Purification method of GLP-1 analogue crude peptide

Abstract

The invention discloses a purification method of a GLP-1 analogue crude peptide. The purification method comprises the following steps: treating a sample, preparing a mobile phase, carrying out purification balance, loading the sample, carrying out post-balance and eluting. The purification method aims at single impurities which are difficult to remove near a main peak, solves the problems of high purification difficulty, low recovery rate and high cost in the prior art, also has an obvious removal effect on front and rear impurities which are difficult to remove in crude stemeglutide or crude tilpoitide, reduces the purification difficulty, and has a great application prospect.

Description

Technical Field

[0001] The present invention relates to a method for purifying a crude peptide, in particular to a method for purifying a crude GLP-1 analog peptide. Background Art

[0002] GLP-1 analogs are an important class of drugs, mainly used to treat type 2 diabetes and have a weight loss effect. Glucagon-like peptide-1 (GLP-1) is a hormone mainly produced by intestinal L cells and belongs to the incretin class. GLP-1 receptor agonists (GLP-1RA) are new hypoglycemic drugs in recent years. They activate GLP-1 receptors, enhance insulin secretion in a glucose concentration-dependent manner, inhibit glucagon secretion, and delay gastric emptying. They reduce food intake through central appetite suppression, thereby achieving the effect of lowering blood sugar and losing weight. GLP-1 analogs are not only used to treat type 2 diabetes, but also show advantages in cardiovascular protection and can reduce the risk of cardiovascular disease.

[0003] At present, the market competition for GLP-1 analog drugs is fierce. In order to reduce costs, major manufacturers produce some products with slightly poor quality and large impurities in the upstream synthesis or modification stage, which are more difficult to purify downstream. Usually, it is shown in the analysis diagram that there are some relatively large single impurities before and after the main peak of the target, which cannot be removed well after multiple rounds of purification. Since the sequences of GLP-1 analogs are similar, the main difference lies in the side chain modification. The impurities introduced during the synthesis are usually missing peptide impurities with polarity very close to the main peak, such as missing peptide Ser and missing peptide Ile, missing peptide Gly and added Gly and other impurities. Therefore, new purification methods for crude GLP-1 analog peptides are urgently needed. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a method for purifying crude GLP-1 analog peptides which is simple to operate, has a high recovery rate and low cost.

[0005] Technical solution: The present invention discloses a method for purifying a crude GLP-1 analog peptide, comprising the following steps:

[0006] (1) Pure sample treatment: Dissolve the crude GLP-1 analog peptide in acetonitrile and water and adjust the pH, then filter and set aside;

[0007] (2) a pure mobile phase: phase A is an acidic solution including TFA, acetic acid or phosphoric acid, and phase B is an organic solvent to which a pH adjuster is added including acetonitrile or methanol. The pH adjuster includes TFA, acetic acid, phosphoric acid, ammonia water or triethylamine;

[0008] (3) A pure purification process: balance, use 80-95% A phase to balance the chromatographic column, preferably 90%; loading, the treated sample is loaded; post-balance, use 80-95% A phase to balance the chromatographic column, preferably 90%; elution, use A phase and B phase as mobile phases, gradually increase the proportion of B phase for gradient elution, and purification is completed.

[0009] In the above purification method, after the first pure gradient elution, a pure eluate sample is taken and a second pure purification is performed, wherein the second pure purification comprises the following steps:

[0010] (4) Treatment of the second pure sample: Take the first pure eluate and mix the sample, adjust the pH, dilute with water, and filter for later use;

[0011] (5) Two pure mobile phases: Phase A is a weak alkaline solution, including ammonium salt or sodium salt, and phase B is an organic solvent with a pH adjuster added, including acetonitrile or methanol. The pH adjuster includes TFA, acetic acid, phosphoric acid, ammonia or triethylamine;

[0012] (6) Secondary purification: equilibration, using a chromatographic column equilibrated with 80-95% phase A, preferably 90%; loading, loading the treated sample; post-equilibration, using a chromatographic column equilibrated with 80-95% phase A, preferably 90%; elution, using phase A and phase B as mobile phases, gradually increasing the proportion of phase B for gradient elution, and purification is completed.

[0013] Wherein, the crude GLP-1 analog peptide includes semaglutide or telpotide.

[0014] The purification method uses a chromatographic column of Sepax Bio-C8 or Sepax BR-C18, a filler particle size of 8 μm or 10 μm, and a pore size of or

[0015] Among them, in step (1), a pure sample is processed, specifically, taking a crude GLP-1 analog peptide, dissolving it in 10% acetonitrile water, adjusting the pH to 8-9 with 10% ammonia water, and the concentration is 5-15 mg / mL, and filtering with a 0.22 μm organic filter membrane for standby use.

