A method for preparing GLP-1 or its analogs

Abstract

The present invention belongs to the field of biopharmaceutical technology, and specifically relates to a method for preparing GLP-1 or its analogs. The present invention provides a leader peptide, and a fusion protein comprising the leader peptide. The leader peptide or fusion protein is used to prepare a semaglutide precursor polypeptide, and the results show that the yield of the prokaryotic expression fusion protein can reach 26g / L, and the purity of the precursor polypeptide obtained after enzyme cleavage is higher than 95%. The precursor polypeptide is further side-chain modified and connected with a dipeptide to efficiently prepare semaglutide.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and in particular relates to a method for preparing GLP-1 or its analogs. Background Art

[0002] Glucagon-like peptide-1 (GLP-1) analogs, as GLP-1 receptor agonists, have excellent glucose-lowering and weight-reducing effects. Currently, a variety of GLP-1 analogs are disclosed in the prior art, including exenatide, lixisenatide, belaglutide, liraglutide, loxenatide, telpotide, semaglutide, albiglutide, dulaglutide and dulaglutide, etc.

[0003] Among them, semaglutide has 94% sequence homology with human GLP-1. Compared with natural GLP-1, semaglutide has the following structural differences: alanine at position 8 is replaced by α-aminoisobutyric acid, which can resist DPP-4 degradation; lysine at position 26 is connected to the C-18 fatty diacid side chain through a spacer, tightly binding to albumin; lysine at position 34 is replaced by arginine to prevent C-18 fatty diacid from binding to the wrong position. Therefore, the half-life of semaglutide is effectively extended, achieving a frequency of injection once a week, greatly reducing the number of injections and improving patient compliance. The metabolism of semaglutide is similar to that of large molecular proteins. It does not have a specific metabolic organ, but is widely metabolized in tissues. Therefore, the medication is not affected by liver and kidney function and has good safety.

[0004] At present, there are two main methods for synthesizing semaglutide: semi-fermentation and chemical synthesis. Published patents CN102875665A, CN112111002A, CN115322250A, CN103848910A, CN106928343A, CN106478806A, etc. all use full chemical synthesis methods. The disadvantage of this type of method is that the steps are cumbersome, and the synthesis of each fragment during the synthesis process requires excessive feeding, resulting in high costs. In addition, the side reactions produced by coupling will affect the yield, and the use of organic reagents will also cause serious environmental pollution, making the scale production of full synthesis more difficult. The original Novo Nordisk (CN101910193A) uses a semi-fermentation method (biosynthesis + chemical synthesis). Through genetic recombination technology, yeast production is used to obtain the precursor polypeptide Arg 34 -GLP-1 9-37 Then, the short peptide His-Aib was first connected by chemical means to form Aib 8 ,Arg 34 -GLP-1 7-37 , attaching a long-lasting modifying group to Lys 26 The side chain amino group of Aib forms a complete semaglutide molecule.8 ,Arg 34 -GLP-1 7-37 The side chain and N-terminal amino group lack protection, resulting in multiple active sites in the connection reaction, which is prone to produce large impurities and large losses.

[0005] Fusion protein expression is a common strategy for eukaryotic and prokaryotic expression systems. The target protein or polypeptide is connected to the partner protein through an endonuclease cleavage site to form a fusion protein, and the fusion protein is cleaved to obtain the target polypeptide. Among them, the sequence of the leader peptide in the fusion protein is very important for the expression of the fusion protein. For example, the patent document (CN114292338B) discloses a fusion protein and a method for preparing a semaglutide intermediate polypeptide. The document discloses a variety of leader peptides. The examples clearly point out that the fusion proteins containing different leader peptides have very different contents of intermediate polypeptides obtained by enzymatic cleavage of inclusion bodies obtained after fermentation and culture in the same culture mode. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a new leader peptide, which is then connected in series with multiple target peptides to form a fusion protein, which significantly improves the yield and purity after biological expression and reduces the production cost. The specific scheme is as follows:

[0007] In a first aspect, the present invention provides a leader peptide, wherein the amino acid sequence of the leader peptide comprises MADEAEKAG (SEQ ID NO: 8).

[0008] Preferably, the C-terminus of the leader peptide also contains an enzyme cleavage site.

[0009] Preferably, the cleavage site is a cleavage site of an amino acid endonuclease and / or an enzyme that recognizes basic amino acids, and more preferably, a cleavage site of a dibasic amino acid endonuclease and / or an enzyme that recognizes C-terminal basic amino acids.

[0010] In a specific embodiment of the present invention, the restriction enzyme cleavage site is the restriction enzyme cleavage site of Kex2 enzyme and / or CPB enzyme.

[0011] Preferably, the restriction site consists of basic amino acids.

[0012] Preferably, the basic amino acid is selected from arginine, lysine and histidine.

[0013] Preferably, the restriction site is 2-5 basic amino acids.

[0014] In a specific embodiment of the present invention, the amino acid sequence of the restriction site includes KR or RR or RK or KK.

[0015] In a specific embodiment of the present invention, the amino acid sequence of the leader peptide comprises MADEAEKAGKR (SEQ ID NO: 1).

[0016] The second aspect of the present invention provides a fusion protein, wherein the fusion protein comprises the above-mentioned leader peptide and target peptide.

[0017] The target peptide includes GLP-1 or its analogs.

[0018] Preferably, the GLP-1 is natural GLP-1 or a truncated sequence thereof, preferably truncated at the N-terminus and / or the C-terminus, and more preferably truncated at the N-terminus by 5-11 (preferably 7-11) amino acids and / or truncated at the C-terminus by 0-1 amino acids.

[0019] The amino acid length of the truncated sequence is 26-33.

[0020] Specifically, the truncated sequence can be GLP-1 5-37 、GLP-1 7-37 、GLP-1 9-37 、GLP-1 11-37 、GLP-1 5 -36 、GLP-1 7-36 、GLP-1 9-36 or GLP-1 11-36 wait.

[0021] Preferably, the GLP-1 analog is a natural GLP-1 or a truncated sequence thereof that has been mutated or structurally modified and has the function of natural GLP-1.

[0022] Preferably, the amino acid sequence of the GLP-1 analog has at least 80% homology with native GLP-1 or its truncated sequence, for example at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% homology.

[0023] Specifically, the mutation site may be position 8, 22, 34 or 36 of the natural GLP-1 sequence.

[0024] Specifically, the mutation includes replacing Ala at position 8 of the natural GLP-1 sequence with a non-natural amino acid (eg, 2-aminoisobutyric acid, Aib).

[0025] Specifically, the mutation includes replacement of Ala at position 8 of the natural GLP-1 sequence by Gly.

[0026] Specifically, the mutation includes replacing Lys at position 34 of the natural GLP-1 sequence with Arg.

[0027] Specifically, the mutation includes replacement of Gly at position 22 of the natural GLP-1 sequence by Glu.

[0028] Specifically, the mutation includes replacement of Arg at position 36 of the natural GLP-1 sequence with Gly.

[0029] Specifically, the structural modification includes but is not limited to fatty acid modification with a chain length of C16-C20, such as fatty acid modification with a chain length of C16, C17, C18, C19 or C20.

[0030] Preferably, the C18 fatty acid includes a monosubstituted octadecane dioic acid, and more preferably includes octadecane dioic acid mono-tert-butyl ester.

[0031] Preferably, the fatty acid modification comprises linkage via a covalent bond or an amino acid (eg, glutamic acid).

[0032] In some embodiments, the GLP-1 analog is a truncated sequence of GLP-1 including a mutation site, such as Arg 34 -GLP-1 5-37 , Arg 34 -GLP-1 7-37 , Arg 34 -GLP-1 9-37 , Arg 34 -GLP-1 11-37 , Arg 34 -GLP-1 5-36 , Arg 34 -GLP-1 7-36 , Arg 34 -GLP-1 9-36 , Arg 34 -GLP-1 11-36 , Gly 8 -GLP-1 7-36 , Gly 8 -Glu 22 -Gly 36 -GLP-1 7-36 , Aib 8 -Arg 34 -GLP-1 5-37 , Aib 8 -Arg 34 -GLP-1 7-37 , Aib 8 -Arg 34 -GLP-1 5-36 or Aib8 -Arg 34 -GLP-1 7-36 wait.

[0033] In some embodiments, the GLP-1 analogue includes one or more of liraglutide, semaglutide, albiglutide or dulaglutide.

[0034] In a specific embodiment of the present invention, the amino acid sequence of the target peptide comprises

[0035] TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 5), HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 9), HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 11), HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG (SEQ ID NO: 12),

[0036] EGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 3) or

[0037] One or more of HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 6).

[0038] In a specific embodiment of the present invention, the GLP-1 analog is liraglutide, and the amino acid sequence of the precursor polypeptide of the liraglutide includes positions 7-37 of the natural GLP-1 sequence in which Lys at position 34 is replaced by Arg, and preferably the amino acid sequence includes that shown in SEQ ID NO: 6. The liraglutide includes a structural modification, preferably adding a 16C fatty acid side chain connected by glutamic acid at the corresponding position of Lys at position 26 of the natural GLP-1 sequence.

[0039] In a specific embodiment of the present invention, the GLP-1 analog is semaglutide, and the amino acid sequence of the precursor polypeptide of semaglutide includes positions 7-37, 9-37 or 11-37 of the natural GLP-1 sequence in which Ala at position 8 is replaced by a non-natural amino acid (e.g., 2-aminoisobutyric acid, Aib), and Lys at position 34 is replaced by Arg, and preferably its amino acid sequence includes SEQ ID NO: 3 or 5. The semaglutide includes a structural modification, preferably a C18 fatty acid (e.g., a monosubstituted octadecane dioic acid) is connected to the corresponding position of Lys at position 26 of the natural GLP-1 sequence.

