Purification process of semeglutide

Abstract

The invention relates to the technical field of medicine purification, in particular to a purification process of semeglutide. Comprising the following steps: S1, by taking an ammonium acetate solution as a mobile phase A and acetonitrile as a mobile phase B, carrying out primary purification on a solution containing the semeglutide according to an elution gradient by adopting a reverse phase chromatography, and collecting a purified eluent; and S2, with a trifluoroacetic acid solution as a mobile phase A and acetonitrile as a mobile phase B, carrying out secondary purification on the eluent obtained in the step S1 according to the elution gradient by adopting the reverse phase chromatography, and collecting the purified eluent containing the semeglutide. According to the method, a solution containing the semeglutide is subjected to specific elution steps, namely, an ammonium acetate solution is taken as a mobile phase A, acetonitrile is taken as a mobile phase B, primary purification is carried out by adopting reversed phase chromatography, a trifluoroacetic acid solution is taken as the mobile phase A, acetonitrile is taken as the mobile phase B, and secondary purification is carried out by adopting reversed phase chromatography; not only is the yield of the semeglutide improved, but also the purity of the semeglutide is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug purification, and in particular to a purification process of semaglutide. Background Art

[0002] Semaglutide is a long-acting GLP-1 (glucagon-like peptide-1) receptor agonist developed by Novo Nordisk. It has a half-life of about 1 week and has good effects in the fields of hypoglycemia and weight loss. Semaglutide was approved for marketing by the US FDA (Food and Drug Administration) in September 2019, which has a significant impact on peptide drugs. Insulin injections are actively developing oral preparations, and oral peptide preparations are also an inevitable trend in the market.

[0003] Semaglutide is a peptide drug administered by subcutaneous injection once a week. Structurally, semaglutide is a GLP-1 (7-37) chain in which Aib (2-aminoisobutyric acid) replaces Ala (alanine) at position 8, Arg (arginine) replaces Lys (lysine) at position 34, and Lys at position 26 is connected to an octadecanoic acid fatty chain. Compared with liraglutide, semaglutide has a longer fatty chain and increased hydrophobicity, but semaglutide has been modified with a short chain of PEG (polyethylene glycol), which greatly enhances its hydrophilicity. The prior art discloses many processes for polypeptide purification, for example, Chinese patent CN109438569A discloses a method for separation and purification of liraglutide, and Chinese patent CN109503705A discloses a method for separation and purification of liraglutide. If the methods in the above patents are used to separate and purify semaglutide, due to the differences in the sequences of the polypeptides, the purity and yield of the semaglutide finally prepared will be low. The prior art also discloses a method for purification of semaglutide, for example, Chinese patent CN117603338A discloses a method for purification of semaglutide, which is purified by two-step reversed-phase high performance liquid chromatography, (1) the first step of purification: using an acidic buffer containing acetate and an ion pairing agent and an organic solvent as the mobile phase, and a reversed-phase filler as the stationary phase; (2) the second step of purification: using an alkaline buffer containing phosphate and an organic solvent as the mobile phase, and a reversed-phase filler as the stationary phase. The yield of semaglutide obtained by the method in this patent is still relatively low. Summary of the invention

[0004] The present invention provides a purification process of semaglutide. By adopting the purification process, not only the yield of semaglutide is improved, but also the purity is improved.

[0005] In order to achieve the above technical objectives, the present invention provides a purification process of semaglutide, comprising the following steps:

[0006] S1. Using ammonium acetate solution as mobile phase A and acetonitrile as mobile phase B, purify the semaglutide-containing solution for the first time by reverse phase chromatography according to the following elution gradient, and collect the purified eluate;

[0007] Time / min Volume percentage of mobile phase A / % Volume percentage of mobile phase B / % 0～3 80 20 3～3.01 80→75 20→25 3.01～43 75→45 25→55 43～63 45→30 55→70

[0008] S2, using trifluoroacetic acid solution as mobile phase A and acetonitrile as mobile phase B, using reverse phase chromatography according to the following elution gradient to purify the eluate obtained in step S1 for a second time, and collecting the purified eluate containing semaglutide;

[0009] Time / min Volume percentage of mobile phase A / % Volume percentage of mobile phase B / % 0～3 80 20 3～3.01 80→65 20→35 3.01～43 65→45 35→55 43～63 45→30 55→70 .

[0010] Furthermore, in step S1, the mass percentage of ammonium acetate in the ammonium acetate solution is 0.1-1.5%; and / or the solvent in the ammonium acetate solution is water.

