A method for separating and purifying liraglutide using reversed-phase chromatography

Abstract

This invention discloses a method for purifying liraglutide using reversed-phase chromatography, belonging to peptide chromatography separation technology in the field of biochemical engineering. The specific steps are as follows: 1) Preparation of crude liraglutide solution; 2) Loading the crude liraglutide solution onto an octadecylsilane-bonded silica gel medium at pH 7.0-9.0, followed by gradient elution and collection of the first eluent; 3) Diluting the first eluent and adding an adjusting agent to obtain a diluted solution; 4) Loading the diluted solution onto the same medium as in the first step of reversed-phase chromatography at pH 6.0-8.0, followed by gradient elution and collection of the second eluent, yielding liraglutide with a purity of over 99% and a total yield of over 80% for both steps. This invention uses only one medium and achieves high-purity liraglutide through two-step reversed-phase chromatography at room temperature and pressure, featuring simple operation, high yield, and easy scale-up.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide separation and purification technology, and relates to a method for separating and purifying liraglutide by reverse phase chromatography. Background Technology

[0002] Liraglutide is a glucagon-like peptide-1 (GLP-1) analog. Based on the natural GLP-1 structure, amino acid substitutions and fatty acid modifications extend its half-life. Liraglutide's main indications are type 2 diabetes and obesity, offering advantages such as blood glucose control, delayed gastric emptying, cardiovascular improvement, and no risk of hypoglycemia.

[0003] During the production and storage of liraglutide, it is susceptible to hydrolysis, oxidation, racemization, and rearrangement reactions due to the influence of oxygen, light, and temperature. These reactions can easily generate structurally similar impurities such as deleted peptides, inserted peptides, mismatched peptides, and deviated peptides, affecting the efficacy and safety of the drug. Therefore, removing these impurities is crucial, and reversed-phase chromatography is a key separation and purification step. Chinese patent CN103275208 discloses a two-step reversed-phase chromatography method for purifying liraglutide. The first step uses octadecylsilane-bonded silica gel as the medium, with 0.1% trifluoroacetic acid / water and 0.1% trifluoroacetic acid / acetonitrile solutions as the elution mobile phase. The second step uses another octadecylsilane-bonded silica gel as the medium, with 1% acetic acid and acetonitrile as the elution mobile phase. The resulting liraglutide purity is 98.6%, with an overall yield of only 20.2% for both steps. Chinese patent CN109503705 also employs a two-step reversed-phase chromatography method to purify liraglutide, using two different media: polymethyl methacrylate polymer and octaalkylsilane-bonded silica gel. In the first step of reversed-phase chromatography, the elution mobile phase A is 30 mM sodium carbonate solution (pH 9.0), and the elution mobile phase B is acetonitrile. In the second step of reversed-phase chromatography, the elution mobile phase A is 90 mM sodium sulfate solution (pH 3.2), and the elution mobile phase B is acetonitrile. The purity of liraglutide is 99.5%, but the yield is only 64.8%. US Patent US20170283478 discloses a two-step reversed-phase chromatography method for separating liraglutide. The first step uses octadecylsilane-bonded silica gel with a particle size of 15 micrometers, with 10 mM Tris (pH 8.0) as the elution mobile phase A and acetonitrile as the elution mobile phase B. The second step uses octadecylsilane-bonded silica gel with a particle size of 5 micrometers, with 0.1% trifluoroacetic acid aqueous solution as the elution mobile phase A and acetonitrile as the elution mobile phase B. The purity of liraglutide is 97.1%, and the yield is only 21.6%. European patent EP2813514 discloses a three-step reversed-phase chromatography method for separating and purifying liraglutide. The first step uses octylsilane-bonded silica gel as the elution mobile phase A, which is a 0.1% trifluoroacetic acid in isopropanol aqueous solution, and the second step uses cyanosilane-bonded silica gel as the elution mobile phase A, which is a 0.15% perchloric acid solution, and the third step uses octylsilane-bonded silica gel with smaller particle size, which is a 0.04% ammonia solution and the fourth step uses acetonitrile. Despite using different media and mobile phases in these three steps, the purity of liraglutide is only 98.7%.

[0004] Among the aforementioned techniques, reversed-phase chromatography achieves a maximum purity of 99.5% for liraglutide, but the yield is only 64.8%. Furthermore, all techniques utilize two or more different media, and industrial production typically requires two chromatography columns and two chromatography systems, increasing equipment costs and floor space requirements.

[0005] Chinese patent CN113024658 discloses a method for purifying liraglutide using reversed-phase chromatography with an octadecylsilane-bonded silica gel medium. The method initially uses 200 mM ammonium bicarbonate and 50 mM sodium bicarbonate (pH 7.5) as elution mobile phase A, and acetonitrile as elution mobile phase B. Then, by switching elution mobile phase A to 200 mM ammonium bicarbonate and 50 mM sodium bicarbonate (pH 9.5), the purity of liraglutide reaches 99.4%, but the yield is only 58.6%. Although this method uses a single medium for chromatographic separation, it requires strict control of pressure and temperature. To prevent carbon dioxide escape and pH fluctuations, the mobile phase is kept in a sealed pressure vessel at a specific pressure. Furthermore, elution mobile phase A uses two salts with different column temperatures, requiring precise temperature control to ensure high resolution and system stability. The operation is complex and not conducive to industrial application.

[0006] In summary, most current reversed-phase chromatography purification methods for liraglutide use two or more media. However, due to insufficient attention to the conformational and structural characteristics of liraglutide, the influence of acid-base environment, and the isoelectric point characteristics of liraglutide, problems such as the inability to simultaneously achieve high levels of purity and yield of the target product, complex processes, and cumbersome operations exist. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention proposes a method for purifying liraglutide using reversed-phase chromatography. This method fully considers the conformational regulation mechanism of liraglutide, using only one chromatographic medium and achieving high-purity liraglutide purification and separation through two steps of reversed-phase chromatography at room temperature and pressure.

