Preparation method of semaglutide

Abstract

The invention relates to the technical field of polypeptide synthesis, and provides a preparation method of semaglutide, which comprises the following steps: gradually coupling a liquid phase and a solid phase to obtain a fragment 1: Boc-His (trt)-Aib and a fragment 3: tBuO-Ste-Glu (AEEA-AEEA-OH)-OtBu, activating the fragment 3, carrying out coupling reaction on the fragment 3 and lysine side chain amino in a fragment 2: Arg34GLP-1 (9-37) protected by a terminal glutamic acid amino group, namely deprotecting P = FMOC to obtain an intermediate I, and purifying to obtain the semaglutide. Coupling the fragment 1 with the intermediate II to obtain an intermediate II, cutting, precipitating, separating, purifying and freeze-drying to obtain a semaglutide product; according to the present invention, based on the reaction rate of the different combination modes of the three fragments and the preparation of the recombinant main chain GLP-1 (9-37), the reaction process of the semaglutide is short, the yield is high, the purity is good, the semaglutide is green and environmentally friendly, and the industrial preparation of the semaglutide is easily achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide synthesis, in particular to a method for preparing semaglutide. Background Art

[0002] Semaglutide, also known as Semaglutide in English, is a new type of long-acting glucagon-like peptide-1 (GLP-1) analog developed and produced by Novo Nordisk of Denmark for the treatment of type 2 diabetes. Semaglutide has the effects of lowering blood sugar, reducing weight and protecting cardiovascular system, and was approved by FDA for marketing in December 2017. After the Lys side chain of Semaglutide is modified by PEG, Glu and 18-carbon dicarboxylic acid, its hydrophilicity is greatly improved and its binding ability with albumin is enhanced; at the same time, after the Ala at the second position of the N-terminus is mutated to Aib, it effectively avoids inactivation by DPP-IV enzymatic hydrolysis, and its half-life reaches 40h. Patients only need to inject once a week. The oral dosage form of the drug is also under development. The CAS number of Semaglutide is 910463-68-2, the molecular formula is C187H291N45O59, the molecular weight is 4113.64g / mol, and the peptide sequence is:

[0003] H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA- γ -Glu-Octadecane dioic)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

[0004] The preparation methods of semaglutide reported so far can be roughly divided into two categories: one is to directly connect Lys containing side chains as fragments to the main chain of semaglutide to complete the synthesis. Patent CN104356224A discloses a method of using a liquid phase method to connect side chains to the ε-NH2 of Lys, and then gradually condense amino acids on a resin to prepare semaglutide. The other is to complete the coupling of the main chain and side chains of semaglutide one by one. Patent CN201511027176 discloses the stepwise synthesis of semaglutide linear peptides in the solid phase, the synthesis of side chain modification groups, the removal of the Lys protection group, and the coupling of side chain modification groups, and finally the cleavage to obtain the polypeptide product. Since the sequence of semaglutide is long and contains many hydrophobic amino acids, it is easy to form folds when synthesized by the method of stepwise condensation of amino acids, resulting in severe shrinkage of the resin, prolonged reaction time, and the production of more impurities in the crude peptide that are very close to the properties of the product; the [D-His] racemic impurity has similar physical and chemical properties to the target peptide of semaglutide, which greatly increases the difficulty of separation and purification of semaglutide products, resulting in a significant reduction in product yield. When the fragment method is used, the reaction control is controlled by using the different trends of the isoelectric point of the amino active site, which can easily cause superimposed side reactions such as insufficient reaction, over-reaction, and inappropriate reaction conditions. Therefore, there is an urgent need for a method for synthesizing semaglutide with high purity and yield and low synthesis cost. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for preparing semaglutide to solve the problems raised in the background technology.

[0006] A method for preparing semaglutide comprises the following steps: obtaining fragment 1: Boc-His(trt)-Aib and fragment 3: tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu by step-by-step liquid-solid coupling; activating fragment 3 and then carrying out coupling reaction with the lysine side chain amino group in fragment 2: Arg34GLP-1(9-37) after protecting the terminal glutamic acid amino group, i.e., deprotecting P=FMOC to obtain intermediate I; coupling fragment 1 with intermediate I to obtain intermediate II; and cutting, precipitating, separating, purifying and freeze-drying to obtain the semaglutide product.

