: The solution was a clear, brownish-yellow solution, with a purity of 78.8% for the GLP-1 precursor protein solution and a yield of 97.8%.

[0021] (2) Enzyme digestion: Dilute the GLP-1 precursor protein solution obtained in step (1) to 13.9 mg / mL, add recombinant enterokinase for digestion, the amount of recombinant enterokinase added is 12 U / g, and 1 mmol / L calcium chloride is added at the same time. Digest at 20℃ for 5 h.

[0022] Experimental results: The solution after enzyme digestion was a clear, brownish-yellow liquid. The HPLC chromatogram of the GLP-1 precursor protein digestion solution is shown below. Figure 1 As shown, the retention time of Arg34GLP-1(9-37) was 26.983 min, the purity was 15.6%, and the enzyme digestion rate was 92.0%.

[0023] (3) High-pressure chromatography 1 Conditions: Mobile phase A was 20 mmol / L phosphate buffer, pH 7.6; mobile phase B was acetonitrile; detection wavelength was 280 nm; polymer packing material was polystyrene / divinylbenzene (UNIPS), specifically UNIPS40-300; flow rate was 3.8 cm / min; injection volume Arg34GLP-1(9-37) ≤24 g / L.

[0024] The specific procedure for high-pressure chromatography is as follows: Equilibration: Equilibrate the column using mobile phase A / mobile phase B (v / v, 9:1) until the baseline is stable; Sample loading: Samples were loaded according to the GLP-1 precursor protein digestion mixture / mobile phase B (v / v, 9:1); Equilibration: Equilibrate the column using mobile phase A / mobile phase B (v / v, 9:1) until the baseline is stable; Sample elution: (a) Over a 2-minute time period, the volume fraction of mobile phase B increased from 10% to 20%; (b) Maintain the volume fraction of mobile phase B at 20% for 18 min; (c) During the 3-minute time period, the volume fraction of mobile phase B increased from 20% to 35%; (d) During the 20 min time period, maintain the volume fraction of mobile phase B at 35%. Following the gradient elution procedure described above, the main peak was collected. The purified GLP-1 precursor protein digestion solution was then purified using a reverse polymer packing material, and the resulting chromatogram is shown below. Figure 2 As shown.

[0025] Experimental results: After purification by high-performance chromatography (HPLC), a light green clear solution was obtained; the HPLC chromatogram of the GLP-1 precursor protease digestion solution after purification by reverse polymer packing is shown below. Figure 3 As shown, the retention time of Arg34GLP-1(9-37) was 26.914 min, the purity was 79.9%, and the yield was 96%.

[0026] (4) High-pressure chromatography 2 Conditions: Mobile phase A was 5 mmol / L phosphate buffer, pH 7.6; mobile phase B was acetonitrile; detection wavelength was 280 nm; packing material was reverse-flow C18; flow rate was 3.8 cm / min; injection volume Arg34GLP-1(9-37) ≤ 16 g / L.

[0027] The specific procedure for high-pressure chromatography 2 is as follows: Equilibration: Equilibrate the column using mobile phase A / mobile phase B (v / v, 9:1) until the baseline is stable; Sample loading: Dilute the main peak solution obtained in step (3) by 3 to 5 times and load it directly; Equilibration: Equilibrate the column using mobile phase A / mobile phase B (v / v, 9:1) until the baseline is stable; Sample elution: (a) Over a 3-minute time period, the volume fraction of mobile phase B increased from 10% to 29%; (b) During the 45 min time period, the volume fraction of mobile phase B increased from 29% to 35%.

[0028] Following the gradient elution procedure described above, the main peak was collected. The purified GLP-1 precursor protein digestion solution was then processed using C18 packing material, and the resulting chromatogram is shown below. Figure 4 As shown.

[0029] Experimental results: After purification by high-performance chromatography (HPLC), a colorless and clear solution was obtained; the HPLC chromatogram of the GLP-1 precursor protease digestion solution after purification with C18 packing material is shown below. Figure 5 As shown, the retention time of Arg34GLP-1(9-37) was 26.885 min, the purity was 96.2%, and the yield was 97%.

[0030] (5) Acid precipitation The main peak obtained in step (4) was diluted 3 times with purified water, the pH was adjusted to 4.8 with acetic acid, and the mixture was placed at low temperature for 6 h. About 60-80% of the supernatant was discarded. The same volume of purified water as the remaining material was added, stirred evenly, and placed at low temperature for 6 h. About 60-80% of the supernatant was discarded again. The remaining material was centrifuged at 6000 rpm for 3 min and the precipitate was collected.