[0008] This invention addresses the generally low yield of liraglutide separation using reversed-phase chromatography. The main reasons for this are: 1. Liraglutide contains a long-chain C16 palmitoyl group, exhibiting strong hydrophobicity. Existing techniques often lack effective conformational protection during the chromatographic gap or elution process, leading to easy intermolecular aggregation of liraglutide, forming difficult-to-elute polymers that remain trapped in the column, thus reducing yield. 2. Some existing techniques use strong acids (such as 0.15% perchloric acid or 0.1% trifluoroacetic acid) or strong bases (pH above 9.5) as the mobile phase. Strong acid environments easily induce aggregation, while strong base environments cause liraglutide to carry excessive charge, disrupting its spontaneously formed stable conformation. Furthermore, under strong base conditions, liraglutide is easily degraded, producing hydrophobic impurities. 3. Liraglutide has an isoelectric point of 4.9. In existing techniques, if the pH is not properly adjusted or the salt concentration fluctuates drastically during the two-step chromatographic switching process, liraglutide is easily precipitated.

[0009] Based on the technical problem to be solved by this invention and the above understanding, the technical solution of this invention is as follows:

[0010] This invention provides a method for separating and purifying liraglutide using reversed-phase chromatography, comprising the following steps:

[0011] 1) Dissolve crude liraglutide in a salt solution containing acetonitrile, and filter the solution through a filter membrane to obtain a crude liraglutide solution;

[0012] 2) Load the crude liraglutide solution into a chromatography column packed with reversed-phase chromatography medium and perform the first step of separation and purification under normal pressure. Elution is performed using a binary mobile phase linear gradient elution. Mobile phase A is a salt solution with a pH of 7.0-9.0, and mobile phase B is acetonitrile. Collect the elution peak to obtain the first eluent.

[0013] 3) Dilute the first eluent with water and add a regulator with a polyhydroxy structure;

[0014] 4) The diluted first eluent obtained in step 3) is loaded onto a chromatography column containing the same reversed-phase chromatography medium as in step 2), and the second separation and purification is carried out under normal pressure. Elution is performed using a binary mobile phase linear gradient elution, where mobile phase A is a salt solution with a pH of 6.0-8.0 and mobile phase B is acetonitrile. The elution peak is collected to obtain the second eluent, which is high-purity liraglutide.

[0015] According to a preferred embodiment of the present invention, the purity of crude liraglutide in step 1) ranges from 75.0 wt% to 90.0 wt%.

[0016] According to a preferred embodiment of the present invention, in step 1), the pH of the acetonitrile-containing salt solution is 7.0-9.0, the volume percentage of acetonitrile in the salt solution is 5%-15%, the mass ratio of liraglutide to acetonitrile is 0.15-0.5:1, and the pore size of the filter membrane is 0.22 µm.

[0017] According to a preferred embodiment of the present invention, in steps 2) and 4), the reverse phase chromatography medium is an alkylsilane-bonded silica gel medium with an alkyl carbon chain length of 8 or 18, a medium particle size of 10 µm, and a medium pore size of 120 Å.

[0018] According to a preferred embodiment of the present invention, in steps 2) and 4), the column temperature is 20-30°C and the operating flow rate is 100-300 cm / h.

[0019] According to a preferred embodiment of the present invention, the salt solution in steps 1) and 2) is a solution prepared by any one of the following salts: ammonium acetate, sodium acetate, potassium acetate, ammonium sulfate, ammonium chloride, diammonium hydrogen phosphate, dipotassium hydrogen phosphate, and disodium hydrogen phosphate, with a salt concentration of 10-110 mM and the pH value adjusted to 7.0-9.0.

[0020] According to a preferred embodiment of the present invention, the mobile phase B in step 2) is pure acetonitrile. During the linear gradient elution process, the proportion of mobile phase B is gradually increased. When the absorbance rises to the threshold in the range of 300-400 mAU, collection begins, and when the absorbance drops to the threshold, collection stops. The elution length is 5-20 column volumes. The threshold in the range of 300-400 mAU can be selected from values ​​within the range or from the endpoints.

[0021] According to a preferred embodiment of the present invention, in step 3), the regulator is one of polyethylene glycol, glycerin, and sorbitol, wherein the molecular weight of polyethylene glycol is 200-1000 Da; the volume percentage of the regulator in the diluent is 0.5%~1.0%; and the mass ratio of liraglutide to the regulator is 1~7:1.

[0022] According to a preferred embodiment of the present invention, the salt solution in step 4) is a solution prepared by any one of dipotassium hydrogen phosphate or disodium hydrogen phosphate, with a concentration of 10-110 mM, and the pH value is adjusted to 6.0-8.0; or it is a solution prepared by any one of sulfuric acid, acetic acid, formic acid, or phosphoric acid, with an acid volume percentage of 0.1%-1%, and the pH value is adjusted to 6.0-8.0.

[0023] According to a preferred embodiment of the present invention, the mobile phase B in step 4) is pure acetonitrile. During the linear gradient elution process, the proportion of mobile phase B is increased. When the absorbance rises to the threshold in the range of 400-500 mAU, collection begins, and when the absorbance drops to the threshold, collection stops. The elution length is 5-20 column volumes. The threshold in the range of 400-500 mAU can be selected from values ​​within the range or from the endpoints.

[0024] Compared with the prior art, the beneficial effects of the present invention include:

[0025] 1) This invention discovers the "conformation regulation" mechanism of liraglutide molecules. A regulator is added to the first eluent diluent. The added regulator has a polyhydroxy structure and wraps around the peptide backbone of liraglutide through hydrogen bonds, forming a hydrated protective layer around the C16 fatty chain of liraglutide, preventing liraglutide from agglomerating due to hydrophobic interactions of the fatty chain between the two chromatography steps. Furthermore, the composite structure of liraglutide and the regulator ensures that liraglutide maintains a suitable conformation in the second reversed-phase chromatography step, enhancing the difference from impurity molecules and thus improving separation. The regulator is hydrophilic and can be washed away by the mobile phase in the second reversed-phase chromatography step without remaining on the medium.

