CN112111002B

CN112111002B - Preparation method of semaglutide

Info

Publication number: CN112111002B
Application number: CN202011038808.0A
Authority: CN
Inventors: 马亚平; 王宇恩; 张凌云; 戴政清; 付信; 周迎春; 王庆磊
Original assignee: Shenzhen Shenchuang Biopharmaceutical Co ltd
Current assignee: Shenzhen Shenchuang Biopharmaceutical Co ltd
Priority date: 2020-09-28
Filing date: 2020-09-28
Publication date: 2021-07-13
Anticipated expiration: 2040-09-28
Also published as: CN112111002A

Abstract

The invention relates to a preparation method of semaglutide, which comprises the following steps: 1) preparing a fully-protected coupling substance of the semaglutide and the solid-phase synthetic resin by a solid-phase synthetic method; 2) adding a cracking agent to crack the solid-phase synthetic resin and the protecting group to obtain a cracking solution; 3) adding a mixed solution of ethyl acetate and cyclohexane into the lysate for crystallization; 4) the crystals were filtered to obtain semaglutide. The method adopts a special reagent combination for crystallization, has uniform crystal particles and less impurity inclusion, and can separate the crystal particles by a filtering mode.

Description

Preparation method of semaglutide

Technical Field

The invention relates to the field of polypeptide synthesis, in particular to a preparation method of semaglutide, and particularly relates to a crystallization method.

Background

Semaglutide (Semaglutide), tradename ozampic, developed by danish norand norde, designated as a dietary and exercise aid to improve glycemic control in type 2 diabetic patients, was marketed in the united states by injection at 12 months and 05 days 2017, and FDA approved oral Semaglutide for marketing as a first oral GLP-1 analogue diabetes drug, which has a promising market prospect.

Semaglutide is also a GLP-1 analog, has 94% sequence homology with human GLP-1, is a second generation fatty chain modified GLP-1 analog developed by norhenode, and has a longer half-life and lower dosage than the first generation fatty chain modified GLP-1 analog liraglutide. The semaglutide keeps the good safety and the blood sugar reduction effectiveness of the liraglutide, and the weight reducing effect is better than the former two.

Simaglutide chemically expressed as Aib⁸,Arg³⁴Lys²⁶- [ N- ε -ODA-ODA- (γ -Glu (N- α -18-octadecanoic acid-1-acyl))]-GLP-1(7-37) of the formula C₁₈₇H₂₉₁N₄₅O₅₉The relative molecular mass is 4113.58, the CAS number is 910463-68-2, and the sequence information is as follows:

H-His⁷-Aib⁸-Glu⁹-Gly¹⁰-Thr¹¹-Phe¹²-Thr¹³-Ser¹⁴-Asp¹⁵-Val¹⁶-Ser¹⁷-Ser¹⁸-Tyr¹⁹-Leu²⁰-Glu²¹-Gly²²-Gln²³-Ala²⁴-Ala²⁵-Lys²⁶(AEEA-AEEA-γ-Glu-OctadecanedioicAcid)-Glu²⁷-Phe²⁸-Ile²⁹-Ala³⁰-Trp³¹-Leu³²-Val³³-Arg³⁴-Gly³⁵-Arg³⁶-Gly³⁷-OH

currently, Novonide produces the backbone Arg of the Semetreuryptin by yeast mainly through gene recombination technology³⁴-GLP-1^9-37Then, the His-Aib is connected; however, only the main chain Arg can be produced by the gene recombination technology³⁴-GLP-1^9-37The method also needs to utilize chemical means and His-Aib reaction to generate the semaglutide main chain Aib⁸,Arg³⁴-GLP-1^7-37(ii) a Then at Lys²⁶The side chain amino is connected with a long-acting modification group. Due to Aib⁸,Arg³⁴-GLP-1^7-37The side chain and the N-terminal amino group are not protected, and a plurality of active sites exist, so that more impurities are generated in the process, and the loss is large.