[0031] Experimental results: After acid precipitation, a white solid containing water was obtained; the HPLC chromatogram of Arg34GLP-1(9-37) after acid precipitation is shown below. Figure 6 As shown, the retention time of Arg34GLP-1(9-37) was 26.889 min, the purity was 96.0%, and the yield was 93%.

[0032] (6) Freeze-drying The precipitate obtained in step (5) was resuspended in water until the Arg34GLP-1(9-37) content was 5.5% (W / V), stirred evenly, and then freeze-dried. The freeze-drying parameters are shown in Table 1. Table 1. Lyophilization parameters of Arg34GLP-1(9-37) intermediate peptide solution

[0033] Experimental results: Arg34GLP-1(9-37) is a white powder with a purity of 96.2%.

[0034] Comparative Example 1 Verify the necessity of ultrafiltration in the purification and preparation of GLP-1 precursor protein and main peptide chain intermediate Arg34GLP-1(9-37). The specific experimental steps are as follows: (1) Centrifugal filtration The fermentation broth was centrifuged using a high-speed refrigerated centrifuge at 7500 rpm for 10 min. The supernatant was filtered through 0.8 μm and 0.45 μm filter membranes.

[0035] Experimental results: The solution was a clear, brownish-yellow solution, with a GLP-1 precursor protein solution purity of 52% and a yield of 91.2%.

[0036] (2) Ultrafiltration Take the solution from step (1), perform ultrafiltration using a 5K ultrafiltration membrane, and wash the concentrate with purified water.

[0037] Experimental results: The solution was a colorless and clear solution, the purity of Arg34GLP-1(9-37) was 76.7%, and the yield was 86.0%.

[0038] (3) Enzyme digestion Take the ultrafiltration concentrate from step (2), dilute it 5 times with pure water, and perform enzymatic digestion according to the enzymatic digestion conditions in Example 1.

[0039] Experimental results: The enzyme digestion rate of the GLP-1 precursor protein solution was 96.4%, and the purity was 17.8%.

[0040] (4) High-pressure chromatography 1 Take the enzyme digestion solution from step (3) and purify it using the polymer UNIPS40-300 packing material. For specific purification conditions, refer to Example 1.

[0041] Experimental results: The purity of Arg34GLP-1(9-37) was 82.0%, and the yield was 80.2%.

[0042] (5) High-pressure chromatography 2 Take the sample solution with a purity of less than 90% from step (4) and further purify it using reverse C18 packing material. For specific purification conditions, refer to Example 1.

[0043] Experimental results: The purity of Arg34GLP-1(9-37) was 95.5%, and the yield was 94.8%.

[0044] Experimental results show that traditional ultrafiltration followed by enzymatic digestion of the fermentation filtrate yields enzymatic digestion results that are essentially consistent with those of this invention. However, one-step reverse polymer packing purification after enzymatic digestion cannot increase the purity of the main peptide intermediate Arg34GLP-1(9-37) to over 90%, still requiring further purification with C18 packing. The resulting samples do not show significant advantages over those of this invention, and ultrafiltration results in losses. Therefore, this invention eliminates the traditional ultrafiltration step, yielding high-purity intermediates with a simple process, high yield, and reduced equipment and production costs, making it suitable for industrial production.

[0045] Example 2 Optimization of GLP-1 precursor protein digestion conditions We optimized the concentration of GLP-1 precursor protein solution, digestion temperature, digestion time, and amount of recombinant enterokinase. The specific optimization conditions and experimental results are shown in Table 2. Table 2 shows that when the concentration of GLP-1 precursor protein solution is 1.7–17 mg / mL, the digestion temperature is 15–25℃, the digestion time is 5–15 h, and the amount of recombinant enterokinase is 6–16 U / g, the digestion rate of the GLP-1 precursor protein solution is approximately 90%, meeting the requirements for GLP-1 precursor protein digestion.

[0046] Table 2 Summary of optimization results for GLP-1 precursor protein solution enzymatic digestion conditions

[0047] Example 3 Optimization of buffer concentration and pH conditions for high-performance chromatography 1 Following the experimental conditions in Implementation 1, the GLP-1 precursor protein solution was centrifuged, filtered, and digested with enzymes. The digested solution was then purified by high-pressure chromatography (HPLC). Only the concentration and pH of mobile phase A (phosphate buffer) were changed; other experimental conditions remained constant. The yields and purity results of HPLC purification under different experimental conditions are shown in Table 3. Table 3 shows that within the phosphate buffer concentration range of 10–30 mmol / L and pH range of 7–8, the purification effect of the main peptide intermediate Arg34GLP-1(9-37) was basically consistent, with yields all exceeding 90% and purity exceeding 70%.