[0026] 2) The liraglutide produced by the method of this invention has high purity and yield. Both reversed-phase chromatography steps optimize the environment between the medium and the liraglutide molecule surface through precise control and synergistic effect of single-salt systems with different pH and salt concentrations, achieving a purity of over 99% and increasing the overall yield of the two reversed-phase chromatography steps to over 80%.

[0027] 3) This invention employs a two-step reversed-phase chromatography separation method using the same medium. Both reversed-phase chromatography steps use octadecylsilane-bonded silica gel media, which is simple, reduces costs and increases efficiency, and is suitable for large-scale industrial production.

[0028] 4) By adding a trace amount of acetonitrile during dissolution, this invention achieves conformational pre-regulation of liraglutide, inducing the appropriate unfolding of its hydrophobic long fatty acyl chain, improving mass transfer and adsorption efficiency, and thus enhancing the stability of the loading stage.

[0029] 5) Both reversed-phase chromatography steps of the present invention are carried out under neutral or weakly acidic / weakly alkaline conditions, which avoids the problems of liraglutide agglomerating due to excessively low pH or degrading to generate hydrophobic impurities due to excessively high pH, ​​as well as the problem that liraglutide is easily precipitated at its isoelectric point due to rapid pH changes.

[0030] 6) The two-step reversed-phase chromatography of the present invention is carried out under room temperature and atmospheric pressure conditions, which has good tolerance to temperature fluctuations, eliminates the need to control column temperature, reduces energy consumption and equipment requirements, and is conducive to industrial production.

[0031] 7) Neither of the two reversed-phase chromatography steps of this invention uses carbonates, eliminating the need for pressure tanks to control the pressure of the mobile phase and preventing drastic pH fluctuations caused by carbon dioxide escaping during desalting or freeze-drying of liraglutide, which could lead to liraglutide precipitation. Attached Figure Description

[0032] Figure 1 This is a comparison of HPLC analysis results of liraglutide after two-step reversed-phase chromatography purification. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention. The scope of protection of the present invention is not limited to the following embodiments.

[0034] The method of this invention does not limit the source or preparation method of crude liraglutide. Crude liraglutide can be produced by full fermentation, semi-fermentation and semi-synthesis, or full chemical synthesis. The purity of crude liraglutide is typically in the range of 75.0 wt% to 90.0 wt%. The crude liraglutide used in this embodiment was obtained by semi-fermentation and semi-synthesis.

[0035] Example 1:

[0036] (1) Sample preparation: Weigh 150 mg of crude liraglutide powder (purity about 83.1 wt%), add 5 mL of 80 mM ammonium acetate solution (pH 9.0) containing 10% (v / v) acetonitrile, filter with a 0.22 micron filter membrane to obtain crude liraglutide solution.

[0037] (2) First step reversed phase chromatography: The crude liraglutide solution was passed through Unisil medium packed with alkylsilane-bonded silica gel. ® Purification was performed using a Revo C8 10-120 chromatography column (alkyl carbon chain length of 8), with a column volume of 4.15 mL. Elution mobile phase A was 80 mM ammonium acetate solution (pH 8.0), and elution mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 180 cm / h, and the column temperature was 25℃. The specific separation process was as follows: Equilibrate with 20% B for 2 column volumes, then load the crude liraglutide solution at a volume of 15.78 mg / mL; wash with 20% B for 2 column volumes, then wash with a linear gradient of 20%-40% B for 2 column volumes, and elute with a linear gradient of 40%-50% B for 12 column volumes. Elution began when the absorbance reached 400 mAU and stopped when the absorbance decreased to 400 mAU, yielding eluent 1; finally, regenerate with acetonitrile:water = 80:20 (v / v) solution for 2 column volumes. The purity of liraglutide in eluent 1 was 98.5%, and the yield was 90.1%.

[0038] (3) Dilute eluent 1 with water by 2 times and add 112 mg of polyethylene glycol (200 Da).

[0039] (4) Second step reversed phase chromatography: Load the diluted eluent 1 onto a container containing the same medium Unisil ® The Revo C810-120 chromatography column was used for separation and purification, with a column volume of 4.15 mL. Elution mobile phase A was 80 mM dipotassium hydrogen phosphate solution (pH 7.0), and mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 250 cm / h, and the column temperature was 25℃. The specific separation process was as follows: equilibration with 20% B for 2 column volumes; loading of a diluted eluent 1 at a volume of 14.13 mg / mL; washing with 20% B for 2 column volumes, followed by a linear gradient wash of 20% B-40% B for 2 column volumes, and then eluting with a linear gradient of 40% B-50% B for 15 column volumes; collection began when the absorbance reached 500 mAU and stopped when the absorbance decreased to 500 mAU, yielding eluent 2; finally, regeneration was achieved with an acetonitrile:water solution of 80:20 (v / v) for 2 column volumes. The purity of liraglutide in eluent 2 was 99.4%, and the overall yield of the two-step reversed-phase chromatography was 80.2%. Figure 1 This is a comparison of HPLC chromatograms of liraglutide after two-step reversed-phase chromatography purification.

[0040] Example 2:

[0041] (1) Sample preparation: Weigh 150 mg of crude liraglutide powder (purity about 75.0 wt%), add 5 mL of 110 mM ammonium sulfate solution (pH 9.0) containing 5% (v / v) acetonitrile to dissolve, filter with a 0.22 micron filter membrane to obtain crude liraglutide solution.

[0042] (2) First step reversed phase chromatography: The crude liraglutide solution was passed through Unisil medium packed with alkylsilane-bonded silica gel. ®Purification was performed using a Revo C8 10-120 chromatography column (alkyl carbon chain length of 8), with a column volume of 4.15 mL. Elution mobile phase A was 110 mM ammonium sulfate solution (pH 9.0), and elution mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 100 cm / h, and the column temperature was 20 °C. The specific separation process was as follows: Equilibrate with 20% B for 2 column volumes, then load the crude liraglutide solution at a volume of 15.06 mg / mL; wash with 20% B for 2 column volumes, followed by a linear gradient wash of 20% B-40% B for 2 column volumes, and then elute with a linear gradient of 40% B-50% B for 20 column volumes. Elution began when the absorbance reached 400 mAU and stopped when the absorbance decreased to 400 mAU, yielding eluent 1; finally, regenerate with acetonitrile:water = 80:20 (v / v) solution for 2 column volumes. The purity of liraglutide in eluent 1 was 98.1%, and the yield was 90.9%.