In the prior art, Fmoc chemical solid-phase synthesis methods for synthesizing Semaglutide (Semaglutide) are provided. US8129343 and US8536122 protection of Lys with Mmt, Mtt, ivDde and Dde²⁶After the main chain is coupled, the side chain protecting group is removed, and the side chain long-acting modification groups are sequentially coupled; CN106928344 protection of Lys by Alloc²⁶After the main chain is coupled, Pd (PPh3) is utilized₄Removing the protecting group, and sequentially coupling the side chain long-acting modification groups; CN106478806 adopts Dde-Lys (Fmoc) -OH as synthetic monomer, and Lys is completed by sequential coupling²⁶The long-acting side chain modifying group of (1) and then Lys is removed²⁶The alpha amino protecting group Dde to complete the coupling of the main chain; in the above synthesis methods, there are difficulties such as various side chain modification steps, complex process, and difficulty in purifying crude products. CN108059666 uses Alloc-Lys (Fmoc) -OH as a synthetic monomer, CTC resin as a solid phase carrier, and uses a solid phase method to synthesize the monomer Alloc-Lys (AEEA-AEEA-gamma-Glu (OtBu) -monobutylococadecanoate) -OH, and then uses the monomer as a monomer to synthesize the solid phase to obtain the semaglutide.

In the process of resin cutting, the crude product crystallization process basically adopts a cracking reagent to cut the resin and carry out ether precipitation to obtain crude peptide; RP-HPLC preparation gave the crude peptide.

At present, no patent of crystallization and precipitation of semaglutide is found, the inventor researches a precipitation method in a semaglutide preparation process, and finds that diethyl ether is used as a solvent to have some problems, the diethyl ether precipitation has poor crystal form, only crude products can be obtained through centrifugation, impurities can be wrapped in the crude products in the centrifugation process to influence the purification and product quality in the later stage, according to the conventional methods in the field, diethyl ether is used as the solvent, when ether solvents such as diethyl ether and the like are selected, the crystallization is basically viscous, the crystal form is inhomogeneous, the filtration is slow, so the product can be obtained only by the centrifugation method generally, as the crystal form is inhomogeneous, the impurities are not easily filtered out by centrifugation, the impurities can be wrapped in the two products, and the example shows that after one-time purification, one main impurity is obviously larger, and a purer finished product can be obtained by more-time purification, the production efficiency, also affecting product quality. Meanwhile, the ether belongs to flammable and explosive dangerous chemicals, certain risks exist in the production operation process, if more environment-friendly organic solvents such as ethyl acetate are adopted for precipitation and crystallization, impurities in a crude product can be better controlled through a filtering mode, and meanwhile, the method has the characteristics of strong controllability, good reproducibility, low energy consumption, environmental friendliness and the like, and has obvious advantages compared with the traditional process.

Disclosure of Invention

Aiming at the requirements of the existing semaglutide synthesis technology, a crystal preparation method of semaglutide is provided.

In view of the above-mentioned drawbacks of crystalline precipitation during the synthesis of semaglutide, the present invention aims to provide a process for the preparation of semaglutide comprising the steps of:

1) preparing a fully-protected coupling substance of the semaglutide and the solid-phase synthetic resin by a solid-phase synthetic method;

2) adding a cracking agent to crack the solid-phase synthetic resin and the protecting group to obtain a cracking solution;

3) adding a mixed solution of ethyl acetate and cyclohexane into the lysate for crystallization;

4) the crystals were filtered to obtain semaglutide.

In an embodiment of the present invention, the temperature of the lysate, the mixed solution of ethyl acetate and cyclohexane, at the time of crystallization in step 3) is 2 to 25 deg.C, preferably 10 to 15 deg.C.

In an embodiment of the present invention, the volume of the mixed solution of ethyl acetate and cyclohexane added in step 3) is 8 to 12 times, preferably 10 times the volume of the lysate.

In the embodiment of the invention, the stirring speed of the mixed solution of ethyl acetate and cyclohexane in the step 3) is 100rmp in the process of adding the lysate, and the stirring speed is increased to 120-130rmp after crystals are formed in the crystallization process.

In an embodiment of the present invention, the ratio of ethyl acetate to cyclohexane in the mixed solution of ethyl acetate and cyclohexane in step 3) is 1:2 to 2:1, preferably 1: 1.

In an embodiment of the invention, the time for crystallization in step 3) is 15 to 60 minutes, preferably 25 to 35 minutes.

In an embodiment of the present invention, the method for filtering in step 4) is reduced pressure filtration, preferably filtration using a reduced pressure funnel.

In an embodiment of the present invention, the filter cake is washed 2 to 5 times with a mixed solution of ethyl acetate and cyclohexane while filtering in step 4).

In an embodiment of the invention, the cleavage agent in step 2) is TFA, TIS, EDT, PhOH and H₂Mixture of O, preferably TFA: and (3) TIS: EDT (electro-thermal transfer coating): PhOH: h₂O-85-95: 2-5:0-3:0-2:1-5 (volume ratio); TFA: and (3) TIS: EDT (electro-thermal transfer coating): PhOH: h₂O-85-95: 2-5:0-3:0-2:1-5 (volume ratio); the cracking time is 1.5-3.5 hours.