[0040] In a specific embodiment of the present invention, the GLP-1 analog is albiglutide, and the amino acid sequence of the precursor polypeptide of the albiglutide includes positions 7-36 of the natural GLP-1 sequence in which Ala at position 8 is replaced by Gly, and preferably its amino acid sequence includes SEQ ID NO: 11. The albiglutide comprises a structural modification, and preferably the structural modification comprises fusing the mutated peptide chain (SEQ ID NO: 11) with serum albumin (HSA).

[0041] In a specific embodiment of the present invention, the GLP-1 analog is dulaglutide, and the amino acid sequence of the precursor polypeptide of dulaglutide includes positions 7-36 of the natural GLP-1 sequence in which Ala at position 8 is replaced by Gly, Gly at position 22 is replaced by Glu, and Arg at position 36 is replaced by Gly, and preferably its amino acid sequence includes SEQ ID NO: 12. The dulaglutide comprises a structural modification, and preferably the structural modification comprises connecting to an IgG4-Fc fragment via a covalent bond.

[0042] Preferably, the target peptides are one or more (eg, 2-10, preferably 2-5 or 3-4) connected in series.

[0043] Preferably, the two or more target peptides have the same sequence or contain different sequences.

[0044] Preferably, the fusion protein comprises A-Bm, wherein A is a leader peptide, B is a target peptide, and m is selected from an integer of 1 to 10. The m is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably, m is selected from an integer of 1 to 5, and further preferably, m is selected from an integer of 1 to 3.

[0045] Preferably, the target peptides are connected to each other via a linker sequence.

[0046] Preferably, the linker sequence contains at least one restriction site.

[0047] Preferably, the cleavage site is a cleavage site of an amino acid endonuclease and / or an enzyme that recognizes basic amino acids, and more preferably a cleavage site of a dual-alkaline amino acid endonuclease and / or an enzyme that recognizes C-terminal basic amino acids.

[0048] In a specific embodiment of the present invention, the restriction enzyme cleavage site is the restriction enzyme cleavage site of Kex2 enzyme and / or CPB enzyme.

[0049] Preferably, the restriction site consists of basic amino acids.

[0050] Preferably, the basic amino acid is selected from arginine, lysine and histidine.

[0051] Preferably, the restriction site is 2-5 basic amino acids.

[0052] In a specific embodiment of the present invention, the amino acid sequence of the restriction site includes KR or RR or RK or KK.

[0053] Preferably, the N-terminus and the C-terminus of the linker sequence contain restriction sites, and the linker sequence further includes DGTDEAEKAG (SEQ ID NO: 7).

[0054] Further preferably, the amino acid sequence of the linker sequence includes KRDGTDEAEKAGKR (SEQ ID NO: 2).

[0055] Preferably, the fusion protein comprises AC n .

[0056] Among them, A is the leader peptide, C is the linker sequence-B m , B is the target peptide.

[0057] In some embodiments, the fusion protein comprises AB m -C n .

[0058] Among them, A is the leader peptide; B is the target peptide; C is the linker sequence-B m .

[0059] m or n is independently selected from an integer of 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0060] Preferably, m is selected from an integer of 1-5, and more preferably, m is selected from an integer of 1-3.

[0061] Preferably, n is an integer selected from 1-6.

[0062] In a specific embodiment of the present invention, m is 1, and n is 2-5.

[0063] In a specific embodiment of the present invention, the target peptide is Arg 34 -GLP-1 9-37 , m is 1, n is 3.

[0064] In a specific embodiment of the present invention, the amino acid sequence of the fusion protein includes SEQ ID NO:4.

[0065] SEQ ID NO: 4:

[0066] MADEAEKAGKREGTFTSDVSSYLEGQAAKEFIAWLVRGRGKRDGTDEAEKAGKREGTFTSDVSSYLEGQAAKEFIAWLVRGRGKRDGTDEAEKAGKREGTFTSDVSSYLEGQAAKEFIAWLVRGRGKRDGTDEAEKAGKREGTFTSDVSSYLEGQAAKEFIAWLVRGRG

[0067] The third aspect of the present invention provides a nucleic acid, which encodes the above-mentioned leader peptide or the above-mentioned fusion protein.

[0068] Preferably, the nucleotide sequence encoding the fusion protein comprises SEQ ID NO:13.

[0069] Preferably, the nucleotide sequence encoding the leader peptide comprises SEQ ID NO:14.

[0070] Further preferably, the nucleotide sequence encoding the leader peptide comprises SEQ ID NO:15.

[0071] SEQ ID NO: 14: ATGGCAGATGAGGCGGAAAAGGCTGGA

[0072] SEQ ID NO: 15: ATGGCAGATGAGGCGGAAAAGGCTGGAAAACGT

[0073] The fourth aspect of the present invention provides an expression vector, wherein the expression vector expresses the above-mentioned leader peptide or the above-mentioned fusion protein; or, the expression vector comprises the above-mentioned nucleic acid.

[0074] Preferably, the expression vector includes a eukaryotic expression vector or a prokaryotic expression vector.

[0075] Preferably, the vector backbone of the expression vector includes but is not limited to pET series, Duet series, pGEX series, pCold series, pBAD series or pTrc series vectors. The pET series vectors include but are not limited to pET-26b(+), pET-24a(+), pET-28a(+), pET-29a(+) or pET-30a(+).

[0076] The fifth aspect of the present invention provides a recombinant cell, wherein the recombinant cell expresses the above-mentioned leader peptide or the above-mentioned fusion protein; or, the recombinant cell contains the above-mentioned nucleic acid or the above-mentioned expression vector.

[0077] Preferably, the recombinant cell comprises a eukaryotic cell or a prokaryotic cell.

[0078] Preferably, the recombinant cell comprises Escherichia coli, Bacillus subtilis or yeast (eg, Pichia pastoris).

[0079] Preferably, the Escherichia coli includes Escherichia coli JM109 (DE3), Escherichia coli BL21 (DE3), Escherichia coli Rosetta (DE3), Escherichia coli Rosetta2 (DE3), Escherichia coli BL21 (DE3) pLysS, Escherichia coli Rosetta2 (DE3) pLysS, Escherichia coli Rosetta-gami (DE3), Escherichia coli HMS174 (DE3), Escherichia coli DH5α, Escherichia coli W3110 and / or Escherichia coli K12.

[0080] The sixth aspect of the present invention provides a method for preparing the above-mentioned leader peptide or the above-mentioned fusion protein, and the preparation method includes chemical synthesis or biological synthesis.

[0081] Preferably, the preparation method comprises:

[0082] 1) obtaining the above-mentioned nucleic acid;

[0083] 2) connecting the nucleic acid obtained in 1) to a vector backbone to obtain an expression vector;

[0084] 3) Transforming the expression vector obtained in 2) into host cells and culturing the host cells.

[0085] Preferably, the culturing of the host cell includes induced expression, such as IPTG induction.

[0086] Further preferably, the culturing of the host cell includes fermentation culture and induced expression.

[0087] Preferably, the culture temperature is 28-37°C, for example 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37°C.

[0088] Preferably, the culture time is 6-50h, for example 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50h.

[0089] Preferably, the pH of the culture system is 7.0-7.2, such as 7, 7.1 or 7.2.

[0090] Preferably, the host cells are fermented to an OD of 600When the OD is 100-130 (e.g., 100, 105, 110, 115, 120, 125, 130), the first induction stage is entered, and the induction time is 6-12h; the host cells are cultured until the OD 600 When the pH value is 150-170 (e.g., 150, 155, 160, 165, 170), the second induction stage is entered, and the expression is induced for 2-10 h (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 h) to end the fermentation.

[0091] Further preferably, the dissolved oxygen in the first induction stage is higher than the dissolved oxygen in the second induction stage.

[0092] In some embodiments, the dissolved oxygen control during the induction first phase is 25-35%, such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35%.

[0093] In some embodiments, the dissolved oxygen control to induce the second stage is 5-15%, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15%.

[0094] Preferably, the host cell comprises Escherichia coli, Bacillus subtilis or yeast (e.g., Pichia pastoris). The Escherichia coli comprises Escherichia coli JM109 (DE3), Escherichia coli BL21 (DE3), Escherichia coli Rosetta (DE3), Escherichia coli Rosetta 2 (DE3), Escherichia coli BL21 (DE3) pLysS, Escherichia coli Rosetta 2 (DE3) pLysS, Escherichia coli Rosetta-gami (DE3), Escherichia coli HMS174 (DE3), Escherichia coli DH5α, Escherichia coli W3110 and / or Escherichia coli K12.

[0095] In a specific embodiment of the present invention, the culturing host cells comprises culturing the host cells at 28-37° C. (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37° C.) for 10-15 h (e.g., 10, 11, 12, 13, 14 or 15 h) to obtain a seed solution; and then culturing the host cells at 0.1%-5% (e.g., 0.1%, 0.5%, 1%, 2%, 3%, 4% or 5%) (v / v) inoculation amount is inoculated into the fermentation medium, and when cultured for 6-8 hours, 0.05-1mM (for example, 0.05, 0.1, 0.5 or 1mM) IPTG is added for induction, and the fermentation is terminated after induction for 6-12h (for example, 6, 7, 8, 9, 10, 11 or 12h) at 28-37°C (for example, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37°C).

[0096] In a specific embodiment of the present invention, the host cell culture includes culturing in a seed culture medium at 28-37°C (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37°C) for 10-15h (e.g., 10, 11, 12, 13, 14 or 15h) to obtain a primary seed solution. The primary seed solution is inoculated into a seed culture medium and further cultured for 3-10h (e.g., 3, 4, 5, 6, 7, 8, 9 or 10h) to obtain a secondary seed solution. Then, the secondary seed solution is inoculated into a fermentation medium for culture, and feed is performed during the fermentation process (glucose 400g / L, yeast powder 150g / L, methionine 1g / L). When the fermentation liquid OD 600 When the value reaches 100-200, add IPTG with a final concentration of 0.05-1mM for induction.