[0043] (3) Dilute eluent 1 with water twice and add 102 mg of glycerol.

[0044] (4) Second step reversed phase chromatography: Load the diluted eluent 1 onto a container containing the same medium Unisil ® The Revo C810-120 chromatography column was used for separation and purification, with a column volume of 4.15 mL. Elution mobile phase A was 80 mM disodium hydrogen phosphate solution (pH 6.0), and mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 100 cm / h, and the column temperature was 20℃. The specific separation process was as follows: equilibration with 20% B for 2 column volumes; loading of a diluted eluent 1 at a sample volume of 13.83 mg / mL column volume; washing with 20% B for 2 column volumes, followed by a linear gradient wash of 20% B-40% B for 2 column volumes, and then eluting with a linear gradient of 40% B-50% B for 20 column volumes; collection began when the absorbance rose to 400 mAU and stopped when the absorbance decreased to 400 mAU, yielding eluent 2; finally, regeneration was achieved with an acetonitrile:water = 80:20 (v / v) solution for 2 column volumes. The purity of liraglutide in eluent 2 was 99.3%, and the overall yield of the two-step reversed-phase chromatography was 80.9%.

[0045] Example 3:

[0046] (1) Sample preparation: Weigh 150 mg of crude liraglutide powder (purity approximately 90.0 wt%), add 5 mL of 10 mM ammonium chloride solution (pH 9.0) containing 15% (v / v) acetonitrile to dissolve, filter with a 0.22 micron filter membrane to obtain crude liraglutide solution.

[0047] (2) First step reversed phase chromatography: The crude liraglutide solution was passed through Unisil medium packed with alkylsilane-bonded silica gel. ®Purification was performed using a Revo C18 10-120 chromatography column (alkyl carbon chain length 18), with a column volume of 4.15 mL. Elution mobile phase A was 10 mM ammonium chloride solution (pH 7.0), and elution mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 300 cm / h, and the column temperature was 30℃. The specific separation process was as follows: Equilibrate with 20% B for 2 column volumes, then load the crude liraglutide solution at a volume of 15.54 mg / mL; wash with 20% B for 2 column volumes, followed by a linear gradient wash of 20% B-40% B for 2 column volumes, and then elute with a linear gradient of 30% B-50% B for 20 column volumes. Elution began when the absorbance reached 300 mAU and stopped when the absorbance decreased to 300 mAU, yielding eluent 1; finally, regenerate with acetonitrile:water = 80:20 (v / v) solution for 2 column volumes. The purity of liraglutide in eluent 1 was 98.8%, and the yield was 88.7%.

[0048] (3) Dilute eluent 1 with water by 2 times and add 17 mg of polyethylene glycol (1000 Da).

[0049] (4) Second step reversed phase chromatography: Load the diluted eluent 1 onto a container containing the same medium Unisil ® The Revo C1810-120 chromatography column was used for separation and purification, with a column volume of 4.15 mL. Elution mobile phase A was 0.1% acetic acid solution (pH 6.0), and mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 300 cm / h, and the column temperature was 30℃. The specific separation process was as follows: equilibration with 20% B for 2 column volumes; loading of a diluted eluent 1 at a volume of 14.03 mg / mL; washing with 20% B for 2 column volumes, followed by a linear gradient wash of 20% B-40% B for 2 column volumes, and then eluting with a linear gradient of 30% B-50% B for 20 column volumes; collection began when the absorbance reached 400 mAU and stopped when the absorbance decreased to 400 mAU, yielding eluent 2; finally, regeneration was achieved with an acetonitrile:water solution of 80:20 (v / v) for 2 column volumes. The purity of liraglutide in eluent 2 was 99.3%, and the overall yield of the two-step reversed-phase chromatography was 80.1%.

[0050] Example 4:

[0051] (1) Sample preparation: Weigh 150 mg of crude liraglutide powder (purity about 85.6 wt%), add 5 mL of 60 mM dipotassium hydrogen phosphate solution (pH 9.0) containing 10% (v / v) acetonitrile to dissolve, filter with a 0.22 micron filter membrane to obtain crude liraglutide solution.

[0052] (2) First step reversed phase chromatography: The crude liraglutide solution was passed through Unisil medium packed with alkylsilane-bonded silica gel.® Purification was performed using a Revo C18 10-120 chromatography column (alkyl carbon chain length 18), with a column volume of 4.15 mL. Elution mobile phase A was 60 mM dipotassium hydrogen phosphate solution (pH 8.0), and elution mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 200 cm / h, and the column temperature was 25℃. The specific separation process was as follows: Equilibrate with 20% B for 2 column volumes, then load the crude liraglutide solution at a volume of 14.94 mg / mL; wash with 20% B for 2 column volumes, followed by a linear gradient wash of 20% B-40% B for 2 column volumes, and then elute with a linear gradient of 40% B-50% B for 15 column volumes. Elution began when the absorbance reached 300 mAU and stopped when the absorbance decreased to 300 mAU, yielding eluent 1; finally, regenerate with acetonitrile:water = 80:20 (v / v) solution for 2 column volumes. The purity of liraglutide in eluent 1 was 98.3%, and the yield was 89.9%.

[0053] (3) Dilute eluent 1 with water by 2 times and add 23 mg of sorbitol.