[0048] Table 3 Results of the optimization test of buffer concentration and pH conditions used in high-performance chromatography 1

[0049] Example 4 Optimization of buffer concentration and pH conditions for high-performance chromatography 2 Following the experimental conditions in Implementation 1, the GLP-1 precursor protein solution was purified by centrifugation, filtration, enzymatic digestion, and high-pressure chromatography (HPLC). The initially purified solution was then further purified by HPLC (HPLC 2), with only the concentration and pH of mobile phase A (phosphate buffer) changed, while other experimental conditions remained constant. The yields and purities after HPLC purification under different experimental conditions are shown in Table 4. Table 4 shows that within the phosphate buffer concentration range of 3–8 mmol / L and pH range of 7–8, the purification effect of the main peptide intermediate Arg34GLP-1(9-37) was basically consistent, with yields all exceeding 97% and purities exceeding 95%.

[0050] Table 4 Results of the optimization test of buffer concentration and pH conditions used in high-performance chromatography 2

[0051] Example 5 Optimization of acid precipitation conditions for Arg34GLP-1(9-37) solution of main peptide intermediate Following the experimental conditions in Implementation 1, the GLP-1 precursor protein solution was purified by centrifugation, filtration, enzymatic digestion, high-pressure chromatography 1, and high-pressure chromatography 2. The purified solution was then subjected to acid precipitation. Only the pH conditions, the two periods of low-temperature settling, and the centrifugation speed were varied during the acid precipitation process; all other experimental conditions remained constant. The yield results of Arg34GLP-1(9-37) under different acid precipitation conditions are shown in Table 5. As shown in Table 5, the yield results of Arg34GLP-1(9-37) were basically consistent within the range of pH value of the acid precipitation solution (4.5–5.5), two periods of low-temperature settling (1–6 h), and centrifugation speed (5000–6000 rpm), with yields all exceeding 90%.

[0052] Table 5. Summary of the yield results of Arg34GLP-1(9-37) under different acid precipitation conditions.

[0053] Example 6 Optimization of lyophilization conditions for Arg34GLP-1(9-37) solution, the intermediate of the main peptide chain Following the experimental conditions in Implementation 1, the GLP-1 precursor protein solution was purified by centrifugation, filtration, enzymatic digestion, high-pressure chromatography 1, high-pressure chromatography 2, and acid precipitation. The resulting precipitates were then resuspended in water to Arg34GLP-1(9-37) content of 4% (w / v) and 8% (w / v), respectively, with other experimental conditions remaining unchanged. The purity results of Arg34GLP-1(9-37) under different lyophilization conditions are shown in Table 6. As can be seen from Table 6, when the Arg34GLP-1(9-37) content is within the range of 4%~8% (w / v) for lyophilization, the purity of the above intermediates obtained is all higher than 95%.

[0054] Table 6 Summary of Arg34GLP-1(9-37) purity results under different freeze-drying conditions

[0055] In summary, the purification and preparation method proposed in this invention involves directly centrifuging and filtering after fermentation to obtain the GLP-1 precursor protein solution, followed by enzymatic digestion, two-step chromatography, acid precipitation, and lyophilization to prepare the main peptide chain intermediate Arg34GLP-1(9-37) or Arg34GLP-1(7-37). This innovatively eliminates complex and cumbersome steps such as ultrafiltration, high-pressure homogenization, inclusion body collection, and handling of metamorphic inclusion bodies. By utilizing a combination of reverse polymer chromatography and reverse silica gel chromatography, the purity of the intermediate is increased to over 95%. This invention simplifies the process, saving costs while obtaining high-purity, high-yield intermediates suitable for direct industrial production and facilitating industrial scale-up.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for purifying and preparing GLP-1 precursor protein and main peptide chain intermediate, characterized in that, Includes the following steps: (1) Centrifugation and filtration: Take the fermentation broth containing the target protein, centrifuge and filter it directly to obtain the GLP-1 precursor protein solution; (2) Enzyme digestion: Take the GLP-1 precursor protein solution obtained in step (1), dilute it, and digest it with recombinant enterokinase to obtain a mixed solution containing tag protein, linker, target intermediate and culture medium; (3) High pressure chromatography 1: Take the mixed solution obtained in step (2), use the reversed polymer packing as the stationary phase, and use mobile phase A and mobile phase B to perform gradient elution on the mixed solution. Collect the target intermediate solution after the first purification at 280 nm. (4) High-pressure chromatography 2: Take the target intermediate solution obtained in step (3), dilute it with water 3 to 5 times, use reverse silica gel chromatography packing as stationary phase, use mobile phase A and mobile phase B to perform gradient elution on the mixed solution, and collect the target intermediate solution after the second purification at 280 nm. (5) Acid precipitation: Take the target intermediate solution obtained in step (4), adjust the pH of the solution to acidic using a pH adjuster, let it stand at low temperature, discard the supernatant, add purified water, stir evenly, let it stand at low temperature, discard the supernatant, and collect the precipitate after centrifugation; (6) Freeze-drying: Resuspend the wet solid in step (5) and freeze-dry to obtain the purified GLP-1 main peptide chain intermediate.