[0097] In a specific embodiment of the present invention, the host cell culture comprises culturing in a seed culture medium at 28-37°C for 10-15 hours to obtain a primary seed solution, further inoculating the primary seed solution into a seed culture medium at 28-37°C for 3-10 hours to obtain a secondary seed solution, and then inoculating the secondary seed solution into a fermentation medium at 28-37°C and controlling the pH at 7.0-7.2 for culturing, and feeding during the fermentation process. When the fermentation liquid OD 600 When the OD value reaches 100-130, add IPTG with a final concentration of 0.05-1mM to start the first stage of induction (dissolved oxygen is controlled at 25-35%) for 6-12h. 600 When the PH value is 150-170, the second induction stage is entered (dissolved oxygen is controlled at 5-15%) and the fermentation is ended after 2-10 hours of induction.

[0098] The seventh aspect of the present invention provides a method for preparing a target peptide, the method comprising enzymatically cleaving the above-mentioned fusion protein or the fusion protein obtained by the above-mentioned preparation method.

[0099] Preferably, the preparation method comprises extracting inclusion bodies in recombinant cells expressing the fusion protein, dissolving the inclusion bodies and then adding enzymes.

[0100] Preferably, the enzyme may be an amino acid endonuclease and / or an enzyme that recognizes basic amino acids, and more preferably may be a dibasic amino acid endonuclease and / or an enzyme that recognizes C-terminal basic amino acids.

[0101] In a specific embodiment of the present invention, the enzyme is Kex2 enzyme and / or CPB enzyme.

[0102] Preferably, the method further comprises a step of washing the inclusion bodies before solubilizing the inclusion bodies.

[0103] Preferably, the extraction of inclusion bodies comprises centrifuging the recombinant cells expressing the fusion protein and resuspending them with a cell wall breaking buffer (preferably at a ratio of 1:5-1:30 (w / v, g / mL)), continuously homogenizing 2-3 times (preferably by a high-pressure homogenizer at a pressure of 800-1000 bar), and collecting by centrifugation.

[0104] Preferably, washing the inclusion bodies comprises washing with a washing buffer.

[0105] Preferably, the cell wall breaking buffer and washing buffer are independently selected from PB solution (containing Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl, KCl) with a pH of 5.5-8.0 (preferably pH 6.0-7.0, such as 5.5, 6, 6.5, 6.9, 7, 7.1, 7.5 or 8) and Tris solution.

[0106] Preferably, the enzymatic digestion comprises adjusting the pH of the dissolved inclusion bodies (preferably to a pH of 8.5-9.0), centrifuging to obtain the supernatant (preferably at a speed of 10000-15000 g for 20-50 min), adding Kex2 enzyme (the mass ratio of Kex2 enzyme to inclusion bodies is 1:(200-500), preferably 1:(250-300)) and CPB enzyme (the mass ratio of CPB enzyme to inclusion bodies is 1:(1000-2000), preferably 1:(1000-1500)), and performing enzymatic digestion with low temperature stirring (preferably stirring at 20-25°C for 8-15 h).

[0107] Preferably, the preparation method further comprises purification.

[0108] Preferably, the purification comprises filtration and / or high pressure reverse purification.

[0109] Preferably, the type of reverse phase polymer filler required in the high pressure reverse phase purification is series, among which UniPS10-300 is preferred. The first purification uses a buffer containing ammonium acetate and ethanol for linear elution gradient elution. The concentration of ammonium acetate in mobile phase A and B is 0.001-1M, and the concentration of ethanol is 0.1-100%, wherein the concentration of ammonium acetate in mobile phase A is 1-500mM, and the concentration of ethanol is 0-10% (v / v), wherein 50mM ammonium acetate-5% (v / v) ethanol solution, pH8.0±0.1 is preferred; the concentration of ammonium acetate in mobile phase B is 1-500mM, and the concentration of ethanol is 70-90% (v / v), wherein 50mM ammonium acetate-80% (v / v) ethanol solution, pH8.0±0.1 is preferred. The elution volume is 10-30CV, wherein 15CV is preferred.

[0110] The eighth aspect of the present invention provides a method for preparing a GLP-1 analog, which comprises modifying its precursor polypeptide.

[0111] Preferably, the GLP-1 analogue includes one or more of liraglutide, semaglutide, albiglutide or dulaglutide.

[0112] Preferably, the modification is a structural modification, including but not limited to a fatty acid modification with a chain length of C16-C20, such as a fatty acid modification with a chain length of C16, C17, C18, C19 or C20.

[0113] Preferably, the C18 fatty acid includes a monosubstituted octadecane dioic acid, and more preferably includes octadecane dioic acid mono-tert-butyl ester.

[0114] Preferably, the fatty acid modification comprises linkage via a covalent bond or an amino acid (eg, glutamic acid).

[0115] In a specific embodiment of the present invention, the GLP-1 analog is liraglutide, and the amino acid sequence of the precursor polypeptide of the liraglutide includes positions 7-37 of the natural GLP-1 sequence in which Lys at position 34 is replaced by Arg, and preferably the amino acid sequence includes that shown in SEQ ID NO: 6. The liraglutide includes a structural modification, preferably adding a 16C fatty acid side chain connected by glutamic acid at the corresponding position of Lys at position 26 of the natural GLP-1 sequence.

[0116] In a specific embodiment of the present invention, the GLP-1 analog is semaglutide, and the amino acid sequence of the precursor polypeptide of semaglutide includes positions 7-37, 9-37 or 11-37 of the natural GLP-1 sequence in which Ala at position 8 is replaced by a non-natural amino acid (e.g., 2-aminoisobutyric acid, Aib), and Lys at position 34 is replaced by Arg, and preferably its amino acid sequence includes SEQ ID NO: 3 or 5. The semaglutide includes a structural modification, preferably a C18 fatty acid (e.g., a monosubstituted octadecane dioic acid) is connected to the corresponding position of Lys at position 26 of the natural GLP-1 sequence.

[0117] In a specific embodiment of the present invention, the GLP-1 analog is albiglutide, and the amino acid sequence of the precursor polypeptide of the albiglutide includes positions 7-36 of the natural GLP-1 sequence in which Ala at position 8 is replaced by Gly, and preferably its amino acid sequence includes SEQ ID NO: 11. The albiglutide comprises a structural modification, and preferably the structural modification comprises fusing the mutated peptide chain (SEQ ID NO: 11) with serum albumin (HSA).

[0118] In a specific embodiment of the present invention, the GLP-1 analog is dulaglutide, and the amino acid sequence of the precursor polypeptide of dulaglutide includes positions 7-36 of the natural GLP-1 sequence in which Ala at position 8 is replaced by Gly, Gly at position 22 is replaced by Glu, and Arg at position 36 is replaced by Gly, and preferably its amino acid sequence includes SEQ ID NO: 12. The dulaglutide comprises a structural modification, and preferably the structural modification comprises connecting to an IgG4-Fc fragment via a covalent bond.

[0119] In a ninth aspect of the present invention, a method for preparing semaglutide is provided, wherein the method comprises modifying the side chains of the target peptide obtained by the above method and then connecting a dipeptide. Fig.11 .

[0120] Preferably, the preparation method comprises performing side chain modification on Lys of the target peptide to obtain semaglutide intermediate 1, and semaglutide intermediate 1 is shown in formula VII.

[0121] Preferably, the side chain modification comprises an acylation reaction.

[0122] Further preferably, the side chain modification comprises performing an acylation reaction, adjusting the pH of the reaction solution to the isoelectric point, and then precipitating and centrifuging to obtain the semaglutide intermediate 1.

[0123]

[0124] In a specific embodiment of the present invention, the side chain modification includes controlling the pH and temperature of the reaction to carry out an acylation reaction, adding a certain amount of pre-cooled water (pure water) to the reaction solution after the acylation reaction, adjusting the pH of the reaction solution to the isoelectric point, precipitating, and centrifuging to obtain semaglutide intermediate 1.

[0125] Preferably, the reaction pH is 10-12 (eg, 10, 11, 11.3 or 12).

[0126] Preferably, the reaction temperature is 0-10°C (eg, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0127] Preferably, the isoelectric point pH of the reaction solution is 4-5 (eg, 4, 4.4, 4.5 or 5).

[0128] Preferably, after the acylation reaction, pre-cooled water in an amount of 4-10 times (eg, 4, 5, 6, 7, 8, 9 or 10) the volume of the reaction solution is added to the reaction solution.

[0129] Preferably, adjusting the pH of the reaction solution to the isoelectric point comprises using one or more of an acidic solution such as hydrochloric acid, trifluoroacetic acid, phosphoric acid or sulfuric acid.

[0130] In a specific embodiment of the present invention, the preparation method comprises synthesizing a side chain, and then connecting it to the Lys of the target peptide through an acylation reaction.

[0131] Preferably, the synthesis method of the side chain comprises:

[0132] (1) Using a monosubstituted octadecane dioic acid as a raw material, an activation reaction is carried out with TSTU under alkaline conditions or in the presence of a condensing agent combination to obtain a side chain intermediate compound 1, and the side chain intermediate compound 1 is shown in Formula I.

[0133] Preferably, the condensing agent combination comprises HOSU and a carbodiimide condensing agent.

[0134] In a specific embodiment of the present invention, step (1) comprises using a monosubstituted octadecane dioic acid as a raw material, reacting it with TSTU and an organic solvent under alkaline conditions, or reacting it with HOSU, a carbodiimide condensation agent and an organic solvent to obtain a side chain intermediate compound 1.

[0135]

[0136] (2) The side chain intermediate compound 1 is used as a raw material and subjected to an acylation coupling reaction with HL-Glu-OR under alkaline conditions to obtain the side chain intermediate compound 2. The side chain intermediate compound 2 is shown in Formula II.

[0137] In a specific embodiment of the present invention, step (2) comprises using the side chain intermediate compound 1 as a raw material, carrying out an acylation coupling reaction with HL-Glu-OR under alkaline conditions, concentrating the reaction solution, adding water, heating and stirring, cooling and precipitating, then adding an organic solvent, heating and dissolving, and precipitating at low temperature.