[0007] Preferably, the steps of preparing the fragment 1 are as follows:

[0008] S101, using liquid phase synthesis to prepare fragment 1: BOC-His(trt)-Aib;

[0009] S102. Using solid phase resin, synthesize and prepare fragment 3 one by one: tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu.

[0010] Preferably, the preparation method of fragment 2 comprises the following steps:

[0011] S201, construction of pET-28a-GLP-1(9-37) recombinant plasmid and engineering strain: artificially synthesize Met-TrxA-DnaB-GLP-1(9-37)-DnaB-GLP-1(9-37)-DnaB-GLP-1(9-37), and clone it into pET-28a by Gibson assembly technology to obtain plasmid pET-28a-CIG1;

[0012] S202, inducing expression of recombinant engineered bacteria: introducing pelleted pET-28a-CIG1 into Escherichia coli BL21 (DE3), pre-inoculating a single colony in 5 mL of LB medium containing 50 μg / mL kanamycin, culturing overnight at 37°C, inoculating 2 mL of the culture into 200 mL of fresh TB medium, culturing at 37°C for 2 hours, adding 0.5 mM isopropyl-β-d-thiogalactopyranoside IPTG after the OD600 value is 0.5-0.8, inducing at 30°C for 10 hours, after expression, collecting the cells by centrifugation at 4000×g, 4°C for 20 min, and then resuspending in 50 mL, 50 mM Tris-HCl, pH 7.5 lysis buffer, and disrupting the cells by ultrasound;

[0013] S203, fermentation culture of recombinant genetically engineered bacteria: the screened genetically engineered bacteria are first streaked and cultured overnight, and then a single colony is picked and placed in an LB liquid culture medium containing Kan+ resistance, cultured at 37°C for 12 hours, and then transferred to a 1000ml Erlenmeyer flask containing 200ml LB culture solution at a ratio of 1%, and cultured at 37°C overnight to become the seed solution for the upper tank. The seed solution for the upper tank is inoculated into a 10L fermenter containing TB culture solution at a ratio of 5%, cultured at 37°C, and the dissolved oxygen is maintained above 25%. The pH is adjusted with ammonia water and maintained at 6.5 until the OD600 of the bacterial solution reaches 50-80, and then isopropyl-BD-thiogalactose is added at a final concentration of 0.2mM. The fermentation is stopped after continuing to culture for 3 hours, the bacterial solution is collected, centrifuged at 8000rpm for 10 minutes, the supernatant is discarded, and the bacterial bodies are collected and stored in a -20°C refrigerator for standby use;

[0014] S204, N-terminal self-cleavage of DnaB: SspDnaB mini-protein was used, and the sample was washed twice with 50 mL of washing buffer at pH 8.0-9.0. After washing, 5 mL of 50 mM Tris-HCl, 1 mM DTT, pH 8.0-9.0 lysis buffer was added to resuspend the sample, and the suspension was kept at 23°C for 24 h for self-cleavage to obtain protein-free target protein;

[0015] S205, C-terminal self-cleavage of DnaB: The samples obtained in S204 were washed twice with 50 mL of pH 6.5 washing buffer, and then 5 mL of 50 mM Tris-HCl, 1 mM DTT, pH 6.5 lysis buffer was added to resuspend the samples. The suspension was kept at 23°C for 24 h. The C-terminal cleavage began to self-cleave at pH 6.5, and the cleavage mixture was centrifuged at 10,000 × g at 4°C for 20 min.

[0016] S206. Purify by chromatography and freeze-dry to obtain Arg34GLP-1(9-37), dissolve Arg34GLP-1(9-37) in alkaline water or ACN, add FMOC-OSU in batches to maintain alkaline reaction, and then obtain FMOC-GLP-1Arg34(9-37) after post-treatment.