In an embodiment of the invention, step 1) may be carried out by any method to prepare a conjugate of the fully protected semaglutide and a solid-phase synthetic resin, for example, by sequentially synthesizing a backbone on the solid-phase synthetic resin from the C-terminus to the N-terminus, then removing the side chain protecting group of Lys, and coupling the side chain. The method for synthesizing the main chain and the side chain can be a polypeptide fragment formed by sequentially coupling amino acids or a plurality of amino acids. It is also possible to use a method in which a part of the main chain is synthesized first, then the main chain is extended to form the main chain, and then the main chain is completed. The conjugates of the fully protected semaglutide and the solid phase synthetic resin may be prepared by any known method.

In one and specific embodiment of the present invention, step 1) comprises the steps of:

1-1) coupling Fmoc-Gly-OH on Wang resin to prepare Fmoc-Gly-Wang resin;

1-2) coupling Fmoc amino acids on Fmoc-Gly-Wang resin in sequence by adopting a proper coupling system according to the peptide sequence of the semaglutide, wherein the specific sequence is as follows: Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Alloc) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH (Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Otbu- (Asp), (Asp, Fmoc-Val) -OH, Fmoc-Leu, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Aib-OH, Boc-His (Trt) -OH;

1-3) removing the Alloc protecting group from Lys (Alloc);

1-4) coupling side chain residues in sequence: Fmoc-AEEA-OH, Fmoc-Glu-OtBu and mono-tert-butyl octadecanedioate.

The substitution degree range of the Wang resin in the step (1) is 0.3-1.0 mmol/g, and more preferably 0.4-0.6 mmol/g; preparing Fmoc-Gly-Wang Resin, specifically Fmoc-Gly-OH and Wang Resin with a proper substitution degree, and coupling amino acids by adopting a proper coupling system, wherein the coupling system comprises HOBt/DIC/DMAP, HOBt/DCC/DMAP, HOAt/DIC/DMAP, HBTU/HOBt/DIPEA, TBTU/HOBt/DIPEA and PyBOP/HOBt/DIPEA; preferably HOBt/DIC/DMAP;

in a preferred embodiment of the present invention, the coupling condensing agent used in the coupling process of the above steps 1-2) and 1-4) is DIC + A or B + A + C, wherein A is at least one of HOBt or HOAt, B is at least one of HBTU, HATU or PyBOP, and C is at least one of DIEA, TMP or DMAP. The consumption of the condensing agent in the coupling system is 0.8-3.0 times of the equivalent of the amino acid. The reaction time is 1-4 hours.

In a preferred embodiment of the present invention, the above step 1-3) selects Pd⁰(Ph₃P)₄/Me₂NH·BH₃Or Pd⁰(Ph₃P)₄/PhSiH₃In DCM solvent, where Pd0 (Ph)₃P)₄In an amount of 0.05 to 1.0 equivalent, preferably 0.1 to 0.5 equivalent, Me on the synthesis scale₂NH·BH₃Or PhSiH₃The amount of (B) is from 10 to 100 equivalents, preferably from 20 to 60 equivalents, on the synthesis scale, and the reaction time is from 0.5 to 4 hours, preferably from 1 to 2 hours. After the reaction was complete, the peptide resin was washed with 0.2M copper reagent for 30 minutes.

In a preferred embodiment of the invention, the crystallization is filtered to obtain semaglutide, which is then further purified, for example by HPLC.

Advantageous effects

1) The crystals obtained by the crystallization method are uniform and moderate in size, can be obtained by a filtration method, and avoids the problem of impurity wrapping when a centrifugation method is used.

2) The crystallization reagent is added into the cracking liquid, and after crystals appear, the crystallization reagent can gradually form crystallized crystals at a constant speed, so that the uniform size of the crystallized crystals is ensured, and the subsequent filtration is facilitated. The crystallization method of the invention actually overcomes the problems that the local crystallization speed of the lysate is high, impurity packages are formed, and crystals are different in size and difficult to filter when diethyl ether, isopropyl ether or methyl tert-butyl ether are used as crystallization reagents and added into the lysate in the traditional method.

3) Cyclohexane is beneficial to separating out more products in the solvent, and the content of impurities in the crude product can be reduced after the cyclohexane is matched with ethyl acetate.