[0138]

[0139] (3) The side chain intermediate compound 2 is used as a raw material, and an activation reaction is carried out with TSTU under alkaline conditions or in the presence of a condensing agent to obtain a side chain intermediate compound 3. The side chain intermediate compound 3 is shown in Formula III.

[0140] Preferably, the condensing agent combination comprises HOSU and a carbodiimide condensing agent.

[0141] In a specific embodiment of the present invention, step (3) comprises using the side chain intermediate compound 2 as a raw material, reacting with TSTU and an organic solvent under alkaline conditions, or reacting with HOSU, a carbodiimide condensation agent and an organic solvent to obtain a side chain intermediate compound 3.

[0142]

[0143] (4) The side chain intermediate compound 3 is used as a raw material and subjected to an acylation coupling reaction with AEEA-AEEA under alkaline conditions to obtain the side chain intermediate compound 4. The side chain intermediate compound 4 is shown in Formula IV.

[0144] In a specific embodiment of the present invention, step (4) comprises using the side chain intermediate compound 3 as a raw material, carrying out an acylation coupling reaction with AEEA-AEEA under alkaline conditions, concentrating the reaction solution, adding hydrochloric acid and ethyl acetate to extract 1-5 times (for example, 1, 2, 3, 4, 5 times), and drying the organic phase by suction filtration and concentrating.

[0145]

[0146] (5) The side chain intermediate compound 4 is used as a raw material, and an activation reaction is carried out with TSTU under alkaline conditions or in the presence of a condensing agent to obtain the side chain intermediate compound 5. The side chain intermediate compound 5 is shown in Formula V.

[0147] Preferably, the condensing agent combination comprises HOSU and a carbodiimide condensing agent.

[0148] In a specific embodiment of the present invention, step (5) comprises using the side chain intermediate compound 4 as a raw material, reacting with TSTU and an organic solvent under alkaline conditions, or reacting with HOSU, a carbodiimide condensation agent (such as DCC) and an organic solvent to obtain the side chain intermediate compound 5.

[0149]

[0150] (6) Using the side chain intermediate compound 5 as a raw material, deprotection is carried out in an acidic solution to obtain a side chain compound, and the side chain compound is shown in Formula VI.

[0151]

[0152] The substituent R is an alkane, alkene or aromatic hydrocarbon group; preferably a methyl group, an ethyl group or a group that can be hydrolyzed under acidic conditions (such as tert-butyl (tBu) or benzyl).

[0153] The schematic diagram of the synthesis process of side chain compounds is as follows Fig.10 shown.

[0154] Preferably, in steps (1), (2), (3) and (5), the organic solvent comprises one or more of DMF, THF, DMSO, DCM, DMA, acetonitrile or water.

[0155] Preferably, the acidic solution in step (6) is selected from one or a combination of two or more of TFA, thioanisole, phenol, triisopropylsilane (TIPS), and water, preferably a mixed solution of TFA and TIPS.

[0156] Preferably, the dipeptide comprises His-Aib.

[0157] Preferably, the connecting dipeptide comprises coupling Fmoc-His-Aib-OH with the N-terminal amino group of semaglutide intermediate 1 to obtain semaglutide intermediate 2, and semaglutide intermediate 2 is shown in formula VIII.

[0158]

[0159] In a specific embodiment of the present invention, the connecting dipeptide comprises activating Fmoc-His-Aib-OH and coupling it with the N-terminal amino group of semaglutide intermediate 1 to obtain semaglutide intermediate 2.

[0160] Preferably, the coupling reaction in the connecting dipeptide is carried out under alkaline conditions.

[0161] Preferably, the coupling reaction of the connecting dipeptide uses a condensing agent.

[0162] Further preferably, the preparation method further comprises deprotecting the semaglutide intermediate 2 to obtain semaglutide.

[0163] Preferably, the deprotection comprises using a lysis solution. Preferably, the lysis solution is one or more of phenol, piperidine, bicyclic amidine or DCM.

[0164] In a specific embodiment of the present invention, a cleavage solution is added to the reaction solution for connecting the dipeptide, so that the semaglutide intermediate 2 is deprotected under the action of the cleavage solution.

[0165] Preferably, after deprotection, a certain amount of pre-cooled water is added to the reaction solution, and semaglutide is obtained by adjusting the pH of the reaction solution to the isoelectric point and then precipitating and centrifuging.

[0166] Preferably, after deprotection, pre-cooled water in an amount of 5 to 12 times the volume of the reaction solution is added to the reaction solution.

[0167] Preferably, the isoelectric point of the reaction solution is pH 4-5 (eg, 4, 4.4, 4.5 or 5).

[0168] Preferably, adjusting the pH of the reaction solution to the isoelectric point comprises using one or more of an acidic solution such as hydrochloric acid, trifluoroacetic acid, phosphoric acid or sulfuric acid.

[0169] Preferably, the preparation method further comprises purification.

[0170] Preferably, the purification comprises first using ethanol as the mobile phase (crude purity) and then using acetonitrile as the mobile phase (fine purity).

[0171] The condensing agent includes one or a combination of two or more of DCC (dicyclohexylcarbodiimide), DIC (N,N'-diisopropylcarbodiimide), EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride), HOSU (N-hydroxysuccinimide), HOBT (1-hydroxybenzotriazole), HOAT (1-hydroxy-7-azabenzotriazole), and PYBOP (benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate).

[0172] The condensing agent used for side chain synthesis is a condensing agent combination, and the condensing agent combination includes HOSU and a carbodiimide condensing agent. Preferably, the carbodiimide condensing agent includes one or more of DIC, DCC or EDCI. In a specific embodiment of the present invention, the condensing agent combination is HOSU and DCC.

[0173] Preferably, the alkaline condition or the base for adjusting pH comprises an inorganic base or an organic base.

[0174] The inorganic base preferably includes one or more of sodium carbonate, potassium carbonate, sodium bicarbonate, sodium hydrogen phosphate, sodium hydroxide or potassium hydroxide.

[0175] The organic base preferably includes one or more of N,N-diisopropylethylamine (DIEA), triethylamine (TEA), DBU, DMF, aniline or pyridine.

[0176] Preferably, the organic base used in the alkaline conditions for connecting the dipeptide includes one or more of N,N-diisopropylethylamine, triethylamine, pyridine, DMF, and aniline, preferably DMF or DIEA.

[0177] Preferably, the inorganic base used in the alkaline conditions for connecting the dipeptide includes one or more of sodium bicarbonate, sodium hydrogen phosphate, sodium hydroxide or potassium hydroxide.

[0178] Preferably, the inorganic base used in the alkaline conditions for synthesizing the side chain includes one or more of sodium carbonate, potassium carbonate, sodium hydroxide or potassium hydroxide.

[0179] Preferably, the organic base used in the alkaline conditions for synthesizing the side chain includes one or more of TEA, DIEA, DBU, DMF, and pyridine, preferably DIEA or DMF.

[0180] Preferably, the inorganic base used to adjust the pH includes one or more of sodium bicarbonate, sodium hydrogen phosphate, sodium hydroxide or potassium hydroxide, preferably sodium hydroxide.

[0181] Preferably, the organic base used to adjust the pH includes one or more of N,N-diisopropylethylamine, triethylamine or pyridine, preferably triethylamine or DIEA.

[0182] In a specific embodiment of the present invention, the preparation method comprises:

[0183] (1) constructing an expression vector comprising a nucleotide sequence encoding the fusion protein, wherein the sequence of the fusion protein comprises: AB m -C n , where A is the leader peptide; B is the target peptide; C is the linker sequence-B m ; m or n is independently selected from an integer of 0-10;

[0184] (2) transforming into a host cell, culturing the host cell (preferably including fermentation culture and induced expression), culturing the host cell including using staged induced fermentation, including a first induction stage and a second induction stage, and obtaining a host cell expressing the fusion protein after induced fermentation;

[0185] (3) extracting inclusion bodies from host cells expressing the fusion protein, dissolving them, and adding enzymes to obtain the target peptide;

[0186] (4) Chemical synthesis of side chain compounds;

[0187] (5) The target peptide is chemically modified (including side chain modification and dipeptide attachment) to prepare semaglutide.

[0188] Preferably, the preparation method further comprises:

[0189] (6) Purifying to obtain high-purity semaglutide, preferably the purification includes crude purification and / or fine purification.

[0190] The tenth aspect of the present invention provides an application of the method for preparing the above-mentioned GLP-1 analog or the method for preparing the above-mentioned semaglutide.

[0191] Preferably, the application includes application in preparing medicines for treating diseases or for weight loss.

[0192] Preferably, the disease comprises diabetes (eg type 2 diabetes) or obesity.

[0193] In the eleventh aspect of the present invention, there is provided a use of the above-mentioned leading peptide, the above-mentioned fusion protein, the above-mentioned nucleic acid, the above-mentioned expression vector, the above-mentioned host cell, the leading peptide, fusion protein or target peptide obtained by the above-mentioned preparation method in the preparation of GLP-1 analogs.

[0194] Preferably, the GLP-1 analogue includes one or more of liraglutide, semaglutide, albiglutide or dulaglutide.

[0195] The twelfth aspect of the present invention provides a method for treating a disease, which comprises using the above-mentioned fusion protein, the fusion protein obtained by the above-mentioned preparation method, the target peptide obtained by the above-mentioned preparation method, the GLP-1 analogue obtained by the above-mentioned preparation method, or semaglutide obtained by the above-mentioned preparation method.

[0196] Preferably, the method comprises administering to the subject an effective amount of the above-mentioned fusion protein, the fusion protein obtained by the above-mentioned preparation method, the target peptide obtained by the above-mentioned preparation method, the GLP-1 analogue obtained by the above-mentioned preparation method, or the semaglutide obtained by the above-mentioned preparation method.