[0011] Furthermore, in step S1, the solvent in the solution containing semaglutide is selected from one or more of acetonitrile, methanol, formic acid, and water; and / or the concentration of the solution containing semaglutide is 10-14 mg / mL.

[0012] Furthermore, in step S1 or step S2, the filler of the chromatographic column is octadecylsilane bonded silica gel with a specification of 50×250 mm, 10 μm; and / or the flow rate is 40-60 mL / min; and / or the column temperature is 24-26°C.

[0013] Furthermore, in step S2, the volume percentage of trifluoroacetic acid in the trifluoroacetic acid solution is 0.08-0.18%; and / or, the solvent in the trifluoroacetic acid solution is water.

[0014] Furthermore, the purification process of semaglutide also includes the steps of converting the semaglutide in the semaglutide-containing eluate after the second purification and drying the semaglutide.

[0015] Furthermore, the salt conversion step includes using an aqueous acetic acid solution as a mobile phase A1, an ammonium acetate solution as a mobile phase A2, and acetonitrile as a mobile phase B, and using a reverse phase chromatography method according to the following elution gradient to convert the semaglutide in the eluate containing semaglutide after the second purification and collect the eluate after the salt conversion;

[0016]

[0017] Furthermore, the filler of the chromatographic column used in the salt conversion step is octadecylsilane bonded silica gel, with a specification of 50×250 mm, 10 μm; and / or, the flow rate is 40-60 mL / min; and / or, the column temperature is 24-26°C.

[0018] Furthermore, the volume percentage of acetic acid in the acetic acid aqueous solution is 0.15-0.25%; and / or the concentration of the ammonium acetate aqueous solution is 9.8-10.2 mM.

[0019] Furthermore, the drying temperature is -20°C to -18°C, and the drying time is 45-50 hours.

[0020] A system used to describe GLP-1 analogs; wherein [Arg34]GLP-1(9-37) indicates a naturally occurring GLP-1(9-37) analog in which lysine at position 34 is replaced by arginine.

[0021] The technical solution of the present invention has the following beneficial effects:

[0022] (1) A purification process of semaglutide provided by the present invention comprises: S1, using ammonium acetate solution as mobile phase A, using acetonitrile as mobile phase B, using reverse phase chromatography to purify the solution containing semaglutide for the first time according to the elution gradient, and collecting the purified eluent; S2, using trifluoroacetic acid solution as mobile phase A, using acetonitrile as mobile phase B, using reverse phase chromatography to purify the eluent obtained in step S1 for the second time according to the elution gradient, and collecting the purified eluent containing semaglutide. The present invention adopts a specific elution step for the solution containing semaglutide, that is, first using ammonium acetate solution as mobile phase A, using acetonitrile as mobile phase B, using reverse phase chromatography to purify for the first time, and then using trifluoroacetic acid solution as mobile phase A, using acetonitrile as mobile phase B, using reverse phase chromatography to purify for the second time, which not only improves the yield of semaglutide, but also improves the purity.

[0023] (2) The present invention provides a purification process for semaglutide, wherein the semaglutide in the semaglutide-containing eluate after the second purification is subjected to salt conversion and drying to prepare semaglutide acetate with better stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1It is the chromatogram of the test solution corresponding to the eluent B after the second purification in Example 1 of Experimental Example 1 of the present invention.

[0026] Figure 2 It is the chromatogram of the test solution corresponding to the eluent B after the second purification of Comparative Example 1 in Experimental Example 1 of the present invention.

[0027] Figure 3 It is a chromatogram of the test solution corresponding to the eluent B after the second purification in Comparative Example 2 in Example 1 of the present invention. DETAILED DESCRIPTION

[0028] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0029] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0030] Table 1 Abbreviations corresponding to their full names

[0031] abbreviation Full name DIC N,N'-Diisopropylcarbodiimide HOSU N-Hydroxysuccinimide DIEA N,N-Diisopropylethylamine O N-Succinimidyl carbonate His Histidine Aib 2-Aminoisobutyric acid Trt Trityl DMF N,N-Dimethylformamide MTBE Methyl tert-butyl ether TFA Trifluoroacetic acid Tis Triisopropylsilane C18 Octadecylsilane bonded silica gel

[0032] The crude semaglutide and semaglutide-containing solutions involved in the following examples, comparative examples and experimental examples were prepared by the following method: (the amount of compound 1 was used as the synthesis scale in step S1 of the following examples and comparative examples; the amount of [Arg34]GLP-1(9-37) was used as the synthesis scale in step S2; the amount of compound 2 was used as the synthesis scale in steps S3-S5):

[0033] S1. Preparation of semaglutide side chain activated ester (synthesis scale 100 mmol): Compound 1 (100 mmol) and DIC (110 mmol) were dissolved in 400 mL of dichloromethane, stirred at 3 °C for 1 h, HOSU (110 mmol) was added to the above solution, reacted at 0 °C for 18 h and then filtered, the filtrate was washed twice with H2O (200 mL), the organic phase was dried with Na2SO4, and then vacuum concentrated to dry the water to obtain 93.2 g of semaglutide side chain activated ester.