[0017] Preferably, the preparation steps of the intermediate I are as follows:

[0018] S301, Activation of fragment 3: Weigh the modifier with a mass ratio of fragment 2 to fragment 3 of 2-3:1, add THF to dissolve it to 100 mg / ml, add TSTU and DIPEA in a molar ratio of 1:1.2-2:2-3, respectively, and stir at room temperature at 200-500 rpm to react until the reaction is basically complete;

[0019] S302. Dissolve fragment 2 in 50% acetonitrile aqueous solution with DIPEA, add the reaction solution prepared in S301, stir and maintain pH 10.6±0.2 until fragment 2 is completely reacted. After the reaction, deprotect and adjust pH to 4-5, centrifuge and wash or freeze-dry to obtain intermediate I: H-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(tBuO-Ste-Glu(AEEA-AEEA-)-OtBu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

[0020] Preferably, the preparation steps of the intermediate II are as follows: fragment 1 is activated, reacted with intermediate I under liquid phase conditions, and then intermediate II is separated.

[0021] Preferably, the entire activation, peptide bond coupling, and protecting group removal are monitored in real time using online Raman spectroscopy to perform the initial reaction.

[0022] Preferably, the recombinant Arg34GLP-1 (9-37) is purified by enzymatic cleavage and then purified by polymer filler with a loading of 25-40 g / L, a yield of >90%, and a purity of >95%.

[0023] Preferably, after obtaining the crude soma product, a polymer is used: PS10-300 with a loading capacity of 5-10 g / L, a yield of >85%, a purity of >98.5%, and a single impurity of <0.2%, which meets the requirements of corresponding indicators for oral administration; and then silica gel: C8 / C18 with a loading capacity of 5-10 g / L, a yield of >85%, a purity of >98.5%, and a single impurity of <0.1%, which meets the requirements of corresponding indicators for injection.

[0024] Preferably, during the 10-hour induction at 30° C., additional 25 mM HEPES, pH 8.5, is added to the culture medium at 0, 2, 4 and 6 h, respectively, to offset the acidification of the culture medium during the cell culture process.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention makes the semaglutide reaction process short, with high yield, good purity, green and environmental protection, and easy industrial preparation of semaglutide through the reaction rate based on different combinations of three fragments and the preparation of recombinant main chain GLP-1 (9-37). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the pET28a-CIG1 bacterial strain construction diagram of the present invention;

[0028] Figure 2 This is a liquid chromatogram of the fragment 2Arg34GLP-1(9-37) of the present invention;

[0029] Figure 3 is the mass spectrum of the fragment 2Arg34GLP-1(9-37) of the present invention;

[0030] Figure 4 This is a diagram of the SDS-PAGE electrophoresis result of pET28a-CIG1 of the present invention;

[0031] Figure 5 This is a diagram showing the results of the self-cleavage SDS-PAGE electrophoresis of GLP-1 (9-37) of the present invention;

[0032] Figure 6 This is the reaction diagram of the purified fragment 2GLP-1ARG (9-37) of the present invention;

[0033] Figure 7 This is the reaction diagram after activation of fragment 3 of the present invention;

[0034] Figure 8 This is the reaction diagram of fragment 3+fragment 2 of the present invention;

[0035] Fig. 9 This is the reaction diagram of fragment 2+fragment 3+fragment 1 of the present invention;

[0036] Fig.10 This is the liquid chromatogram of the finished product of semaglutide;

[0037] Fig.11 This is the mass spectrum of semaglutide; Fig.12 This is the cracking reaction diagram of the crude Soma product; Fig.13 This is the detection spectrum of the crude Soma product after one purification; Fig.14 This is the detection spectrum after secondary purification of the crude Soma product. DETAILED DESCRIPTION

[0038] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0039] As attached Figure 1 To Attachment Fig.11 As shown:

[0040] Embodiment 1: The present invention provides a method for preparing semaglutide, comprising: obtaining fragment 1: Boc-His(trt)-Aib and fragment 3: tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu by liquid-solid phase stepwise coupling, activating fragment 3, and then coupling it with the terminal glutamic acid amino group-protected fragment 2: Arg34GLP-1(9-37) lysine side chain amino group, i.e., deprotecting P=FMOC to obtain intermediate I, coupling fragment 1 with intermediate I to obtain intermediate II, and cutting, precipitating, separating, purifying, and freeze-drying to obtain the semaglutide product.