[0197] Preferably, the disease comprises diabetes (eg type 2 diabetes) or obesity.

[0198] The thirteenth aspect of the present invention provides a method for weight loss, which comprises using the above-mentioned fusion protein, the fusion protein obtained by the above-mentioned preparation method, the target peptide obtained by the above-mentioned preparation method, the GLP-1 analogue obtained by the above-mentioned preparation method, or the semaglutide obtained by the above-mentioned preparation method.

[0199] Preferably, the weight loss method comprises administering to the subject an effective amount of the above-mentioned fusion protein, the fusion protein obtained by the above-mentioned preparation method, the target peptide obtained by the above-mentioned preparation method, the GLP-1 analogue obtained by the above-mentioned preparation method, or the semaglutide obtained by the above-mentioned preparation method.

[0200] Compared with the prior art, the present invention has the following advantages:

[0201] 1) The fusion protein described in the present application contains a leader peptide (SEQ ID NO: 8) and a linker sequence (SEQ ID NO: 2) of a specific sequence, which changes the isoelectric point and hydrophilicity of the fusion protein, facilitates the extraction, enzyme digestion and purification of inclusion bodies, and the purity of the obtained precursor polypeptide can reach more than 95%.

[0202] 2) The present application uses staged induction fermentation to cultivate host cells expressing fusion proteins, including a first stage of induction for rapid bacterial growth and rapid protein expression under high dissolved oxygen conditions, and a second stage of induction for reducing bacterial aging and maintaining bacterial viability under low dissolved oxygen conditions, which is beneficial to the accumulation of products and greatly increases the yield of fusion protein, reaching 26 g / L.

[0203] 3) In the process of enzymatic digestion of inclusion bodies, the present application found through comparative experiments of adding Kex2 enzyme and CPB enzyme in different proportions that the effects of adding enzymes in different proportions were different. When Kex2 enzyme and CPB enzyme were added in ratios of 1:250 and 1:1000 respectively, rapid and accurate enzymatic digestion of inclusion bodies could be achieved.

[0204] 4) The present application controls the order of coupling, that is, the main chain (precursor polypeptide) is first connected to the side chain and then to the dipeptide, and only two steps are required to obtain a crude semaglutide with a purity of more than 85%, which greatly reduces the difficulty of subsequent purification; the coupling reaction of the dipeptide with Fmoc-His-Aib-OH is simple to operate, the reaction is thorough, and the racemization of the amino acid can be avoided; the post-treatment process of the main chain side chain and the dipeptide deprotection is mainly carried out by adding water and adjusting the pH, which saves costs to a great extent; the deprotection is directly carried out after the dipeptide is connected, which is simple to operate, will not introduce new impurities and can avoid the racemization of the amino acid.

[0205] 5) The purification of crude semaglutide in this application includes "rough purification" and "fine purification". First, ethanol is used as the mobile phase for "rough purification", which can save reagent costs. Further, acetonitrile is used as the mobile phase for "fine purification", which can collect the target product (such as semaglutide) with high purity, and the purity can reach more than 99%. This combination of "rough purification" and "fine purification" ensures the yield while reducing the purification cost.

[0206] 6) The present application provides a sustainable production process for producing semaglutide with higher efficiency, lower cost, simple purification and higher purity, and easy production and scale-up.

[0207] The "GLP-1 analogue" described in the present invention refers to a mutation or structural modification of natural GLP-1 or its truncated sequence, and has the function of simulating the physiological effects of natural GLP-1, promoting insulin secretion and inhibiting glucagon release, thereby regulating blood sugar levels. In some embodiments, the mutation includes but is not limited to substitution, deletion and / or insertion of one or more natural or non-natural amino acids. In some embodiments, the structural modification is, for example, fatty acid chain modification. In some embodiments, the mutation or structural modification can extend the half-life.

[0208] The “non-natural amino acids” described in the present invention include 2-aminoisobutyric acid (Aib), imidazole-4-acetate (IA), imidazole propionic acid (IPA), α-aminobutyric acid (Abu), tert-butylglycine (Tle), 3-aminomethylbenzoic acid, anthranilic acid, deaminohistidine, β-alanine, 2-aminohistidine, β-hydroxy-histidine, homohistidine, Nα-acetyl-histidine, α-fluoro-methyl-histidine, α-methyl-histidine, α,α-dimethyl-glutamic acid, m-CF3-phenylalanine, α,β-diaminopropionic acid, 3-pyridylalanine, 2-pyridylalanine, 4-pyridylalanine, (1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)-carboxylic acid, (1-aminocyclopentyl)carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic acid or (1-aminocyclooctyl)carboxylic acid. One or more of the group.

[0209] The "sites" described in the present invention, such as mutation sites or modification sites of GLP-1, are all described based on the amino acid sequence of natural GLP-1. It can be understood by those skilled in the art that the sites in the truncated sequence are also the sites corresponding to the amino acid sequence of natural GLP-1 after alignment. Among them, the amino acid sequence of natural GLP-1 is HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 10). For example, semaglutide or liraglutide are both Lys 26 The fatty acid modification is linked to the above, wherein 26 is the 26th position of SEQ ID NO:10.

[0210] The "GLP-1 x-xx " represents the truncated sequence from position x to position xx of the amino acid sequence of natural GLP-1, for example, GLP-1 5-37 The sequence is from position 5 to position 37 of the amino acid sequence of natural GLP-1.

[0211] The "X" of the present invention n -GLP-1 x-xx " represents that the sequence of the analog is from position x to position xx of the amino acid sequence of natural GLP-1, and position n is mutated to X, for example, "Arg 34 -GLP-1 9-37 The sequence of " is SEQ ID NO: 3.

[0212] The "precursor polypeptide" of the present invention refers to the amino acid sequence of a GLP-1 drug before structural modification and which can be encoded by a nucleic acid. The GLP-1 drug includes liraglutide, semaglutide, albiglutide or dulaglutide. For example, the precursor polypeptide of semaglutide contains Arg 34 -GLP-15-37 , Arg 34 -GLP-1 7-37 , Arg 34 -GLP-1 9-37 , Arg 34 -GLP-1 11-37 , Arg 34 -GLP-1 5-36 , Arg 34 -GLP-1 7-36 , Arg 34 -GLP-1 9-36 , Arg 34 -GLP-1 11-36 , Gly 8 -GLP-1 7 -36 , Gly 8 -Glu 22 -Gly 36 -GLP-1 7-36 .

[0213] The term “include” or “comprising” described in the present invention is an open description, which includes the specified components or steps described and other specified components or steps that will not be substantially affected.

[0214] The term "and / or" used in the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately in the present application. For example, "A and / or B" includes "A", "A and B" and "B"; another example is "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0215] "Treatment" as used herein means to slow, interrupt, prevent, control, stop, alleviate or reverse the progression or severity of a sign, symptom, disorder, condition or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions or disorders.

[0216] The "homology" mentioned in the present invention refers to the description of protein sequence or nucleotide sequence. Those skilled in the art can adjust the sequence according to actual work needs so that the sequence has (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41% compared with the sequence obtained in the prior art. , 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity, but still have the same or similar activity as the original sequence.

[0217] An "effective amount" as used herein refers to an amount or dosage of the product of the present invention that provides the desired treatment after administration to a patient in single or multiple doses.

[0218] The "subject" described in the present invention can be a human or a non-human animal (e.g., a non-human mammal), and the non-human mammal can be a wild animal, a zoo animal, an economic animal, a pet, an experimental animal, etc. Preferably, the non-human mammal includes, but is not limited to, pigs, cattle, sheep, horses, donkeys, foxes, minks, jackals, camels, dogs, cats, rabbits, mice (e.g., rats, mice, hamsters, gerbils, chinchillas, squirrels), monkeys, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0219] Figure 1 .Example 1 Fusion protein expression recombinant plasmid map.

[0220] Figure 2 .Example 2 SDS-Page electrophoresis detection of fermentation expression products.

[0221] Figure 3 Example 3 SDS-Page electrophoresis detection of samples after bacterial cell disruption and inclusion body dissolution.

[0222] Figure 4 .Liquid chromatogram of the side chain intermediate compound 1 obtained in Example 5.

[0223] Figure 5 .Liquid chromatogram of the side chain intermediate compound 2 obtained in Example 5.

[0224] Figure 6 .Liquid chromatogram of the side chain intermediate compound 3 obtained in Example 5.

[0225] Figure 7 .Mass spectrum of the side chain compound obtained in Example 5.

[0226] Figure 8 .Chromatographic spectrum of crude purified semaglutide obtained in Example 11.

[0227] Fig. 9 .Chromatographic spectrum of purified semaglutide obtained in Example 11.

[0228] Fig.10 .Schematic diagram of the synthesis process of the side chain compounds of semaglutide.

[0229] Fig.11 .Schematic diagram of the preparation process of semaglutide. DETAILED DESCRIPTION

[0230] The following specific examples are convenient for those skilled in the art to understand the present invention, but should not limit the scope of protection of the present invention in any way. According to the present invention, changing the feed ratio of raw materials, or the reaction solvent or condensing agent, etc., are all within the scope of protection of the present invention. In the following examples, unless otherwise specified, the methods used are conventional methods, and the materials and reagents can be purchased from conventional biochemical reagent suppliers.

[0231] Some of the culture media, feed ingredients, buffer formulations and parameters used in the examples are as follows:

[0232] Seed culture medium: yeast powder 15g / L, sodium chloride 10g / L, glucose 1g / L.

[0233] Basic fermentation medium: glucose 10g / L, yeast powder 35g / L, potassium dihydrogen phosphate 5g / L, disodium hydrogen phosphate dodecahydrate 10g / L, calcium chloride dihydrate 0.15g / L, ammonium sulfate 4g / L, magnesium sulfate 1.5g / L.

[0234] The feed ingredients are: glucose 400g / L, yeast powder 150g / L, methionine 1g / L.