[0054] (4) Second step reversed phase chromatography: Load the diluted eluent 1 onto a container containing the same medium Unisil ® The Revo C1810-120 chromatography column was used for separation and purification, with a column volume of 4.15 mL. Elution mobile phase A was 0.1% sulfuric acid solution (pH 6.0), and mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 200 cm / h, and the column temperature was 20℃. The specific separation process was as follows: equilibration with 20% B for 2 column volumes; loading of a diluted eluent 1 at a volume of 13.59 mg / mL; washing with 20% B for 2 column volumes, followed by a linear gradient wash of 20% B-40% B for 2 column volumes, and then eluting with a linear gradient of 40% B-50% B for 15 column volumes; collection began when the absorbance reached 500 mAU and stopped when the absorbance decreased to 500 mAU, yielding eluent 2; finally, regeneration was achieved with an acetonitrile:water solution of 80:20 (v / v) for 2 column volumes. The purity of liraglutide in eluent 2 was 99.3%, and the overall yield of the two-step reversed-phase chromatography was 81.6%.

[0055] Example 5:

[0056] (1) Sample preparation: Weigh 150 mg of crude liraglutide powder (purity about 83.4 wt%), add 5 mL of 100 mM disodium hydrogen phosphate solution (pH 9.0) containing 10% (v / v) acetonitrile, filter with a 0.22 micron filter membrane to obtain crude liraglutide solution.

[0057] (2) First step reversed phase chromatography: The crude liraglutide solution was passed through Unisil medium packed with alkylsilane-bonded silica gel. ® Purification was performed using a Revo C8 10-120 chromatography column (alkyl carbon chain length of 8), with a column volume of 4.15 mL. Elution mobile phase A was 100 mM disodium hydrogen phosphate solution (pH 8.0), and elution mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 250 cm / h, and the column temperature was 20℃. The specific separation process was as follows: Equilibrate with 20% B for 2 column volumes, then load the crude liraglutide solution at a volume of 15.26 mg / mL; wash with 20% B for 2 column volumes, followed by a linear gradient wash of 20% B-40% B for 2 column volumes, and then elute with a linear gradient of 40% B-60% B for 20 column volumes. Elution began when the absorbance reached 300 mAU and stopped when the absorbance decreased to 300 mAU, yielding eluent 1; finally, regenerate with acetonitrile:water = 80:20 (v / v) solution for 2 column volumes. The purity of liraglutide in eluent 1 was 98.1%, and the yield was 91.6%.

[0058] (3) Dilute eluent 1 with water by 2 times and add 115 mg of polyethylene glycol (500 Da).

[0059] (4) Second step reversed phase chromatography: Load the diluted eluent 1 onto a container containing the same medium Unisil ® The Revo C810-120 chromatography column was used for separation and purification, with a column volume of 4.15 mL. Elution mobile phase A was 110 mM disodium hydrogen phosphate solution (pH 8.0), and mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 250 cm / h, and the column temperature was 20℃. The specific separation process was as follows: equilibration with 20% B for 2 column volumes; loading of a diluted eluent 1 at a volume of 14.19 mg / mL; washing with 20% B for 2 column volumes, followed by a linear gradient wash of 20% B-40% B for 2 column volumes, and then eluting with a linear gradient of 40% B-60% B for 20 column volumes; collection began when the absorbance reached 500 mAU and stopped when the absorbance decreased to 500 mAU, yielding eluent 2; finally, regeneration was achieved with an acetonitrile:water solution of 80:20 (v / v) for 2 column volumes. The purity of liraglutide in eluent 2 was 99.5%, and the overall yield of the two-step reversed-phase chromatography was 80.4%.

[0060] Example 6:

[0061] (1) Sample preparation: Weigh 150 mg of crude liraglutide powder (purity about 81.6 wt%), add 5 mL of 70 mM diammonium hydrogen phosphate solution (pH 9.0) containing 10% (v / v) acetonitrile, filter with a 0.22 micron filter membrane to obtain crude liraglutide solution.

[0062] (2) First step reversed phase chromatography: The crude liraglutide solution was passed through Unisil medium packed with alkylsilane-bonded silica gel. ® Purification was performed using a Revo C8 10-120 chromatography column (alkyl carbon chain length of 8), with a column volume of 4.15 mL. Elution mobile phase A was 70 mM diammonium hydrogen phosphate solution (pH 9.0), and elution mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 250 cm / h, and the column temperature was 30℃. The specific separation process was as follows: Equilibrate with 20% B for 2 column volumes, then load the crude liraglutide solution at a volume of 14.96 mg / mL; wash with 20% B for 2 column volumes, followed by a linear gradient wash of 20% B to 40% B for 2 column volumes, and then elute with a linear gradient of 40% B to 45% B for 5 column volumes. Elution began when the absorbance reached 400 mAU and stopped when the absorbance decreased to 400 mAU, yielding eluent 1; finally, regenerate with acetonitrile:water = 80:20 (v / v) solution for 2 column volumes. The purity of liraglutide in eluent 1 was 98.3%, and the yield was 88.7%.

[0063] (3) Dilute eluent 1 with water twice and add 58 mg of glycerol.

[0064] (4) Second step reversed phase chromatography: Load the diluted eluent 1 onto a container containing the same medium Unisil ® The Revo C810-120 chromatography column was used for separation and purification, with a column volume of 4.15 mL. Elution mobile phase A was 1.0% formic acid solution (pH 8.0), and mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 250 cm / h, and the column temperature was 30℃. The specific separation process was as follows: equilibration with 20% B for 2 column volumes; loading of a diluted eluent 1 at a volume of 14.19 mg / mL; washing with 20% B for 2 column volumes, followed by a linear gradient wash of 20% B-40% B for 2 column volumes, and then eluting with a linear gradient of 40% B-45% B for 5 column volumes; collection began when the absorbance reached 500 mAU and stopped when the absorbance decreased to 500 mAU, yielding eluent 2; finally, regeneration was achieved with an acetonitrile:water solution of 80:20 (v / v) for 2 column volumes. The purity of liraglutide in eluent 2 was 99.5%, and the overall yield of the two-step reversed-phase chromatography was 81.5%.

[0065] Example 7:

[0066] (1) Sample preparation: Weigh 150 mg of crude liraglutide powder (purity approximately 79.1 wt%), add 5 mL of 50 mM sodium acetate solution (pH 9.0) containing 10% (v / v) acetonitrile to dissolve, filter with a 0.22 micron filter membrane to obtain crude liraglutide solution.