2. The purification and preparation method according to claim 1, characterized in that, When GLP-1 is semaglutide, the main peptide chain intermediate is Arg34GLP-1(9-37); when GLP-1 is liraglutide, the main peptide chain intermediate is Arg34GLP-1(7-37).

3. The purification and preparation method according to claim 1, characterized in that, In step (1), the solid content of the waste residue after centrifugation is ≥75%, and it is filtered using a 0.45 μm hollow fiber membrane.

4. The purification and preparation method according to claim 1, characterized in that, In step (2), the concentration of the GLP-1 precursor protein solution after dilution is 1.7~17 mg / mL, the amount of recombinant enterokinase added is 6~16 U / g, the enzyme digestion temperature is 15~25℃, the enzyme digestion time is 5~15 h, and 1 mmol / L of calcium chloride is added during enzyme digestion.

5. The purification and preparation method according to claim 1, characterized in that, The reverse polymer packing material in step (3) is one of polystyrene / divinylbenzene, polystyrene / polymethyl methacrylate, and polystyrene / divinylbenzene, and the reverse silica gel chromatography packing material in step (4) is one of C18, C4, and C8.

6. The purification and preparation method according to claim 1, characterized in that, In steps (3) and (4), mobile phase A is one or more of phosphate buffer, citrate buffer, acetate buffer, tris(hydroxymethyl)aminomethane buffer, and glycine buffer, and the pH of mobile phase A is 7-8; mobile phase B is one or more of methanol, anhydrous ethanol, and acetonitrile.

7. The purification and preparation method according to claim 1, characterized in that, In step (3), mobile phase A is 10~30 mmol / L phosphate buffer, mobile phase B is acetonitrile, the mobile phase velocity is 3.8 cm / min, and the injection volume of the mixed solution is Arg34GLP-1(9-37) or Arg34GLP-1(7-37) ≤24 g / L; The gradient elution procedure is as follows: (a) Over a 2-minute time period, the volume fraction of mobile phase B increased from 10% to 20%; (b) Maintain the volume fraction of mobile phase B at 20% for 18 min; (c) During the 3-minute time period, the volume fraction of mobile phase B increased from 20% to 35%; (d) During the 20 min time period, maintain the volume fraction of mobile phase B at 35%.

8. The purification and preparation method according to claim 1, characterized in that, In step (4), mobile phase A is 3-8 mmol / L phosphate buffer, mobile phase B is acetonitrile, the mobile phase velocity is 3.8 cm / min, and the injection volume of mixed solution is ≤16 g / L for Arg34GLP-1(9-37) or Arg34GLP-1(7-37). The gradient elution procedure is as follows: (a) Over a 3-minute time period, the volume fraction of mobile phase B increased from 10% to 29%; (b) During the 45-minute time period, the volume fraction of mobile phase B increased from 29% to 35%.

9. The purification and preparation method according to claim 1, characterized in that, In step (5), the pH adjuster is one of glacial acetic acid, hydrochloric acid, phosphoric acid, or citric acid, and the solution pH is 4.5 to 5.

5.

10. The purification and preparation method according to claim 1, characterized in that, In step (5), the two low-temperature standing times are 1-6 h each time, the volume of supernatant discarded each time is 60%-80% of the original solution volume, the ratio of the volume of purified water added to the volume of the remaining supernatant is 1:1, the centrifugation speed is 5000-6000 rpm, and the centrifugation time is 3-5 min.

11. The purification and preparation method according to claim 1, characterized in that, In step (6), the mass-volume ratio of Arg34GLP-1(9-37) or Arg34GLP-1(7-37) after resuspending in purified water is 4%~8%.

12. The main peptide intermediate Arg34GLP-1(9-37) or Arg34GLP-1(7-37) obtained by the purification and preparation method according to any one of claims 1 to 11.

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