[0235] PB solution: Na2HPO4·12H2O: 1.517 g / L, NaH2PO4·2H2O: 0.276 g / L, NaCl: 4.385 g / L, KCl: 0.2 g / L;

[0236] Inclusion body solubilization buffer: 50 mM Tris solution.

[0237] Example 1: Construction of a recombinant engineered bacterium expressing semaglutide precursor polypeptide fusion protein

[0238] Design a fusion protein sequence suitable for E. coli expression: leader peptide-Arg 34 -GLP-1 9-37 -(Linking sequence-Arg 34 -GLP-1 9-37 ) 3 .

[0239] The amino acid sequences of the appropriate leader peptide and linker sequence can not only enhance the expression of the fusion protein, but also prevent degradation by host proteases, balance the isoelectric point of the fusion protein, and improve the solubility of the fusion protein.

[0240] The sequence of the leader peptide is MADEAEKAGKR (SEQ ID NO: 1), and the linker sequence is KRDGTDEAEKAGKR (SEQ ID NO: 2). The C-terminus of the leader peptide is a KR residue, which is linked to the target peptide (including Arg 34 -GLP-1 5-37 , Arg 34 -GLP-1 7 -37 , Arg 34 -GLP-1 9-37 , Arg 34 -GLP-1 11-37 , Arg 34 -GLP-1 5-36 , Arg 34 -GLP-1 7-36 , Arg 34 -GLP-1 9-36 , Arg 34 -GLP-1 11-36 , Gly 8 -GLP-1 7-36 , Gly 8 -Glu 22 -Gly 36 -GLP-1 7-36 ), the N-terminus and C-terminus of the connecting sequence both contain KR sites, and the connecting sequence and the target peptide are arranged at intervals.

[0241] Semaglutide precursor peptide Arg 34 -GLP-1 9-37 As an example of the target peptide, the fusion protein sequence is

[0242] MADEAEKAG KREGTFTSDVSSYLEGQAAKEFIAWLVRGRG KR DGTDEAEKAG KR EGTFTSDVSSYLEGQAAKEFIAWLVRGRG KR DGTDEAEKAG KR EGTFTSDVSSYLEGQAAKEFIAWLVRGRG KR DGTDEAEKAG KR EGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 4), the isoelectric point of this sequence is 5.80, and the average hydrophilicity is -0.827.

[0243] The DNA sequence encoding the above fusion protein was obtained by total gene synthesis and cloned into the Nde I / Xho I restriction site of the pET-26b(+) vector. The recombinant plasmid map is shown in Figure 1 The recombinant plasmid was transferred into the E. coli host cell BL21 (DE3) by heat shock method to obtain the recombinant engineered bacteria expressing the fusion protein. The gene sequence is shown in SEQ ID NO: 13:

[0244] ATGGCAGATGAGGCGGAAAAGGCTGGAAAACGTGAGGGCACCTTCACCTCCGACGTGAGCAGCTATCTGGAAGGTCAGGCTGCGAAAGAATTCATTGCCTGGCTGGTTCGTGGCCGTGGTAAAAGAGA TGGTACTGACGAGGCAGAAAAGGCGGGTAAGCGCGAGGGCACCTTTACCAGCGACGTTAGCTCCTATTTGGAAGGCCAGGCGGCGAAAGAGTTCATCGCCTGGCTGGTTCGTGGACGTGGCAAGCGCGA TGGTACGGATGAAGCGGAGAAGGCGGGTAAGCGTGAGGGCACCTTTACCTCCGATGTGTCGAGCTACTTGGAGGGTCAAGCGGCTAAAGAATTTATCGCATGGCTTTGTGCGTGGTCGTGGTAAGCGCG ACGGCACGGACGAAGCAGAAAAAGCAGGCAAACGCGAAGGTACGTTTACCTCTGATGTCAGCTCTTACCTGGAGGGCCAAGCCGCTAAAGAGTTCATTGCGTGGCTGGTGCGCGGTCGTGGTTAA(SEQ ID NO: 13).

[0245] Example 2: High-density fermentation culture and induced expression of recombinant engineered bacteria

[0246] The recombinant engineered bacteria obtained in Example 1 were cultured at 30°C for 15 hours to obtain a first-level seed solution; the first-level seed solution was inoculated into 100 mL of seed culture medium at a 2% (v / v) inoculation amount, and cultured for 5 hours to obtain a second-level seed solution; then the second-level seed solution was inoculated into 5 L of basic fermentation medium and cultured at 34°C and pH 7.0-7.2. At the same time, feed was added during the fermentation process. When the fermentation liquid OD 600 When the OD value reached 120, IPTG was added at a final concentration of 1 mM for the first stage of induction. At 34°C, the dissolved oxygen parameter was controlled at 33±2% and the induction culture was carried out for 7 h. At this stage, E. coli was in a high metabolic state to achieve rapid expression of the product. 600 When the concentration reaches 165, the second stage of induction begins. In the second stage of induction, the dissolved oxygen is controlled at about 8±2% at 30°C and culture is continued for 8 hours. At this stage, the oxygen supply state of the fermenter can be reduced, and the amount of carbon and nitrogen source feed can be reduced, so that the bacteria are in a lower metabolic state, the accumulation of acetic acid is reduced, the vitality of the bacteria is maintained, and the further expression of the product is achieved. Stop the fermentation, collect the fermentation broth, centrifuge at 4°C and 6500rpm for 20 minutes, discard the supernatant, and collect the bacteria.

[0247] Semaglutide precursor peptide Arg 34 -GLP-1 9-37 Taking the target peptide as an example, the SDS-Page detection of the expression product induced by the recombinant engineering bacteria is as follows Figure 2 As shown in Figure 2, the yield of the fusion protein can reach 26 g / L.

[0248] Example 3: Precursor polypeptide Arg containing semaglutide 34 -GLP-1 9-37 Extraction, washing and enzyme digestion of fusion protein inclusion bodies

[0249] Step 1: Bacteria disruption and inclusion body extraction

[0250] The bacteria collected in Example 2 were poured into the prepared cell-breaking buffer (50 mM PB solution, pH 7.0 ± 0.1) at a ratio of 1:10 (bacteria: cell-breaking buffer, w / v, g / mL) and resuspended, and stirred with a stirrer until there were no lumps in the stirred liquid observed with the naked eye; the resuspended bacterial liquid was homogenized 3 times in a high-pressure homogenizer at a pressure of 950 bar (suspension after cell-breaking); the broken bacterial liquid was centrifuged at 13000g for 30 min for solid-liquid separation, the supernatant (centrifugal supernatant after cell-breaking) was discarded, and the crude inclusion body precipitate was collected.

[0251] Step 2: Inclusion body washing

[0252] The collected crude inclusion body precipitate was added to the washing buffer (50mM PB solution) at a ratio of 1:10 (inclusion body: washing buffer, w / v, g / mL) to the wet weight of the inclusion body, and resuspended until there were no obvious lumps. Centrifuge at 13000g for 30 minutes, discard the supernatant and collect the precipitate. Repeat the washing, centrifugation and discard the supernatant (the centrifugation supernatant after washing) to collect the precipitate (the inclusion body precipitate after washing). The results of the electrophoresis test of the inclusion body sample after washing are shown in Figure 3 .

[0253] Step 3: Solubilization and digestion of inclusion bodies

[0254] After washing, the inclusion bodies were added with pure water at a ratio of 1:8 (w / v, g / mL) by wet weight, and mechanically stirred until the sample was stirred into tiny particles. Inclusion body dissolution buffer was added to the total volume of pure water to a concentration of 50 mM, the pH was adjusted to 10-11, and stirred at 10°C overnight until the sample was completely dissolved (see the results of electrophoresis detection of the inclusion body sample after dissolution). Figure 3 ). Adjust the pH to between 8.5-9.0, centrifuge at 13000g for 30 minutes, and discard the precipitate (centrifuge the precipitate after re-dissolution). Take the supernatant (centrifuge the supernatant after re-dissolution), add Kex2 enzyme and CPB enzyme in a ratio of 1: (200-500) and 1: (1000-2000), respectively, and stir at low speed for 9 hours at 25℃. Take samples for RP-HPLC detection.

[0255] In addition, through the comparative experiment of adding different ratios of Kex2 enzyme and CPB enzyme, it was found that when Kex2 enzyme and CPB enzyme were added at a ratio of 1:250 and 1:1000 respectively, rapid and accurate enzymatic digestion of inclusion bodies could be achieved. 34 -GLP-1 9-37 The purity is 95.51%.

[0256] Example 4: Semaglutide precursor polypeptide Arg 34 -GLP-1 9-37 Purification

[0257] The digested sample obtained in Example 3 was centrifuged at 13000g for 20 minutes and the supernatant was taken. The centrifuged supernatant was clarified and filtered using a deep filter. The clarified semaglutide precursor polypeptide was purified using UniPS10-300 filler. Mobile phase A: 50mM ammonium acetate-5% (v / v) ethanol pH8.0±0.1, mobile phase B: 50mM ammonium acetate-80% (v / v) ethanol pH8.0±0.1. Elution volume 15CV. Linear flow rate: 150cm / h, loading capacity: 8g / L filler.

[0258] The purification procedure is shown in Table 1 below:

[0259] Table 1

[0260] T(min) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 100 0 20 55 45 160 35 65

[0261] The column was equilibrated with the initial gradient of the purification method for 30 minutes. The sample to be purified was measured according to the loading amount, and 50% ethanol was added to the sample solution until the ethanol content in the sample solution was about 5%. The sample was injected with a loading pump at a linear flow rate of 150 cm / h. The purification method program was run for elution. The collected eluate was tested by RP-HPLC. The results showed that the purified precursor polypeptide Arg 34 -GLP-1 9 -37 The purity is 96.49%.