4) The invention overcomes the prejudice of the prior art that the lower the temperature is, the better the crystallization effect is, and the crystal particles formed are too small to be beneficial to filtration due to the too low temperature when the frozen ether in the prior art is adopted to separate out crystals, so that the filtration speed is very slow, the efficiency is low, and if the crystal particles are separated out at room temperature, the crystal particles are too large to form impurity packages, and meanwhile, the solvent is partially dissolved in the product due to the high temperature, so that the yield is reduced. The invention adopts the crystallization temperature of 10-15 ℃ to improve the crystallization efficiency and simultaneously ensures the particle size of the crystal and the purity of the product.

5) The crystallization solvent adopted by the invention is preferably a mixed solvent with the volume ratio of ethyl acetate to cyclohexane being 1:1, and the ethyl acetate and the cyclohexane are more environment-friendly compared with diethyl ether, isopropyl ether and methyl tert-butyl ether.

6) The crystallization and precipitation method of semaglutide provided by the invention can easily complete the synthesis of semaglutide by adopting the simplest and economic method and obtain a good synthesis effect, and the preparation method has the advantages of simple operation, low production cost, high yield, environmental protection and the like.

Drawings

FIG. 1: example 6 crude sample after crystallization chromatographed result (purity: 87.72%, content of key impurity (RT ═ 21.100): 2.77%).

FIG. 2: example 11 the crude sample after crystallization was chromatographed (purity: 92.72%, content of key impurities (RT 21.164): 1.54%).

FIG. 3: the sample of example 6 was purified by one HPLC and then chromatographed (content of key impurity (RT ═ 20.835): 1.13%).

FIG. 4: the sample of example 11 was purified by one HPLC and then chromatographed (content of key impurity (RT ═ 20.927): 0.73%).

FIG. 5: example 6 results of chromatography on final product (purity: 98.79%, content of key impurity (RT ═ 20.837): 0.68%).

FIG. 6: example 11 chromatographic analysis of the final product (purity: 99.12%, no key impurities were detected).

Detailed Description

The invention discloses a preparation method of polypeptide, which can be realized by appropriately improving process parameters by the technical personnel in the field with reference to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Abbreviations used in the specification and claims have the following specific meanings:

the polypeptide provided by the invention, the preparation method and the raw materials, auxiliary materials and reagents used in the application can be purchased from the market or produced by the user.

The invention is further illustrated by the following examples:

EXAMPLE 1 preparation of Fmoc-Gly-Wang resin

Weighing 100 g of Wang resin with the substitution degree of 0.8mmol/g into a solid phase reaction column, adding DMF, and carrying out bubbling swelling for 60 minutes by nitrogen; weighing 29.8 g (100mmol) of Fmoc-Gly-OH, 16.2 g (120mmol) of HOBt and 1.2 g (10mmol) of DMAP, dissolving the mixture with DMF, adding 20.3mL DIC (120mmol) in an ice-water bath at 0 ℃, activating the mixture for 5 minutes, adding the mixture into a reaction column, reacting the mixture for 1.5 hours, adding 70mL of acetic anhydride and 60mL of pyridine, mixing and sealing the mixture for 24 hours, washing the mixture for three times by DCM, draining the Resin after methanol shrinkage to obtain 108 g of Fmoc-Gly-Wang Resin in total, wherein the detection substitution degree is 0.35 mmol/g.

Example 2 preparation of a Semetreuptade backbone peptide resin

28.6 g (10mmol) of the Fmoc-Gly-Wang resin obtained in example 1 (Sub ═ 0.35mmol/g) were weighed into a reaction column, washed 3 times with DCM and swollen with DMF for 30 min. The Fmoc protecting group was then removed with DBLK and washed 6 times with DMF. Fmoc-Arg (Pbf) -OH 19.35 g (30mmol), HOBt 4.86 g (36mmol) were weighed, dissolved in DMF, 6.1mL DIC (36mmol) were added in an ice water bath at 0 deg.C, activated for 5 minutes, charged to a reaction column, reacted for 2 hours, and then the Fmoc protecting group was removed with DBLK. Repeating the above procedure by coupling Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (alloc) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (Val-tBu) -OH, (Fmoc-OH, Fmoc-Asp (Otbu) -OH, Fmoc-, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Aib-OH, Boc-His (Trt) -OH; after the reaction was completed, the peptide resin was washed with DMF.