[0034] S2. Preparation of compound 2 (synthesis scale 40 mmol): DIEA was added to a mixed solvent of acetonitrile: water = 1: 1.5 (2800 ml: 4200 ml) and the pH was adjusted to 10.5 to obtain solvent 2. [Arg34] GLP-1 (9-37) (40 mmol) was then slowly added in an ice-water bath; a solution containing the side chain activated ester of semaglutide (40 mmol) (solvent: acetonitrile, 1400 ml) was then slowly added and the reaction was stirred at 5° C. for 2 0h later, the pH was adjusted to 5.2 with glacial acetic acid, and then purified water with a volume 4 times that of solvent 2 was added, mixed and centrifuged, the solid after centrifugation was taken, acetonitrile was added to slurry twice, filtered, and the filtered solid was dried; the dried solid was then dissolved with acetonitrile: water = 1: 1 (V: V) to prepare a sample solution with a concentration of 1 mg / ml, and the sample solution was then mixed with mobile phase A of 0.1% phosphoric acid aqueous solution (V: V), mobile phase B of acetonitrile, and eluted with an elution column (filler C18, particle size 10 μm, pore size ) was eluted according to the elution gradient in Table 2 at a flow rate of 800 mL / min, and the eluted effluent was lyophilized to obtain 132 g of compound 2.

[0035] Table 2 Elution gradient

[0036] time Volume percentage of mobile phase A Volume percentage of mobile phase B 0~10min 95％ 5％ 10~10.1min 95→90％ 5％→10％ 10.1~70.1min 90％→35％ 10％→65％

[0037] S3. Preparation of compound 3 (synthesis scale 20 mmol): Compound 2 (20 mmol) and Boc-His(Trt)-Aib-OSu (30 mmol) were dissolved in 400 mL of DMF, and 7 mL (40 mmol) of DIEA was added. The mixture was stirred at 18°C ​​for 20 h for condensation reaction. After the reaction was completed, acetic acid was added to adjust the pH to 5.0, and then water with a volume 5 times that of DMF was added and stirred for 1 h. The mixture was centrifuged. 600 mL of acetonitrile was added to the target precipitate after centrifugation, and the mixture was slurried twice. The mixture was centrifuged again, and the solid after centrifugation was vacuum dried to obtain 96.3 g of compound 3.

[0038] S4. Preparation of crude semaglutide: 963 mL of lysis solution (TFA: Tis: H2O = 95: 2.5: 2.5, 5°C) was added to 96.3 g of compound 3 obtained in step S3, and the reaction was stirred at room temperature for 3 h, and the reaction product was added to 4000 mL of pre-cooled (-4°C) MTBE for precipitation, centrifuged, and the centrifuged precipitate was washed twice with 1500 mL of MTBE, centrifuged again, and the target precipitate after centrifugation was vacuum dried to obtain 83.5 g of crude semaglutide. The crude semaglutide was dissolved in an acetonitrile aqueous solution with a volume percentage concentration of 20% to prepare a solution containing semaglutide with a concentration of 13 mg / mL.

[0039] Compound 1 is (S)-22-(tert-butyloxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxo-3,6,12,15,42-pentaoxa-9,18,23-triazatetradecanoic acid.

[0040] The structure of the side chain activated ester of semaglutide is:

[0041]

[0042] The structure of compound 2:

[0043]

[0044] The structure of compound 3:

[0045]

[0046] Example 1

[0047] This embodiment provides a purification process of semaglutide, comprising the following steps:

[0048] S1. First purification: The solution containing semaglutide was passed through a C18 chromatographic column ( 10μm, 50×250mm), with ammonium acetate solution (solvent: water) with a mass percentage of 0.2% ammonium acetate as mobile phase A, and acetonitrile as mobile phase B, at a flow rate of 50mL / min and a column temperature of 25°C, the first purification was carried out according to the gradient in Table 3, and the detection wavelength was 220nm, and the eluate A containing semaglutide was collected after the main peak appeared.