[0262] The collected eluate was added to 2-10°C pre-cooled water at a ratio of 1:2 (v / v) and stirred to mix. The pH was adjusted to 4.8±0.2 with 2M HCl solution. The mixture was refrigerated at 2-10°C and allowed to stand for 12-14 hours. The clear stratification of the supernatant and the precipitate of the semaglutide precursor polypeptide was observed. The siphon method was used to remove the sedimentation supernatant, and the remaining sedimentation sample was centrifuged at 13000g and 4°C for 10-30 minutes. The precipitated sample was collected, which was the semaglutide precursor polypeptide.

[0263] Example 5: Synthesis of semaglutide side chain

[0264] Using octadecane dioic acid mono-tert-butyl ester as the starting material, semaglutide side chain compounds were synthesized through 6 steps of reaction. The schematic diagram of the synthesis process of the side chain compound is shown in Fig.10 shown.

[0265] Among them, octadecane dioic acid mono-tert-butyl ester, HL-Glu-OtBu, AEEA-AEEA and other conventional reagents can be obtained through commercial channels. The specific steps are as follows:

[0266] Step 1: Preparation of side chain intermediate compound 1

[0267] Weigh 30.00 g of 18-alkyl diacid mono-tert-butyl ester into a three-necked reaction bottle, add 150 mL of THF to dissolve it completely, then add 29.25 g of TSTU, 17 mL of DIEA and 30 mL of DMF in sequence and react overnight at room temperature. Concentrate the reaction solution, add the remaining concentrated solution into 300 mL of cold acetonitrile to precipitate solids, filter by suction, scrape off the filter cake and dry to obtain a white solid, i.e., the side chain intermediate compound 1. Figure 4 The liquid phase spectrum showed that the purity of compound 1 was 99.29%.

[0268] Step 2: Preparation of side chain intermediate compound 2

[0269] Weigh 35.00g of the side chain intermediate compound 1 into a three-necked reaction bottle, and add 175mL of THF to dissolve. Weigh 16.73g of HL-Glu-OtBu, add 175mL (DMF / H2O=1 / 1) solvent to mix, and add to the above reaction and stir evenly. After heating, it becomes a clear solution, and it is cooled to room temperature to react for 3h. Concentrate the reaction solution, add 350mL of water, stir, and heat to 60℃ and stir for 10min, and precipitate after cooling. Heat the precipitate with 350mL of acetonitrile to dissolve it, precipitate it at low temperature overnight, filter it with suction, scrape out the filter cake and dry it to obtain a white solid, i.e., the side chain intermediate compound 2. Figure 5 The liquid phase spectrum showed that the purity of the side chain intermediate compound 2 was 97.53%.

[0270] Step 3: Preparation of side chain intermediate compound 3

[0271] Weigh 38.00g of the side chain intermediate compound 2 and place it in a three-necked reaction bottle, add 380mL of THF to dissolve it completely, and add 13mL of DIEA. Weigh 22.64g of TSTU and dissolve it in 150mL of acetonitrile, add it to the above reaction solution, and react at room temperature overnight. The reaction solution is directly concentrated, dissolved in 380mL of acetonitrile, and precipitated at low temperature overnight. Filter by suction, scrape out the filter cake and dry it to obtain a white solid, i.e., the side chain intermediate compound 3. Figure 6 The liquid phase spectrum showed that the purity of the side chain intermediate compound 3 was 97.54%.

[0272] Step 4: Preparation of side chain intermediate compound 4

[0273] Weigh 40.00g of side chain intermediate compound 3 and 24.56g of AEEA-AEEA into a three-necked reaction bottle, add 200mL of ethanol to dissolve, and stir. Add 21mL of DIEA to the above reaction and react at room temperature overnight. The reaction solution is concentrated, 400mL of 0.1M HCl and 200mL of ethyl acetate are added to extract 3 times, the organic phases are combined and washed once with saturated brine, and the organic phase is dried. Filter by suction, and concentrate the filtrate to obtain side chain intermediate compound 4. The purity of side chain intermediate compound 4 is 94.52%.

[0274] Step 5: Preparation of side chain intermediate compound 5

[0275] Weigh 50.00g of side chain intermediate compound 4 and place it in a three-necked reaction bottle. Add 300mL of THF to dissolve it completely, add 7.14g of HOSU, and control the temperature at 0-5℃ in an ice-water bath. Weigh 12.80g of DCC and dissolve it in 100mL of THF. Add it dropwise into the above reaction solution at 0-5℃, and stir at 0-5℃ for 15min. Remove the ice-water bath and react at room temperature for 3h. Filter and concentrate the filtrate to obtain side chain intermediate compound 5. The purity of side chain intermediate compound 5 is 83.27%.

[0276] Step 6: Preparation of semaglutide side chain compounds

[0277] Weigh 55.00g of the side chain intermediate compound 5 into a reaction bottle, add 110mL of TFA / TIPS = 97 / 3, and react at room temperature for 1.5h. Add the above reaction solution dropwise into 2.2L of ice isopropyl ether, stir and crystallize for 1h. Filter with suction, add 2.2L of ethyl acetate to the filter cake and beat for 1h. Filter with suction, scrape out the filter cake and vacuum dry to obtain the side chain compound. The purity of the side chain compound is 91.14%. Figure 7 The mass spectrum (MS) showed that the molecular weight of the side chain compound was: MS: m / z = 831.5 (M+H + ).

[0278] Example 6: Semaglutide precursor polypeptide Arg 34 -GLP-1 9-37 Lys side chain

[0279] 1.5 g of semaglutide precursor polypeptide Arg 34 -GLP-1 9-37 Place in a reaction bottle, add 100 mL of pure water, stir and dissolve, adjust the pH to 11.3 with 2%-5% NaOH solution, and cool the reaction solution to 0-10°C in a water bath; dissolve 0.59 g of the side chain compound with 11.8 mL of DMF, and slowly drop it into the above reaction solution, the dropwise addition time is 1.5 hours, react at 0-10°C for 20 minutes, add 140 mL of pure water to the reaction solution after the reaction is completed, adjust the pH of the reaction solution to 4.4 with hydrochloric acid, precipitate, and centrifuge to obtain semaglutide intermediate 1. The purity after detection is 89.11%.

[0280] Example 7: Semaglutide precursor polypeptide Arg 34 -GLP-1 9-37 Lys side chain

[0281] 1.5 g of semaglutide precursor polypeptide Arg 34 -GLP-1 9-37 Place in a reaction bottle, add 100 mL of pure water, stir and dissolve, adjust the pH to 11.3 with 2%-5% NaOH solution, and cool the reaction solution to 0-10°C in a water bath; dissolve 0.59 g of the side chain with 11.8 mL of DMF, and slowly drop it into the above reaction solution, the dropwise addition time is 5 hours, and react at 0-10°C for 20 minutes. After the reaction is completed, add 140 mL of pure water to the reaction solution, adjust the pH of the reaction solution to 4.4 with hydrochloric acid, precipitate, and centrifuge to obtain semaglutide intermediate 1. The purity after detection is 88.97%.

[0282] Example 8: Semaglutide precursor polypeptide Arg 34 -GLP-19-37 Lys side chain

[0283] 1.5 g of semaglutide precursor polypeptide Arg 34 -GLP-1 9-37 Place in a reaction bottle, add 100 mL of pure water, stir and dissolve, adjust the pH to 11.3 with triethylamine, and cool the reaction solution to 0-10°C in a water bath; dissolve 0.59 g of the side chain with 11.8 mL of DMF, and slowly drop it into the above reaction solution, the dropwise addition time is 5 hours, and react at 0-10°C for 20 minutes. After the reaction is completed, add 140 mL of pure water to the reaction solution, adjust the pH of the reaction solution to 5.0 with trifluoroacetic acid for precipitation, and centrifuge to obtain semaglutide intermediate 1. The purity after detection is 90.23%.

[0284] Example 9: Preparation of semaglutide by connecting semaglutide intermediate 1 to a dipeptide

[0285] Weigh 1.5g of the semaglutide intermediate 1 prepared in Example 8 in a reaction flask, add a small amount of water, adjust the pH with DIEA, the pH does not exceed 11, stir and dissolve it, add 5-9 times the volume of DMF, weigh 0.71g of Fmoc-His-Aib-OH, dissolve it with a small amount of DMF, add 0.54mL DIEA, freeze for 10min, add 1.04g of PyBOP and shake to activate for 20min. Add the activation solution to the reaction flask, adjust the pH to about 9 with DIEA and react for 3h. After the substrate reaction is completed by HPLC detection, add 5-10% (volume ratio with the reaction solution) of piperidine for deprotection reaction for 2h, and perform post-treatment after HPLC detection of deprotection. Add pre-cooled water with a volume of 5-10 times the reaction volume, adjust the pH to 4.4 with hydrochloric acid for precipitation, and centrifuge to obtain crude semaglutide. The purity after detection is 83.39%.

[0286] Example 10: Preparation of semaglutide by connecting semaglutide intermediate 1 to a dipeptide

[0287] Weigh 1.5g of the semaglutide intermediate 1 prepared in Example 7 in a reaction flask, add a small amount of water, adjust the pH with triethylamine, the pH does not exceed 11, stir and dissolve it, add 5-9 times the volume of DMF, weigh Fmoc-His-Aib-OH 0.71g, HOAt 0.21g, dissolve with a small amount of DMF, freeze for 10min, add 0.24mL DIC and shake well, put it in the refrigerator for freezing and activation for 10min. Add the activation solution to the reaction flask, adjust the pH to about 9 with triethylamine and react for 3h. After the HPLC detection substrate reaction is completed, add 5-10% (to the volume ratio of the reaction solution) of piperidine for deprotection reaction for 2h, and perform post-treatment after the HPLC detection of deprotection is completed. Add pre-cooled water with a volume of 5-10 times the reaction volume, adjust the pH to 4.4 with hydrochloric acid for precipitation, and centrifuge to obtain crude semaglutide. The purity after detection is 84.79%.