[0067] (2) First step reversed phase chromatography: The crude liraglutide solution was passed through Unisil medium packed with alkylsilane-bonded silica gel. ® Purification was performed using a Revo C8 10-120 chromatography column (alkyl carbon chain length of 8), with a column volume of 4.15 mL. Elution mobile phase A was 50 mM sodium acetate solution (pH 9.0), and elution mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 300 cm / h, and the column temperature was 20℃. The specific separation process was as follows: Equilibrate with 20% B for 2 column volumes, then load the crude liraglutide solution at a volume of 15.46 mg / mL; wash with 20% B for 2 column volumes, followed by a linear gradient wash of 20% B-40% B for 2 column volumes, and then elute with a linear gradient of 40% B-50% B for 15 column volumes. Elution began when the absorbance reached 300 mAU and stopped when the absorbance decreased to 300 mAU, yielding eluent 1; finally, regenerate with acetonitrile:water = 80:20 (v / v) solution for 2 column volumes. The purity of liraglutide in eluent 1 was 98.0%, and the yield was 90.1%.

[0068] (3) Dilute eluent 1 with water by 2 times and add 150 mg of sorbitol.

[0069] (4) Second step reversed phase chromatography: Load the diluted eluent 1 onto a container containing the same medium Unisil ® The Revo C810-120 chromatography column was used for separation and purification, with a column volume of 4.15 mL. Elution mobile phase A was 0.5% phosphoric acid solution (pH 6.0), and mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 300 cm / h, and the column temperature was 20℃. The specific separation process was as follows: equilibration with 20% B for 2 column volumes; loading of a diluted eluent 1 at a volume of 14.59 mg / mL; washing with 20% B for 2 column volumes, followed by a linear gradient wash of 20% B-40% B for 2 column volumes, and then eluting with a linear gradient of 40% B-50% B for 15 column volumes; collection began when the absorbance reached 400 mAU and stopped when the absorbance decreased to 400 mAU, yielding eluent 2; finally, regeneration was achieved with an acetonitrile:water solution of 80:20 (v / v) for 2 column volumes. The purity of liraglutide in eluent 2 was 99.4%, and the overall yield of the two-step reversed-phase chromatography was 80.1%.

[0070] Example 8:

[0071] (1) Sample preparation: Weigh 150 mg of crude liraglutide powder (purity about 86.1 wt%), add 5 mL of 50 mM potassium acetate solution (pH 7.0) containing 10% (v / v) acetonitrile to dissolve, filter with a 0.22 micron filter membrane to obtain crude liraglutide solution.

[0072] (2) First step reversed phase chromatography: The crude liraglutide solution was passed through Unisil medium packed with alkylsilane-bonded silica gel. ® Purification was performed using a Revo C8 10-120 chromatography column (alkyl carbon chain length of 8), with a column volume of 4.15 mL. Elution mobile phase A was 50 mM potassium acetate solution (pH 9.0), and elution mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 300 cm / h, and the column temperature was 25℃. The specific separation process was as follows: Equilibrate with 20% B for 2 column volumes, then load the crude liraglutide solution at a volume of 15.06 mg / mL; wash with 20% B for 2 column volumes, then wash with a linear gradient of 20% B-40% B for 2 column volumes, and elute with a linear gradient of 40% B-50% B for 15 column volumes. Elution began when the absorbance reached 300 mAU and stopped when the absorbance decreased to 300 mAU, yielding eluent 1; finally, regenerate with acetonitrile:water = 80:20 (v / v) solution for 2 column volumes. The purity of liraglutide in eluent 1 was 98.6%, and the yield was 89.6%.

[0073] (3) Dilute eluent 1 with water by 2 times and add 30 mg of polyethylene glycol (600 Da).

[0074] (4) Second step reversed phase chromatography: Load the diluted eluent 1 onto a container containing the same medium Unisil ® The Revo C810-120 chromatography column was used for separation and purification, with a column volume of 4.15 mL. Elution mobile phase A was 10 mM dipotassium hydrogen phosphate solution (pH 7.0), and mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 300 cm / h, and the column temperature was 25℃. The specific separation process was as follows: equilibration with 20% B for 2 column volumes; loading of a diluted eluent 1 at a volume of 14.19 mg / mL; washing with 20% B for 2 column volumes, followed by a linear gradient wash of 20% B-40% B for 2 column volumes, and then eluting with a linear gradient of 40% B-50% B for 15 column volumes; collection began when the absorbance reached 400 mAU and stopped when the absorbance decreased to 400 mAU, yielding eluent 2; finally, regeneration was achieved with an acetonitrile:water solution of 80:20 (v / v) for 2 column volumes. The purity of liraglutide in eluent 2 was 99.4%, and the overall yield of the two-step reversed-phase chromatography was 81.2%.

[0075] Comparative Example 1:

[0076] This comparative example is similar to Example 1, except that no "adjuster" is added during the dilution step.

[0077] (1) Sample preparation: Weigh 150 mg of crude liraglutide powder (purity about 83.1 wt%), add 5 mL of 80 mM ammonium acetate solution (pH 9.0) containing 10% (v / v) acetonitrile, filter with a 0.22 micron filter membrane to obtain crude liraglutide solution.

[0078] (2) First step: Reversed-phase chromatography: The crude liraglutide solution was purified by chromatography using a Unisil® Revo C8 10-120 column packed with alkylsilane-bonded silica gel medium (alkyl carbon chain length of 8). The column volume was 4.15 mL. Elution mobile phase A was 80 mM ammonium acetate solution (pH 8.0), and elution mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 180 cm / h, and the column temperature was 25 °C. The specific separation process is as follows: Equilibrate with 20% B for 2 column volumes, then load the crude liraglutide solution at a volume of 15.28 mg / mL; wash with 20% B for 2 column volumes, then wash with a linear gradient of 20% B-40% B for 2 column volumes, and finally elute with a linear gradient of 40% B-50% B for 12 column volumes. Collect the eluent when the absorbance reaches 400 mAU and stop collecting when the absorbance decreases to 400 mAU, obtaining eluent 1; finally, regenerate with acetonitrile:water = 80:20 (v / v) solution for 2 column volumes. The purity of liraglutide in eluent 1 is 98.2%, and the yield is 90.3%.