[0049] Table 3 First purification gradient

[0050] Time / min Volume percentage of mobile phase A / % Volume percentage of mobile phase B / % 0～3 80 20 3～3.01 80→75 20→25 3.01～43 75→45 25→55 43～63 45→30 55→70

[0051] S2, second purification: eluent A was passed through a C18 chromatographic column ( 10μm, 50×250mm), with 0.15% trifluoroacetic acid solution (solvent water, V:V) as mobile phase A, acetonitrile as mobile phase B, at a flow rate of 50mL / min, a column temperature of 25°C, a second purification was performed according to the gradient in Table 4, and the detection wavelength was 220nm, and the eluate B containing semaglutide was collected after the main peak appeared.

[0052] Table 4 Second purification gradient

[0053] Time / min Volume percentage of mobile phase A / % Volume percentage of mobile phase B / % 0～3 80 20 3～3.01 80→65 20→35 3.01～43 65→45 35→55 43～63 45→30 55→70

[0054] S3, salt transfer: The semaglutide-containing eluate B obtained in step S3 was passed through a C18 chromatographic column ( 10μm, 50×250mm), 0.2% acetic acid aqueous solution (V:V) as mobile phase A1, 10mM ammonium acetate aqueous solution as mobile phase A2, acetonitrile as mobile phase B, semaglutide was trans-salted according to the gradient in Table 5 at a flow rate of 50mL / min and a column temperature of 25°C, and the eluate containing semaglutide acetate was collected at a detection wavelength of 220nm after the main peak appeared.

[0055] Table 5 Elution gradient - salt transfer

[0056]

[0057]

[0058] S4. Drying: The eluate containing semaglutide acetate obtained in step S4 is dried at -20°C for 48 hours to obtain pure semaglutide acetate.

[0059] Comparative Example 1

[0060] This comparative example provides a purification process for semaglutide. The process parameters differ from those in Example 1 only in that the mobile phases in steps S1 and S2 are different. In step S1 of this comparative example, 0.1% trifluoroacetic acid solution (solvent water, V:V) is used as mobile phase A; in step S2, 0.1% trifluoroacetic acid solution (solvent water, V:V) is used as mobile phase A.

[0061] Comparative Example 2

[0062] This comparative example provides a purification process for semaglutide. The process parameters differ from those in Example 1 only in that the mobile phases in steps S1 and S2 are different. In step S1 of this comparative example, 0.15% trifluoroacetic acid solution (solvent water, V:V) is used as mobile phase A; in step S2, 0.15% trifluoroacetic acid solution (solvent water, V:V) is used as mobile phase A.

[0063] Experimental Example 1

[0064] Pretreatment: Purify the crude semaglutide according to the purification process of Example 1 and Comparative Examples 1-2 and collect eluent A, eluent B, and eluent containing semaglutide acetate respectively. Eluent A, eluent B, and eluent containing semaglutide acetate are freeze-dried by a freeze dryer to prepare solids, which are weighed and recorded as M1, M2, and M3 respectively.

[0065] 1. Purity detection

[0066] Preparation of test solution: The pure semaglutide acetate prepared in Example 1 and Comparative Examples 1-2 and the solid obtained by the above pretreatment were dissolved in 20% acetonitrile aqueous solution (V:V) to prepare a 16.8 mg / ml test solution.

[0067] The HPLC conditions used for purity testing are shown in Table 6.

[0068] Table 6 Test conditions

[0069]

[0070]

[0071] The purity of the chromatogram information collected by high performance liquid chromatography was calculated by area normalization method. The purity results are shown in Table 7 below. Figure 1-3 As shown, compared with Comparative Examples 1-2, the purity of the purification process and the purified product in Example 1 is higher.

[0072] Table 7 Test results - Purity

[0073] sample Example 1 Comparative Example 1 Comparative Example 2 Semaglutide crude product 57.68％ 57.68％ 57.68％ Solid obtained by pretreatment with eluent A 90.23％ 85％ 87.64％ Solid obtained by pretreatment with eluent B 99.556％ 95.718％ 96.628％ Solid obtained by pretreatment of the eluate containing semaglutide acetate 99.685％ 96.653％ 97.267％ Pure semaglutide acetate 99.670％ 96.85％ 97.30％

[0074] 2. Concentration detection

[0075] Preparation of test solution: The semaglutide acetate prepared in Example 1 and Comparative Examples 1-2 and the solid obtained by the above pretreatment were dissolved in 20% acetonitrile aqueous solution (V:V) to prepare a 16.8 mg / ml test solution.

[0076] Preparation of semaglutide reference solution: dissolve semaglutide standard in 20% acetonitrile-water solvent (V:V) to prepare a 1 mg / ml standard solution.