[0288] Example 11: Purification of semaglutide after conjugation

[0289] The first step of purification (crude purification): 1 g of crude semaglutide obtained in Example 9 was added to 150 mL of pure water for dissolution, stirred at 200 rpm for 2 h, and the pH was adjusted to 7.50 ± 0.2, and then filtered using 3 μm and 0.45 μm secondary filtration. The chromatographic column of the packing was balanced with mobile phase 1 (10% ethanol) and set aside. Ethanol was added to the filtered sample to a final ethanol concentration of 5%, and the sample was loaded for purification at a linear flow rate of 150 cm / h. Mobile phase 2 was pre-washed, and mobile phase 2 (0.2M acetic acid-20% ethanol) and mobile phase 3 (0.2M acetic acid-80% ethanol) were used for gradient washing and elution at a linear flow rate of 150 cm / h and a loading of 10 g / L packing. The chromatogram is shown in Figure 8 , collect the target peak, and the collected samples are tested by RP-HPLC, with a purity of 97.66%.

[0290] The first step of purification procedure is shown in Table 2.

[0291] Table 2

[0292] Time (min) Mobile phase 2 (%) Mobile phase 3 (%) 0 100 0 50 100 0 60 62 38 260 42 58

[0293] The second step of purification (fine purification): the crude pure sample was diluted with an equal volume of pure water, and the pH value was adjusted to 7.0-7.5 with 2M NaOH. The column was washed with mobile phase A (10% acetonitrile solution), equilibrated with mobile phase B (50mM ammonium acetate-10% acetonitrile solution), and then loaded for purification. The linear flow rate was 150cm / h and the loading capacity was 6-8g / L. Mobile phase B and mobile phase C (85% acetonitrile solution) were used for gradient washing and elution. The chromatogram is shown in Figure 1. Fig. 9, collect the target peak, add the collected target peak sample solution to 2-10℃ pre-cooled water at a ratio of 1:3 (v / v), stir quickly, and adjust the pH to 4.9-5.1 with 2M HCl solution. Let it stand in a 2-10℃ refrigerator for 15h to settle, and remove the sedimentation supernatant by siphoning. Then centrifuge at 13000g and 4℃ for 10min, and collect the precipitate after centrifugation to obtain a high-purity semaglutide sample. RP-HPLC detection showed a purity of 99.78%.

[0294] The second step purification procedure is shown in Table 3.

[0295] Table 3

[0296]

[0297]

[0298] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0299] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A leader peptide, characterized in that The amino acid sequence of the leader peptide comprises MADEAEKAG (SEQ ID NO: 8).

2. The leader peptide according to claim 1, characterized in that The C-terminus of the leader peptide further comprises an enzyme cleavage site, preferably an enzyme cleavage site for Kex2 enzyme and / or CPB enzyme; Further preferably, the amino acid sequence of the restriction site includes KR or RR or RK or KK; Preferably, the amino acid sequence of the leader peptide comprises MADEAEKAGKR (SEQ ID NO: 1).

3. A fusion protein, characterized in that The fusion protein comprises the leader peptide and the target peptide according to claim 1 or 2, and the target peptide comprises GLP-1 or its analogs.

4. The fusion protein according to claim 3, characterized in that The GLP-1 analogs include one or more of liraglutide, semaglutide, albiglutide or dulaglutide.

5. The fusion protein according to claim 3, characterized in that The amino acid sequences of the target peptides include TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 5), HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 9), HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 11), HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG (SEQ ID NO: 12), EGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 3) or One or more of HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 6).

6. The fusion protein according to any one of claims 3 to 5, characterized in that: The target peptide is one or more than two in series; Preferably, the two or more target peptides have the same sequence or contain different sequences.

7. The fusion protein according to claim 6, characterized in that The target peptides are connected to each other through a linker sequence; Preferably, the linker sequence comprises at least one restriction site, such as a restriction site for Kex2 enzyme and / or CPB enzyme; Further preferably, the amino acid sequence of the restriction site includes KR or RR or RK or KK.

8. The fusion protein according to claim 7, characterized in that The N-terminus and C-terminus of the linker sequence contain restriction sites, and the linker sequence also includes DGTDEAEKAG (SEQ ID NO: 7); Preferably, the amino acid sequence of the linker sequence includes KRDGTDEAEKAGKR (SEQ ID NO: 2).

9. The fusion protein according to any one of claims 3 to 8, characterized in that: The fusion protein includes AB m or AC n , where A is the leader peptide, B is the target peptide, and C is the linker sequence-B m , m or n is independently selected from an integer of 1-10.

10. The fusion protein according to any one of claims 3 to 9, characterized in that: The fusion protein includes AB m -C n , where A is the leader peptide; B is the target peptide; C is the linker sequence-B m ; m or n is independently selected from an integer of 1-10; Preferably, m is 1 and n is 2-5; More preferably, the amino acid sequence of the fusion protein includes SEQ ID NO:

4.

11. A nucleic acid, characterized in that The nucleic acid encodes the leader peptide according to any one of claims 1-2 or the fusion protein according to any one of claims 3-10; Preferably, the nucleotide sequence encoding the fusion protein comprises SEQ ID NO: 13; Preferably, the nucleotide sequence encoding the leader peptide comprises SEQ ID NO: 14; Further preferably, the nucleotide sequence encoding the leader peptide comprises SEQ ID NO:

15.

12. An expression vector or recombinant cell, characterized in that: The expression vector or recombinant cell expresses the leader peptide according to any one of claims 1 to 2 or the fusion protein according to any one of claims 3 to 10; or, the expression vector or recombinant cell contains the nucleic acid according to claim 11; Preferably, the expression vector comprises a eukaryotic expression vector or a prokaryotic expression vector; Preferably, the recombinant cell comprises a eukaryotic cell or a prokaryotic cell.

13. A method for preparing the leader peptide according to any one of claims 1 to 2 or the fusion protein according to any one of claims 3 to 10, characterized in that: The preparation method includes chemical synthesis or biological synthesis; Preferably, the preparation method comprises: 1) obtaining the nucleic acid according to claim 11; 2) connecting the nucleic acid obtained in 1) to a vector backbone to obtain an expression vector; 3) Transforming the expression vector obtained in 2) into host cells and culturing the host cells.

14. The preparation method according to claim 13, characterized in that: The culturing of host cells includes inducing expression; Preferably, the culturing of the host cell includes fermentation culture and induced expression; Preferably, the culture temperature is 28-37°C; Preferably, the culture time is 6-50h; Preferably, the pH of the culture system is 7.0-7.2; Preferably, the host cells are fermented to an OD of 600 When the OD is 100-130, the first stage of induction begins, and the induction time is 6-12 hours. The host cells are cultured until the OD 600 When the pH is 150-170, the second induction stage begins, and the fermentation ends after 2-10 hours of induction expression. Further preferably, the dissolved oxygen in the first stage of induction is controlled to be 25-35%; Further preferably, the dissolved oxygen in the second stage of induction is controlled to be 5-15%.

15. A method for preparing a target peptide, characterized in that: The preparation method comprises enzymatically digesting the fusion protein according to any one of claims 3 to 10 or the fusion protein obtained by the preparation method according to any one of claims 13 to 14; Preferably, the preparation method comprises extracting inclusion bodies in recombinant cells expressing the fusion protein, dissolving them and then adding enzymes; Preferably, the enzyme is Kex2 enzyme and / or CPB enzyme.

16. A method for preparing semaglutide, characterized in that: The preparation method comprises modifying the side chain of the target peptide obtained by the preparation method of claim 15 and then connecting a dipeptide.

17. The preparation method according to claim 16, characterized in that: The preparation method comprises performing side chain modification on Lys of the target peptide, and the side chain modification preferably comprises performing an acylation reaction to obtain a semaglutide intermediate 1, and the semaglutide intermediate 1 is shown in Formula VII; 18. The preparation method according to any one of claims 16-17, characterized in that: The dipeptide includes His-Aib; Preferably, the connecting dipeptide comprises coupling Fmoc-His-Aib-OH with the N-terminal amino group of semaglutide intermediate 1 to obtain semaglutide intermediate 2, and semaglutide intermediate 2 is shown in formula VIII; 19. The preparation method according to claim 18, characterized in that: The preparation method further comprises deprotecting the semaglutide intermediate 2 to obtain semaglutide.

20. The preparation method according to any one of claims 16 to 19, characterized in that: The synthesis method of the side chain comprises: (1) using a monosubstituted octadecane dioic acid as a raw material, and performing an activation reaction with TSTU under alkaline conditions or in the presence of a condensing agent combination to obtain a side chain intermediate compound 1, wherein the side chain intermediate compound 1 is shown in Formula I; (2) using the side chain intermediate compound 1 as a raw material, and carrying out an acylation coupling reaction with HL-Glu-OR under alkaline conditions to obtain the side chain intermediate compound 2, the side chain intermediate compound 2 is shown in Formula II; (3) using the side chain intermediate compound 2 as a raw material, and performing an activation reaction with TSTU under alkaline conditions or in the presence of a condensing agent combination to obtain a side chain intermediate compound 3, wherein the side chain intermediate compound 3 is shown in Formula III; (4) Using the side chain intermediate compound 3 as a raw material, an acylation coupling reaction is carried out with AEEA-AEEA under alkaline conditions to obtain the side chain intermediate compound 4, and the side chain intermediate compound 4 is shown in Formula IV; (5) Using the side chain intermediate compound 4 as a raw material, an activation reaction is carried out with TSTU under alkaline conditions or in the presence of a condensing agent combination to obtain the side chain intermediate compound 5, wherein the side chain intermediate compound 5 is shown in Formula V; (6) Using the side chain intermediate compound 5 as a raw material, deprotection is performed in an acidic solution to obtain a side chain compound, and the side chain compound is shown in Formula VI; The substituent R is an alkane, alkene or aromatic hydrocarbon group; preferably a methyl group, an ethyl group or a group that can be hydrolyzed under acidic conditions (such as tert-butyl (tBu) or benzyl).

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