EXAMPLE 3 removal of Lys side chain protecting groups

The fully protected peptide resin obtained in example 4 was washed 3 times with DCM. 24.5 g of dimethyl ether are weighed outAmine borane was measured in 800mL of DCM and added to the reaction column, after 10 minutes of reaction, 1.21 g of Pd was added⁰(Ph₃P), reaction for 2 hours. The resin was then washed 3 times with DCM, the peptide resin was washed with 0.2M copper reagent in DMF for 30 minutes, the resin was washed 3 times with DMF, and the resin was washed 3 times with DCM to give the peptide resin with selectively removed Alloc for use.

Example 4 coupling of the Semetreuptade side chain

12.0 g (20mmol) of Fmoc-AEEA-OH, 4.86 g (36mmol) of HOAt were weighed out, dissolved in DMF, and 6.1mL of DIC (36mmol) were added in an ice-water bath at 0 ℃ to activate for 5 minutes, the peptide resin of example 10 was added, reacted for 2 hours, and then the Fmoc protecting group was removed with DBLK. Repeating the operation, and coupling Fmoc-AEEA-OH, Fmoc-Glu-OtBu and tert-butyl octadecanedioate according to the sequence; after the reaction was completed, the reaction mixture was washed 6 times with DMF, again 3 times with DCM, then washed 3X 10 minutes with methanol and dried under vacuum to give 121.6 g of a peptide resin of semaglutide.

Example 5 cleavage of S-Metroglucide

121.6 g of the peptide resin obtained in example 16 was charged into a 2000mL single-neck flask, and lysate 1200mL of TFA was prepared in advance: and (3) TIS: EDT (electro-thermal transfer coating): PhOH: h₂The lysate was added to the flask and allowed to react at room temperature for 2.5 hours (vol/vol) with O90: 3:3:2:2, the resin was filtered off, the resin was washed with 50mL TFA, and the filtrates were combined.

Example 6 crystallization of Semetreuptade

Adding 100mL of lysate into a round-bottom flask, controlling the temperature in the round-bottom flask solution to be 10-15 ℃, slowly adding diethyl ether through a separating funnel while stirring at the stirring speed of 100rmp, continuing to stir for 30 minutes after the addition is finished, filtering by using a sand core funnel, washing three times by using a proper amount of diethyl ether, draining, and drying to obtain 3.6g of crude semaglutide, wherein the yield is 85.7%, and is lower than that of example 11. After crystallization, the crude product was viscous, the filtration time was 90 minutes, the filtration rate was much slower than in example 11, the crude purity after crystallization was 87.7%, the content of key impurity (RT-21.100) was 2.77% (see fig. 1), and the purity was 5% lower than in example 11, and the content of key impurity was significantly higher. The secondary HPLC purification conditions are given in example 17. The key impurity (RT ═ 20.835) content of the sample of example 6 was 1.13% after one HPLC purification (see fig. 3), and the purity of the sample of example 6 was the final product after two HPLC purifications: 98.79% and the content of key impurity (RT ═ 20.837) was 0.68% (see fig. 5).

Although purification can be performed by means of HPLC and the content of impurities can be reduced by means of multiple times of HPLC, not only is the purification efficiency extremely low, but also the yield of the product is affected, and the method is not environment-friendly.

Example 7 crystallization of Semetreuptade

Adding 100mL of lysate into a round-bottom flask, controlling the temperature in the solution in the round-bottom flask to be 10-15 ℃, slowly adding isopropyl ether through a separating funnel, stirring while adding, wherein the stirring speed is 100rmp, continuously stirring for 30 minutes after adding, then filtering by using a sand core funnel, washing for three times by using a proper amount of isopropyl ether, draining, and drying to obtain 3.7g of crude semaglutide, wherein the yield is 88.1%, the crude semaglutide is viscous after crystallization, the filtering time is 80 minutes, the purity of the crude semaglutide after crystallization is 85.86%, and the content of key impurities is 2.55%.

Example 8 crystallization of Semetreuptade

Adding 100mL of lysate into a round-bottom flask, controlling the temperature in the solution in the round-bottom flask to be 10-15 ℃, slowly adding methyl tert-butyl ether through a separating funnel, stirring while adding, wherein the stirring speed is 100rmp, continuously stirring for 30 minutes after adding, filtering by using a sand core funnel, washing three times by using a proper amount of methyl tert-butyl ether, draining, and drying to obtain 3.5g of crude semaglutide, wherein the yield is 83.3%, the yield is far lower than that of example 11, the crude semaglutide is viscous after crystallization, the filtering time is 85 minutes, the filtering speed is far slower than that of example 11, the purity of the crude semaglutide after crystallization is 80.86%, the content of key impurities is 2.87%, the purity of the crude semaglutide.