[0041] The steps of preparing fragment 1 are as follows:

[0042] S101, using liquid phase synthesis to prepare fragment 1: BOC-His(trt)-Aib;

[0043] S102. Using solid phase resin, synthesize and prepare fragment 3 one by one: tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu.

[0044] The steps of preparing fragment 2 are as follows:

[0045] S201, construction of pET-28a-GLP-1(9-37) recombinant plasmid and engineering strain: artificially synthesize Met-TrxA-DnaB-GLP-1(9-37)-DnaB-GLP-1(9-37)-DnaB-GLP-1(9-37), and clone it into pET-28a by Gibson assembly technology to obtain plasmid pET-28a-CIG1;

[0046] S202, induce expression of recombinant engineered bacteria: introduce pelleted pET-28a-CIG1 into Escherichia coli BL21 (DE3), pre-inoculate a single colony in 5 mL of LB medium containing 50 μg / mL kanamycin, culture overnight at 37°C, inoculate 2 mL of the culture into 200 mL of fresh TB medium, culture at 37°C for 2 hours, add 0.5 mM isopropyl-β-d-thiogalactopyranoside IPTG after the OD600 value is 0.5-0.8, induce at 30°C for 10 hours, after expression, collect cells by centrifugation at 4000×g, 4°C for 20 min, then resuspend in 50 mL, 50 mM Tris-HCl, pH 7.5 lysis buffer, and disrupt cells by ultrasound; wherein during the 10-hour induction at 30°C, additional 25 mM HEPES, pH 8.5, was added to the culture at 0, 2, 4 and 6 h, respectively, to offset the acidification of the culture medium during cell culture;

[0047] S203, fermentation culture of recombinant genetically engineered bacteria: the screened genetically engineered bacteria are first streaked and cultured overnight, and then a single colony is picked and placed in an LB liquid culture medium containing Kan+ resistance, cultured at 37°C for 12 hours, and then transferred to a 1000ml Erlenmeyer flask containing 200ml LB culture solution at a ratio of 1%, and cultured at 37°C overnight to become the seed solution for the upper tank. The seed solution for the upper tank is inoculated into a 10L fermenter containing TB culture solution at a ratio of 5%, cultured at 37°C, and the dissolved oxygen is maintained above 25%. The pH is adjusted with ammonia water and maintained at 6.5 until the OD600 of the bacterial solution reaches 50-80, and then isopropyl-BD-thiogalactose is added at a final concentration of 0.2mM. The fermentation is stopped after continuing to culture for 3 hours, the bacterial solution is collected, centrifuged at 8000rpm for 10 minutes, the supernatant is discarded, and the bacterial bodies are collected and stored in a -20°C refrigerator for standby use;

[0048] S204, N-terminal self-cleavage of DnaB: SspDnaB mini-protein was used, and the sample was washed twice with 50 mL of washing buffer at pH 8.0-9.0. After washing, 5 mL of 50 mM Tris-HCl, 1 mM DTT, pH 8.0-9.0 lysis buffer was added to resuspend the sample, and the suspension was kept at 23°C for 24 h for self-cleavage to obtain protein-free target protein;

[0049] S205, C-terminal self-cleavage of DnaB: The samples obtained in S204 were washed twice with 50 mL of washing buffer at pH 6.5, and then 5 mL of 50 mM Tris-HCl, 1 mM DTT, pH 6.5 lysis buffer was added to resuspend the samples. The suspension was kept at 23°C for 24 h. The C-terminal cleavage began to self-cleave at pH 6.5, and the cleavage mixture was centrifuged at 10,000 × g at 4°C for 20 min.