[0077] Concentration detection method: Take the standard solution, and after elution by Shimadzu analytical instrument according to the following elution procedure (Table 8), obtain semaglutide peak 1; take the test solution, and after elution by Shimadzu analytical instrument according to the following elution procedure, obtain semaglutide peak 2; record the peak areas of peak 1 and peak 2, and calculate the concentration according to the following formula:

[0078] The concentration of the test solution = the peak area of ​​Peak 2 × the concentration of the standard solution (1 mg / ml) / the peak area of ​​Peak 1.

[0079] Table 8 Gradient elution program

[0080]

[0081] The concentration test results are shown in Table 9. Compared with Comparative Examples 1-2, the concentration of the product during the purification process of Example 1 is higher.

[0082] Table 9 Test results - concentration mg / mL

[0083] sample Example 1 Comparative Example 1 Comparative Example 2 Test solution - solution containing semaglutide 13 13 13 Test solution - eluate A after the first purification 14.8 13.9 14.2 Test solution - eluate B after the second purification 15.6 14.1 14.7 Test solution - eluate containing semaglutide acetate 16.7 14.4 14.8 Test solution-semaglutide acetate 16.8 14.5 14.9

[0084] 3. Yield calculation

[0085] The yield of the purification process of Example 1 and Comparative Examples 1-2 was calculated, and the yield calculation formula is as follows:

[0086] Eluent A: yield = M1 / weight of crude semaglutide × 100%;

[0087] Eluent B: yield = M2 / M1 × 100%;

[0088] Semaglutide acetate: total yield = weight of semaglutide acetate / weight of crude semaglutide × 100%.

[0089] The calculation results of the yield are shown in Table 10. Compared with Comparative Examples 1-2, the yield of the product in the purification process of Example 1 is higher.

[0090] Table 10 Calculation results - yield %

[0091]

[0092] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A purification process for semaglutide, characterized in that: The following steps are involved: S1. Using ammonium acetate solution as mobile phase A and acetonitrile as mobile phase B, purify the semaglutide-containing solution for the first time by reverse phase chromatography according to the following elution gradient, and collect the purified eluate; S2, using trifluoroacetic acid solution as mobile phase A and acetonitrile as mobile phase B, using reverse phase chromatography according to the following elution gradient to purify the eluate obtained in step S1 for a second time, and collecting the purified eluate containing semaglutide; 。 2. The purification process of semaglutide according to claim 1, characterized in that: In step S1, the mass percentage of ammonium acetate in the ammonium acetate solution is 0.1-1.5%; and / or the solvent in the ammonium acetate solution is water.

3. The purification process of semaglutide according to claim 1, characterized in that: In step S1, the solvent in the solution containing semaglutide is selected from one or more of acetonitrile, methanol, formic acid, and water; and / or the concentration of the solution containing semaglutide is 10-14 mg / mL.

4. The purification process of semaglutide according to any one of claims 1 to 3, characterized in that: In step S1 or step S2, the filler of the chromatographic column is octadecylsilane bonded silica gel with a specification of 50×250 mm, 10 μm; and / or the flow rate is 40-60 mL / min; and / or the column temperature is 24-26° C.

5. The purification process of semaglutide according to any one of claims 1 to 4, characterized in that: In step S2, the volume percentage of trifluoroacetic acid in the trifluoroacetic acid solution is 0.08-0.18%; and / or the solvent in the trifluoroacetic acid solution is water.

6. The purification process of semaglutide according to any one of claims 1 to 5, characterized in that: The purification process of semaglutide further comprises the steps of converting salt and drying the semaglutide in the semaglutide-containing eluate after the second purification.

7. The purification process of semaglutide according to claim 6, characterized in that: The salt conversion step comprises: using acetic acid aqueous solution as mobile phase A1, ammonium acetate aqueous solution as mobile phase A2, and acetonitrile as mobile phase B, using reverse phase chromatography according to the following elution gradient to convert semaglutide in the semaglutide-containing eluate after the second purification, and collecting the eluate after salt conversion; 8. The purification process of semaglutide according to claim 7, characterized in that: The filler of the chromatographic column used in the salt conversion step is octadecylsilane bonded silica gel, with a specification of 50×250 mm, 10 μm; and / or, the flow rate is 40-60 mL / min; and / or, the column temperature is 24-26° C.

9. The purification process of semaglutide according to claim 7, characterized in that: The volume percentage of acetic acid in the acetic acid aqueous solution is 0.15-0.25%; and / or the concentration of the ammonium acetate aqueous solution is 9.8-10.2 mM.

10. The purification process of semaglutide according to claim 6, characterized in that: The drying temperature is -20°C to -18°C, and the drying time is 45-50 hours.

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