Example 9 crystallization of Semetreuptade

Adding 100mL of lysate into a round-bottom flask, controlling the temperature in the solution in the round-bottom flask to be 10-15 ℃, slowly adding ethyl acetate through a separating funnel, stirring while adding, wherein the stirring speed is 100rmp, continuously stirring for 30 minutes after adding, filtering by using a sand core funnel, washing three times by using a proper amount of ethyl acetate, draining, and drying to obtain 3.8g of crude semaglutide, wherein the yield is 90.4%, the cracking yield is lower than that of example 11, after crystallization, the crystal form is better, the filtering time is 40 minutes, the filtering speed is slightly lower than that of example 11, the purity of the crude semaglutide after crystallization is 90.86%, the content of key impurities is 1.97%, the purity of the crude semaglutide is slightly lower, and the content of the key impurities is.

Example 10 crystallization of Semetreuptade

Adding 100mL of lysate into a round-bottom flask, controlling the temperature in the solution in the round-bottom flask to be 10-15 ℃, slowly adding cyclohexane through a separating funnel, stirring while adding, wherein the stirring speed is 100rmp, continuously stirring for 30 minutes after adding, filtering by using a sand core funnel, washing three times by using an appropriate amount of cyclohexane, draining, and drying to obtain 3.9g of crude semaglutide, wherein the yield is 92.8%, the cracking yield is lower than that of example 11, the crude semaglutide is viscous after crystallization, the crystal is not shaped, the filtering time is 75 minutes, the filtering speed is far slower than that of example 11, the purity of the crude semaglutide after crystallization is 88.35%, the content of key impurities is 2.08%, the purity of the crude semaglutide is lower, and.

Example 11 crystallization of Semetreuptade

Adding 100mL of lysate into a round-bottom flask, controlling the temperature in the round-bottom flask solution to be 10-15 ℃, mixing ethyl acetate and cyclohexane in a volume ratio of 1:1 in advance, slowly adding the mixed solution of ethyl acetate and cyclohexane through a separating funnel while stirring at a stirring speed of 100rmp, continuously stirring after the addition, increasing the stirring speed to 120-130rmp when crystals begin to generate, and stirring for 30 minutes in total. And then filtering by using a sand core funnel, washing three times by using a proper amount of mixed solution of ethyl acetate and cyclohexane, draining, and drying to obtain 4.1 g of crude semaglutide, wherein the yield is 97.6%, the yield is higher than that of other examples, after crystallization, crystals are uniform, the filtering speed is high, the filtering time is 30 minutes, the filtering speed is obviously higher than that of other examples, the purity of the crude semaglutide after crystallization is 92.72%, the content of key impurities (RT (21.164)) is 1.54% (see figure 2), and compared with other examples, the purity is high, and the content of the key impurities is low. Conditions for secondary HPLC purification example 17. After one-time HPLC purification, the content of the key impurity (RT ═ 20.927) was 0.73% (see fig. 4), which was lower than that of example 11, and after two-time HPLC purification, the purity of the final product was 99.12% and the key impurity was not detected (see fig. 6), and compared to example 6, the purity was high and the key impurity was not detected.

Example 12 crystallization of Semetreuptade

Adding 100mL of lysate into a round-bottom flask, controlling the temperature in the round-bottom flask solution to be 10-15 ℃, mixing ethyl acetate and cyclohexane in a volume ratio of 2:1 in advance, slowly adding the mixed solution of ethyl acetate and cyclohexane through a separating funnel while stirring at a stirring speed of 100rmp, continuously stirring after the addition, increasing the stirring speed to 120-130rmp when crystals begin to generate, and stirring for 30 minutes in total. And then filtering by using a sand core funnel, washing three times by using a proper amount of mixed solution of ethyl acetate and cyclohexane, draining, and drying to obtain 3.9g of crude semaglutide, wherein the yield is 92.8%, the cracking yield is lower than that of example 11, the filtering time is 45 minutes, the filtering speed is slightly slower than that of example 11, the purity of the crude semaglutide after crystallization is 90.38%, the content of key impurities is 1.88%, and compared with example 11, the purity of the crude semaglutide is slightly lower, and the content of the key impurities is slightly higher.