[0050] S206. Purify by chromatography and freeze-dry to obtain Arg34GLP-1(9-37), dissolve Arg34GLP-1(9-37) in alkaline water or ACN, add FMOC-OSU in batches to maintain alkaline reaction, and then obtain FMOC-GLP-1Arg34(9-37) after post-treatment.

[0051] The preparation steps of intermediate I are as follows:

[0052] S301, Activation of fragment 3: Weigh the modifier with a mass ratio of fragment 2 to fragment 3 of 2-3:1, add THF to dissolve to 100 mg / ml, add TSTU and DIPEA at a molar ratio of 1:1.2-2:2-3, respectively, calculated at 1 g / ml, and react at room temperature with stirring at 200-500 rpm until the reaction is substantially complete;

[0053] S302. Dissolve fragment 2 in 50% acetonitrile aqueous solution with DIPEA, add the reaction solution prepared in S301, stir and maintain the pH at 10.6±0.2 until fragment 2 is completely reacted. After the reaction, adjust the pH to 4-5, wash by centrifugation or freeze-dry to obtain intermediate I: H-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Al a-Lys(tBuO-Ste-Glu(AEEA-AEEA-)-OtBu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

[0054] The preparation steps of intermediate II are as follows: Fragment 1 BOC-His(trt)-Aib is activated and reacted with intermediate I under liquid phase conditions to obtain intermediate II. The progress of the synthesis is detected online by Raman spectroscopy during the entire process to ensure the reaction rate and terminate the reaction in time to obtain a good reaction effect.

[0055] During the entire activation and coupling of peptide bonds, online Raman spectroscopy was used for real-time monitoring to achieve the earliest reaction. The resulting soma crude product cleavage reaction diagram is shown in the attached figure. Fig.12 As shown, the external standard peptide content was 75.6%.

[0058] Purification of recombinant Arg34GLP-1 (9-37): After enzyme cleavage, use polymer filler for purification, with a loading capacity of 25-40g / L, a yield of >90%, a purity of >95%, and a long service life.

[0059] After obtaining the crude soma product, the polymer: PS10-300 loading capacity 5-10g / L, yield>85%, purity>98.5%, single impurity<0.2%, meeting the corresponding index requirements for oral administration; then silica gel: C8 / C18 loading capacity 5-10g / L, yield>85%, purity>98.5%, single impurity<0.1%, meeting the corresponding index requirements for injection.

[0060] After the crude product of Soma was purified once, its detection spectrum was as follows Fig.13 The specific data are shown in the following table:

[0063]

[0064] After secondary purification of the crude product of Soma, its detection spectrum is as shown in the attached Fig.14 The specific data are shown in the following table:

[0067]

[0068] Example 2: In the preparation process of fragment 2, a mini-protein SspDnaB widely used in protein purification is used, which can cut the target protein fused at its N-terminus and C-terminus, respectively referred to as N-terminal and C-terminal cutting. The first step of SspDnaB N-terminal cleavage is the rearrangement of the NS acyl group at the N-terminus (Cys1) of the protein, resulting in the formation of a thioester connection between the target protein and the protein. Then, a thiol reagent such as 1,4-dithiothreitol (DTT) or 2-thiolethanolsulfonic acid is added to induce the cleavage of the thioester bond to obtain a protein-free target protein. The C-terminal cleavage of SspDnaB starts with a pH change. The C-terminal cleavage starts at a pH of 6.5, inducing the cyclization of the C-terminal Asn154 side chain to form a succinimidyl group, and the peptide bond is broken to obtain a protein-free target protein. Finally, the target protein is obtained by cutting twice in 2.

[0069] Example 3: This example is different from the previous two examples in the process of preparing semaglutide:

[0070] 1. Take 3287.45 g of the fragment and dissolve it in THF to 100 mg / ml. Add 153.47 g of TSTU and 131.73 g of DIPEA respectively. Stir and react at room temperature (25°C) for 4±1 hours. Add 1000 g of GLP-1ARG (9-37) dissolved in an equal volume of acetonitrile dropwise.