[0079] (3) Dilute eluent 1 with water by 2 times without adding any "modifier".

[0080] (4) Second step: Reversed-phase chromatography: The diluted eluent 1 was loaded onto a Unisil® Revo C810-120 chromatography column packed with the same medium for separation and purification. The column volume was 4.15 mL. The elution mobile phase A was 80 mM dipotassium hydrogen phosphate solution (pH 7.0), and the mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 250 cm / h, and the column temperature was 25 °C. The specific separation process is as follows: Equilibrate with 20% B for 2 column volumes; load the diluted eluent 1 at a volume of 13.83 mg / mL; wash with 20% B for 2 column volumes, then wash with a linear gradient of 20% B-40% B for 2 column volumes, followed by elution with a linear gradient of 40% B-50% B for 15 column volumes; start collecting when the absorbance reaches 500 mAU and stop collecting when the absorbance drops to 500 mAU, obtaining eluent 2; finally, regenerate with acetonitrile:water = 80:20 (v / v) solution for 2 column volumes. The purity of liraglutide in eluent 2 is 99.1%, and the overall yield of the two-step reversed-phase chromatography is 75.4%. Compared to Example 1, the yield is significantly lower. This demonstrates that the "regulator" plays a crucial conformational regulating role in the system of this invention.

[0081] Comparative Example 2:

[0082] The operation of this comparative example is similar to that of Example 1, except that only salt is used to prepare the solution in the sample preparation.

[0083] (1) Sample preparation: Weigh 150 mg of crude liraglutide powder (purity about 83.1 wt%), add 5 mL of 80 mM ammonium acetate solution (pH 9.0) to dissolve, filter with a 0.22 micron filter membrane to obtain crude liraglutide solution.

[0084] (2) First step: Reversed-phase chromatography: The crude liraglutide solution was purified by chromatography using a Unisil® Revo C8 10-120 column packed with alkylsilane-bonded silica gel medium (alkyl carbon chain length of 8). The column volume was 4.15 mL. Elution mobile phase A was 80 mM ammonium acetate solution (pH 8.0), and elution mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 180 cm / h, and the column temperature was 25 °C. The specific separation process is as follows: Equilibrate with 20% B for 2 column volumes, then load the crude liraglutide solution at a volume of 15.18 mg / mL; wash with 20% B for 2 column volumes, then wash with a linear gradient of 20% B-40% B for 2 column volumes, and finally elute with a linear gradient of 40% B-50% B for 12 column volumes. Collect the eluent when the absorbance reaches 400 mAU and stop collecting when the absorbance decreases to 400 mAU, obtaining eluent 1; finally, regenerate with acetonitrile:water = 80:20 (v / v) solution for 2 column volumes. The purity of liraglutide in eluent 1 is 94.6%, with a yield of 78.1%.

[0085] (3) Dilute eluent 1 with water by 2 times and add 97 mg of polyethylene glycol (200 Da).

[0086] (4) Second step: Reversed-phase chromatography: The diluted eluent 1 was loaded onto a Unisil® Revo C810-120 chromatography column packed with the same medium for separation and purification. The column volume was 4.15 mL. The elution mobile phase A was 80 mM dipotassium hydrogen phosphate solution (pH 7.0), and the mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 250 cm / h, and the column temperature was 25 °C. The specific separation process is as follows: Equilibrate with 20% B for 2 column volumes; load the diluted eluent 1 at a volume of 13.83 mg / mL; wash with 20% B for 2 column volumes, then wash with a linear gradient of 20% B-40% B for 2 column volumes, followed by elution with a linear gradient of 40% B-50% B for 15 column volumes; start collecting when the absorbance reaches 500 mAU and stop collecting when the absorbance drops to 500 mAU, obtaining eluent 2; finally, regenerate with acetonitrile:water = 80:20 (v / v) solution for 2 column volumes. The purity of liraglutide in eluent 2 is 98.5%, and the overall yield of the two-step reversed-phase chromatography is 65.4%. Compared to Example 1, both purity and yield decreased, with the yield decreasing by approximately 20%, demonstrating that trace amounts of acetonitrile play a crucial and indispensable role in conformational pre-regulation in the system of this invention.

[0087] Comparative Example 3:

[0088] The operation of this comparative example is similar to that of Example 1, except that the pH of mobile phase A is adjusted to 9.5 in the second step of reversed-phase chromatography.

[0089] (1) Sample preparation: Weigh 150 mg of crude liraglutide powder (purity approximately 83.1 wt%), add 5 mL of 80 mM ammonium acetate solution (pH 9.0) containing 10% (v / v) acetonitrile to dissolve, filter with a 0.22 micron filter membrane to obtain crude liraglutide solution.

[0090] (2) First step: Reversed-phase chromatography: The crude liraglutide solution was purified by chromatography using a Unisil® Revo C8 10-120 column packed with alkylsilane-bonded silica gel medium (alkyl carbon chain length of 8). The column volume was 4.15 mL. Elution mobile phase A was 80 mM ammonium acetate solution (pH 8.0), and elution mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 180 cm / h, and the column temperature was 25 °C. The specific separation process is as follows: Equilibrate with 20% B for 2 column volumes, then load the crude liraglutide solution at a volume of 15.48 mg / mL; wash with 20% B for 2 column volumes, then wash with a linear gradient of 20% B-40% B for 2 column volumes, and finally elute with a linear gradient of 40% B-50% B for 12 column volumes. Collect the eluent when the absorbance reaches 400 mAU and stop collecting when the absorbance decreases to 400 mAU, obtaining eluent 1; finally, regenerate with acetonitrile:water = 80:20 (v / v) solution for 2 column volumes. The purity of liraglutide in eluent 1 is 98.1%, and the yield is 90.5%.