Example 13 crystallization of Semetreuptade

Adding 100mL of lysate into a round-bottom flask, controlling the temperature in the round-bottom flask solution to be 10-15 ℃, mixing ethyl acetate and cyclohexane in a volume ratio of 1:2 in advance, slowly adding the mixed solution of ethyl acetate and cyclohexane through a separating funnel while stirring at a stirring speed of 100rmp, continuously stirring after the addition, increasing the stirring speed to 120-130rmp when crystals begin to generate, and stirring for 30 minutes in total. And then filtering by using a sand core funnel, washing three times by using a proper amount of mixed solution of ethyl acetate and cyclohexane, draining, and drying to obtain 4.0g of crude semaglutide, wherein the yield is 95.2%, the cracking yield is slightly lower than that of example 11, the crude semaglutide is viscous after crystallization, the filtering time is 60 minutes, the filtering speed is far slower than that of example 11, the purity of the crude semaglutide after crystallization is 87.38%, the content of key impurities is 2.07%, and compared with example 11, the purity of the crude semaglutide is lower and the content of the key impurities is higher.

Example 14 crystallization of Semetreuptade

Adding 100mL of lysate into a round-bottom flask, controlling the temperature in the round-bottom flask solution to be 5-10 ℃, mixing ethyl acetate and cyclohexane in a volume ratio of 1:1 in advance, slowly adding the mixed solution of ethyl acetate and cyclohexane through a separating funnel while stirring at a stirring speed of 100rmp, continuously stirring after the addition, increasing the stirring speed to 120-130rmp when crystals begin to generate, and stirring for 30 minutes in total. And then filtering by using a sand core funnel, washing the filter cake three times by using a proper amount of mixed solution of ethyl acetate and cyclohexane, draining, and drying to obtain 4.0g of crude semaglutide, wherein the yield is 95.2%, the cracking yield is slightly lower than that of example 11, the crude semaglutide is viscous after crystallization, the filtering time is 90 minutes, the filtering speed is far slower than that of example 11, the purity of the crude semaglutide after crystallization is 90.38%, the content of key impurities is 1.85%, and compared with example 11, the purity of the crude semaglutide is lower and the content of the key impurities is higher.

Example 15 crystallization of Semetreuptade

Adding 100mL of lysate into a round-bottom flask, controlling the temperature in the round-bottom flask solution to be 15-20 ℃, mixing ethyl acetate and cyclohexane in a volume ratio of 1:1 in advance, slowly adding the mixed solution of ethyl acetate and cyclohexane through a separating funnel while stirring at a stirring speed of 100rmp, continuously stirring after the addition, increasing the stirring speed to 120-130rmp when crystals begin to generate, and stirring for 30 minutes in total. Then filtering by a sand core funnel, washing the filter cake three times by using a proper amount of mixed solution of ethyl acetate and cyclohexane, draining, and drying to obtain 3.4g of crude semaglutide with the yield of 80.9 percent, which is far lower than that of the example 11. After crystallization, the filtration time was 35 minutes, the filtration rate was much slower than that of example 11, and the purity of the crude product after crystallization was 90.88% and the content of the key impurities was 1.74%, compared with example 11, the purity of the crude product was slightly lower and the content of the key impurities was slightly higher.

Example 16 crystallization of Semetreuptade

Adding 1000mL of mixed ethyl acetate and cyclohexane with a volume ratio of 1:1 in advance into a round-bottom flask, controlling the temperature in the solution in the round-bottom flask to be 15-20 ℃, slowly adding 100mL of lysate into the mixed solution of the ethyl acetate and the cyclohexane through a separating funnel while stirring at a stirring speed of 100rmp, continuously stirring after the addition, increasing the stirring speed to 120-130rmp when crystals begin to generate, and stirring for 30 minutes. Then filtering by using a sand core funnel, washing the filter cake three times by using a proper amount of mixed solution of ethyl acetate and cyclohexane, draining, and drying to obtain 3.5g of crude semaglutide, wherein the yield is 83.3%, the yield is far lower than that of example 11, the crystal is blocky and is not easy to stir and disperse, the filtering time is 80 minutes, the filtering speed is far slower than that of example 11, the purity of the crude semaglutide after crystallization is 86.88%, the content of key impurities is 2.16%, and compared with example 11, the purity of the crude semaglutide is lower and the content of the key impurities is higher.