[0071] The pH was maintained at 10.6±0.2 and the temperature was 25°C±3. The reaction was stirred for 1.5±0.5 hours, and Raman spectroscopy was monitored online. After the reaction was completed, the final pH was adjusted to 4.8±0.5. After stirring for 1 hour at room temperature (25°C), the intermediate I was obtained by centrifugation and filtration.

[0072] 2. Take 1MOL of protected dipeptide, dissolve it in NMP to 0.1g / ml, add 3937.27g of DIPEA under stirring, control the temperature at 25±3°C, stir for 10 minutes, then add 579.83g of HATU, stir and react for 3±0.5 hours; add to the acetonitrile solution of intermediate I;

[0073] The reaction was stirred at room temperature and the Raman spectrum was monitored online. The pH was adjusted to 4.8±0.5, stirred evenly, stirred at room temperature for 1.5±0.5 hours, centrifuged, filtered, lysed, precipitated, and centrifuged to obtain a crude Soma product.

[0074] Importantly, it should be noted that the construction and arrangement of the present application shown in a plurality of different exemplary embodiments is merely illustrative. Although only a few embodiments are described in detail in this disclosure, it should be readily understood by those who refer to this disclosure. The order or sequence of any process or method steps may be changed or reordered according to alternative embodiments. Therefore, the present invention is not limited to specific embodiments, but extends to a variety of modifications that still fall within the scope of the appended claims.

[0075] It will be appreciated that in the development of any actual implementation, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort may be complex and time-consuming, but will be a routine task of design, fabrication, and production for those of ordinary skill having the benefit of this disclosure without undue experimentation.

[0076] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing semaglutide, Features: Fragment 1: Boc-His(trt)-Aib and fragment 3: tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu are obtained by step-by-step liquid-solid coupling. After fragment 3 is activated, it is coupled with the lysine side chain amino group in fragment 2: Arg34 GLP-1(9-37) after the terminal glutamic acid amino group is protected, that is, P=FMOC is deprotected to obtain intermediate I, and fragment 1 is coupled with it to obtain intermediate II, which is cut, precipitated, separated, purified and freeze-dried to obtain the semaglutide product.

2. A method for preparing semaglutide according to claim 1, Features: The steps of preparing the fragment 1 are as follows: S101, using liquid phase synthesis to prepare fragment 1: BOC-His(trt)-Aib; S102. Using solid phase resin, synthesize and prepare fragment 3 one by one: tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu.

3. A method for preparing semaglutide according to claim 1, Features: The steps of preparing the fragment 2 are as follows: S201, construction of pET-28a-GLP-1(9-37) recombinant plasmid and engineering strain: artificially synthesize Met-TrxA-DnaB-GLP-1(9-37)-DnaB-GLP-1(9-37)-DnaB-GLP-1(9-37), and clone it into pET-28a by Gibson assembly technology to obtain plasmid pET-28a-CIG1; S202, inducing expression of recombinant engineered bacteria: introducing pelleted pET-28a-CIG1 into Escherichia coli BL21 (DE3), pre-inoculating a single colony in 5 mL of LB medium containing 50 μg / mL kanamycin, culturing overnight at 37°C, inoculating 2 mL of the culture into 200 mL of fresh TB medium, culturing at 37°C for 2 hours, adding 0.5 mM isopropyl-β-d-thiogalactopyranoside IPTG after the OD600 value is 0.5-0.8, inducing at 30°C for 10 hours, after expression, collecting the cells by centrifugation at 4000×g, 4°C for 20 min, and then resuspending in 50 mL, 50 mM Tris-HCl, pH7.5 lysis buffer, and disrupting the cells by ultrasound; S203, fermentation culture of recombinant genetically engineered bacteria: the screened genetically engineered bacteria were first streaked and cultured overnight, and then a single colony was picked and placed in an LB liquid culture medium containing Kan+ resistance, cultured at 37°C for 12 hours, and then transferred to a 1000ml Erlenmeyer flask containing 200ml LB culture solution at a ratio of 1%, and cultured at 37°C overnight to become the seed solution for the upper tank. The seed solution for the upper tank was inoculated into a 10L fermenter containing TB culture solution at a ratio of 5%, cultured at 37°C, and the dissolved oxygen was maintained above 25%. The pH was adjusted with ammonia water and maintained at 6.5 until the OD600 of the bacterial solution reached 50-80, and then isopropyl-BD-thiogalactose was added at a final concentration of 0.2mM. The culture was continued for 3 hours to stop the fermentation, the bacterial solution was collected, centrifuged at 8000rpm for 10 minutes, the supernatant was discarded, and the bacterial bodies were collected and stored in a -20°C refrigerator for standby use; S204, N-terminal self-cleavage of DnaB: Using Ssp DnaB mini-protein, wash twice with 50 mL of washing buffer at pH 8.0-9.