[0091] (3) Dilute eluent 1 with water by 2 times and add 114 mg of polyethylene glycol (200 Da).

[0092] (4) Second step: Reversed-phase chromatography: The diluted eluent 1 was loaded onto a Unisil® Revo C8 10-120 column packed with the same medium for separation and purification. The column volume was 4.15 mL. The elution mobile phase A was 80 mM dipotassium hydrogen phosphate solution (pH 10), and the mobile phase B was acetonitrile. The detection wavelength was 214 nm, the linear flow rate was 250 cm / h, and the column temperature was 25℃. The specific separation process is as follows: Equilibrate with 20% B for 2 column volumes; load the diluted eluent 1 at a volume of 14.13 mg / mL; wash with 20% B for 2 column volumes, then wash with a linear gradient of 20% B-40% B for 2 column volumes, followed by elution with a linear gradient of 40% B-50% B for 15 column volumes; start collecting when the absorbance reaches 500 mAU and stop collecting when the absorbance drops to 500 mAU, obtaining eluent 2; finally, regenerate with acetonitrile:water = 80:20 (v / v) solution for 2 column volumes. The purity of liraglutide in eluent 2 is 98.7%, and the overall yield of the two-step reversed-phase chromatography is 61.2%. Compared to Example 1, both purity and yield decreased. Specifically, the purity in the second step did not reach above 99%, and the overall yield decreased by approximately 20%. This indicates that excessively high pH not only causes liraglutide degradation and impurities but also disrupts its conformational stability, resulting in a significant decrease in purity and yield.

[0093] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make several equivalent substitutions, modifications, and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A method for separating and purifying liraglutide using reversed-phase chromatography, characterized in that, Includes the following steps: 1) Dissolve crude liraglutide in a salt solution containing acetonitrile, and filter the solution through a filter membrane to obtain a crude liraglutide solution; 2) Load the crude liraglutide solution into a chromatography column packed with reversed-phase chromatography medium and perform the first step of separation and purification under normal pressure. Elution is performed using a binary mobile phase linear gradient elution. Mobile phase A is a salt solution with a pH of 7.0-9.0, and mobile phase B is acetonitrile. Collect the elution peak to obtain the first eluent. 3) Dilute the first eluent with water and add a regulator with a polyhydroxy structure; 4) The diluted first eluent obtained in step 3) is loaded onto a chromatography column containing the same reversed-phase chromatography medium as in step 2), and the second separation and purification is carried out under normal pressure. Elution is performed using a binary mobile phase linear gradient elution, where mobile phase A is a salt solution with a pH of 6.0-8.0 and mobile phase B is acetonitrile. The elution peak is collected to obtain the second eluent, which is high-purity liraglutide.

2. The method for separating and purifying liraglutide by reversed-phase chromatography according to claim 1, characterized in that, The purity of crude liraglutide in step 1) ranges from 75.0 wt% to 90.0 wt%.

3. The method for separating and purifying liraglutide by reversed-phase chromatography according to claim 1, characterized in that, In step 1), the pH of the acetonitrile-containing salt solution is 7.0-9.0, the volume percentage of acetonitrile in the salt solution is 5%-15%, the mass ratio of liraglutide to acetonitrile is 0.15-0.5:1, and the pore size of the filter membrane is 0.22 µm.

4. The method for separating and purifying liraglutide by reversed-phase chromatography according to claim 1, characterized in that, In steps 2) and 4), the reversed-phase chromatography medium is an alkylsilane-bonded silica gel medium with an alkyl carbon chain length of 8 or 18, a medium particle size of 10 µm, and a medium pore size of 120 Å.

5. The method for separating and purifying liraglutide by reversed-phase chromatography according to claim 1, characterized in that, In steps 2) and 4), the column temperature is 20-30℃ and the flow rate is 100-300 cm / h.

6. The method for separating and purifying liraglutide by reversed-phase chromatography according to claim 1, characterized in that, The salt solution in steps 1) and 2) is a solution prepared from any one of the following salts: ammonium acetate, sodium acetate, potassium acetate, ammonium sulfate, ammonium chloride, diammonium hydrogen phosphate, dipotassium hydrogen phosphate, and disodium hydrogen phosphate, with a salt concentration of 10-110 mM and the pH value adjusted to 7.0-9.

0.

7. The method for separating and purifying liraglutide by reversed-phase chromatography according to claim 1, characterized in that, In step 2), the mobile phase B is pure acetonitrile. During the linear gradient elution process, the proportion of mobile phase B is gradually increased. When the absorbance rises to the threshold in the range of 300-400 mAU, collection begins, and when the absorbance drops to the threshold, collection stops. The elution length is 5-20 column volumes.

8. The method for separating and purifying liraglutide by reversed-phase chromatography according to claim 1, characterized in that, In step 3), the regulator is one of polyethylene glycol, glycerol, and sorbitol, with the molecular weight of polyethylene glycol being 200-1000 Da; the volume percentage of the regulator in the diluent is 0.5%~1.0%; and the mass ratio of liraglutide to the regulator is 1~7:

1.

9. The method for separating and purifying liraglutide by reversed-phase chromatography according to claim 1, characterized in that, The salt solution in step 4) is a solution prepared from any one of dipotassium hydrogen phosphate or disodium hydrogen phosphate, with a concentration of 10-110 mM, and the pH value is adjusted to 6.0-8.0; or a solution prepared from any one of sulfuric acid, acetic acid, formic acid, or phosphoric acid, with an acid volume percentage of 0.1%-1%, and the pH value is adjusted to 6.0-8.

0.

10. The method for separating and purifying liraglutide by reversed-phase chromatography according to claim 1, characterized in that, In step 4), the mobile phase B is pure acetonitrile. During the linear gradient elution process, the proportion of mobile phase B is increased. When the absorbance rises to the threshold in the range of 400-500 mAU, collection begins, and when the absorbance drops to the threshold, collection stops. The elution length is 5-20 column volumes.

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