Example 17 Secondary HPLC purification of Semetreuptatide

Taking the crude product, and carrying out two-step purification on the crude peptide by using a reversed-phase high-performance liquid-phase color boiling method: a first purification step and a second purification step, wherein the first purification step comprises: mobile phase A was 0.1% TFA-H by volume fraction₂O solution, and the mobile phase B is a TFA-ACN solution with the volume fraction of 0.1 percent; the second step of purification is to convert the pure product into salt: mobile phase A is 0.1% acetic acid-H₂O solution, and the mobile phase B is acetic acid-ACN solution with the volume fraction of 0.1 percent; both purification steps were gradient elution: the elution gradient is 10-60% of phase B, C18 is used for preparing a column (50 multiplied by 250mm, 10 mu m), the elution time is 60min, the flow rate is 80mL/min, and the ultraviolet detection wavelength is 220 nm. And concentrating and freeze-drying to obtain the finished semaglutide.

Example 18 detection conditions for crystalline Semetreuptade product

The detection method of the filtered sample comprises the following steps: 1. the instrument comprises the following steps: DIONEX170, 2 column: Welch-C18-120A-5 μm 4.6X 250mm,3. mobile phase: phase A: 2% triethylamine solution adjusted to pH 3.0 with phosphoric acid, phase B: chromatographically pure acetonitrile, 4. flow rate: 1mL/min,5. wavelength: 220nm, 6. gradient (B%): 22% -42% (40 min).

From the above examples, it is clear that crystallization with different reagents gives very different product states. Even the same kind of organic reagent may produce different product states. Different product states have a great influence on the final separation, purity and post-treatment of the product. For example, products with small or non-shaped crystalline particles can be filtered out or filtered at too low a rate for commercial use, which significantly affects the implementation of the solution, but can only be separated by centrifugation, which in turn leads to the introduction of impurities. The method of the invention is simple, and can obtain good crystallization state, so the product can be obtained quickly by decompression filtration, and the purity is very high. The filtration speed of the method is at least 2 times of that of other methods, more importantly, the content of key impurities in the RT-21 min vicinity can be obviously reduced if the crystallization condition of the invention is adopted, and the impurities can be completely removed by HPLC. Although HPLC is an important means of purification, and most impurities can be removed by multiple purifications, HPLC purification time is long, organic solvent is consumed, and yield is also reduced. The method adopts a method for adjusting crystallization conditions, and unexpectedly finds that the crystallization conditions can be used for preliminary purification in a crystallization step, the purification method is simple, the adopted equipment is simple, and the efficiency is higher.

Claims

1. A preparation method of semaglutide comprises the following steps:

4) filtering the crystals to obtain semaglutide;

the temperature of the lysate and the mixed solution of ethyl acetate and cyclohexane during crystallization in the step 3) is 10-15 ℃;

the ratio of ethyl acetate to cyclohexane in the mixed solution of ethyl acetate and cyclohexane in step 3) was 1: 1.

2. The method according to claim 1, wherein the volume of the mixed solution of ethyl acetate and cyclohexane added in step 3) is 8 to 12 times the volume of the lysate.

3. The method according to claim 2, wherein the volume of the mixed solution of ethyl acetate and cyclohexane added in step 3) is 10 times the volume of the lysate.

4. The preparation process according to claim 1, wherein the stirring speed of the mixed solution of ethyl acetate and cyclohexane in the step 3) is 100rmp during the addition of the lysate, and the stirring speed is increased to 120-130rmp after the formation of crystals during the crystallization.

5. The method according to claim 1, wherein the crystallization time in the step 3) is 15 to 60 minutes.

6. The method according to claim 5, wherein the crystallization time in the step 3) is 25 to 35 minutes.

7. The method according to claim 1, wherein the filtration in step 4) is performed under reduced pressure.

8. The method according to claim 7, wherein the filtration in step 4) is performed by using a reduced pressure funnel.

9. The method according to claim 1, wherein the filtration in step 4) is carried out by washing the cake 2 to 5 times with a mixed solution of ethyl acetate and cyclohexane.

10. The method of claim 1, wherein the cleavage agent of step 2) is TFA, TIS, EDT, PhOH or H₂A mixture of O.

11. The method of claim 10, wherein the cleavage agent of step 2) is TFA: and (3) TIS: EDT (electro-thermal transfer coating): PhOH: h₂O-85-95: 2-5:0-3:0-2: 1-5; the cracking time is 1.5-3.5 hours.

12. The preparation method according to claim 1, wherein in step 1), the main chain is sequentially synthesized from the C end to the N end on the solid-phase synthetic resin, then the side chain protecting group of Lys is removed, and the side chain is coupled to obtain the fully-protected coupling product of the semaglutide and the solid-phase synthetic resin.