0. After washing, add 5 mL of 50 mM Tris-HCl, 1 mM DTT, pH 8.0-9.0 lysis buffer to resuspend the sample, and keep the suspension at 23°C for 24 h for self-cleavage to obtain protein-free target protein; S205, C-terminal autocleavage of DnaB: The samples obtained in S204 were washed twice with 50 mL of pH 6.5 washing buffer, and after washing, 5 mL of 50 mM Tris-HCl, 1 mM DTT, pH 6.5 lysis buffer was added to resuspend the sample, and the suspension was kept at 23°C for 24 h. The C-terminal cleavage began to autocleave at pH 6.5, and the cleavage mixture was centrifuged and incubated at 10,000 × g, 4°C for 20 min; S206. Purify by chromatography and freeze-dry to obtain Arg34 GLP-1 (9-37), dissolve Arg34 GLP-1 (9-37) in alkaline water or ACN, add FMOC-OSU in batches to maintain alkaline reaction, and then post-treat to obtain fragment 2FMOC-GLP-1Arg34 (9-37).

4. A method for preparing semaglutide according to claim 1, Features: The preparation steps of the intermediate I are as follows: S301, Activation of fragment 3: Weigh the modifier with a mass ratio of fragment 2 to fragment 3 of 2-3:1, add THF to dissolve it to 100 mg / ml, add TSTU and DIPEA in a molar ratio of 1:1.2-2:2-3, respectively, and stir at room temperature at 200-500 rpm to react until the reaction is basically complete; S302. Dissolve fragment 2 in 50% acetonitrile aqueous solution with DIPEA, add the reaction solution prepared in S301, stir and maintain pH 10.6±0.2 until fragment 2 is completely reacted. After the reaction, deprotect and adjust pH 4-5, centrifuge and wash or freeze-dry to obtain intermediate I: H-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(tBuO-Ste-Glu(AEEA-AEEA-)-OtBu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

5. A method for preparing semaglutide according to claim 1, Features: The preparation steps of the intermediate II are as follows: fragment 1 is activated, reacted with intermediate I under liquid phase conditions, and intermediate II is separated.

6. A method for preparing semaglutide according to any one of claims 1 to 5, Features: During the entire activation, coupling peptide bond, and removal of protecting groups, online Raman spectroscopy is used for real-time monitoring to carry out the earliest reaction.

7. A method for preparing semaglutide according to claim 3, Features: Purification of recombinant Arg34GLP-1(9-37): After enzyme cleavage, purification was performed using polymer filler with a loading of 25-40 g / L, a yield of >90%, and a purity of >95%.

8. A method for preparing semaglutide according to claim 1, Features: After obtaining the crude soma product, the polymer: PS10-300 loading capacity 5-10g / L, yield>85%, purity>98.5%, single impurity<0.2%, meeting the corresponding index requirements for oral administration; then silica gel: C8 / C18 loading capacity 5-10g / L, yield>85%, purity>98.5%, single impurity<0.1%, meeting the corresponding index requirements for injection.

9. A method for preparing semaglutide according to claim 1, Features: During the 10-hour induction of S202 at 30°C, additional 25 mM HEPES, pH 8.5, was added to the culture at 0, 2, 4, and 6 h to counteract medium acidification during cell culture.

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