[0016] Wherein, in step (2), the volume fraction of the pH adjuster in the phase B in the organic solvent is 0.1% to 1%.

[0017] Wherein, in step (3), the gradient elution is specifically:

[0018] Time (min) Mobile phase B (%) Mobile phase A (%) 0 10 90 10 15 85 135 40 60 135.01 80 20 145 80 20

[0019] Or:

[0020] Time (min) Mobile phase B (%) Mobile phase A (%) 0 10 90 10 20 80 210 40 60

[0021] Or:

[0022] Time (min) Mobile phase B (%) Mobile phase A (%) 0 10 90 5 34 66 6 34 66 88 39 61

[0023] Among them, in step (4), the two pure samples are processed specifically as follows: a pure eluent is combined, the pH is adjusted to 8-9 with 10% ammonia water, and then diluted to half with purified water, and filtered with a 0.22 μm organic filter membrane for standby use.

[0024] Wherein, in step (5), the volume fraction of the pH adjuster in the phase B in the organic solvent is 0.1% to 1%.

[0025] Wherein, in step (6), the gradient elution is specifically:

[0026] Time (min) Mobile phase B (%) Mobile phase A (%) 0 10 90 10 15 85 135 40 60 135.01 80 20 145 80 20

[0027] Or:

[0028] Time (min) Mobile phase B (%) Mobile phase A (%) 0 10 90 5 10 90 6 38 62 66 44 56

[0029] Principle of the invention: The purification method of the crude GLP-1 analog peptide of the present invention achieves an improvement in the purification effect by reasonably setting and configuring the mobile phase and adding a pH regulator as a modifier in phase B. In the industrial production of reversed-phase chromatography, the prior art mainly adjusts the system of phase A, including parameters such as the type, concentration and pH of the buffer salt, and phase B is a pure organic phase. The inventors found in actual research that adding buffer salts or modifiers to phase B is of great help in improving the purification effect; specifically, as a zwitterionic compound, the pH has a great influence on the retention of the polypeptide in reversed-phase chromatography. The method of the present invention uses a simple organic solvent for elution, while shielding the negative effects caused by changes in other forces, or using an obvious pH gradient to help change the elution situation, thereby achieving more effective polypeptide purification; Example 1 and Comparative Example 1 show that after adding some modifiers to the organic phase, the front impurities that are difficult to remove are significantly controlled, and the purification effect is improved by nearly 96%, and the removal effect of the back impurities is close; Example 2 and Comparative Example 2 show that after adding some modifiers to the organic phase B, the front and back impurities that are difficult to remove are also significantly removed, and the purification effect of the front impurities is improved by nearly 45%, and the purification effect of the back impurities is improved by 17%.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the purification method of the crude GLP-1 analog peptide of the present invention has an extremely strong purification effect, wherein, after the first purification, the front and back impurities that are difficult to remove in the crude semaglutide peptide are also significantly removed, and the purification effect of the front impurities is improved by nearly 45%, and the purification effect of the back impurities is improved by 17%; after the second purification, the front impurities that are difficult to remove in the crude semaglutide peptide are significantly controlled, and the purification effect is improved by nearly 96%, and the removal effect of the back impurities is close; the front and back impurities that are difficult to remove in the crude telportide peptide are also significantly removed. After two-step purification, the single impurity can be controlled within 0.1%. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the analysis diagram of a pure eluted sample from Example 1;

[0032] Figure 2 This is the analysis diagram of the second pure elution sample of Example 1;

[0033] Figure 3 This is the analysis diagram of the pure eluted sample of Comparative Example 1;

[0034] Figure 4 This is the analysis diagram of a pure eluted sample from Example 2;

[0035] Figure 5 This is the analysis diagram of the pure eluted sample of Comparative Example 2;

[0036] Figure 6 This is an enlarged image of the crude peptide analysis of semaglutide;

[0037] Figure 7 This is an enlarged picture of the analysis of crude telportipeptide in Example 3;

[0038] Figure 8 This is an enlarged view of the pure elution analysis of tepoxetine in Example 3;

[0039] Fig. 9 This is an enlarged view of the dipure elution analysis of telportopeptide in Example 3. DETAILED DESCRIPTION

[0040] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0041] Embodiment 1:

[0042] Sample type: Crude semaglutide peptide. Semaglutide obtained by capturing, enzymatically cleaving, and side chain modification of semaglutide fusion protein expressed in E. coli fermentation supernatant.

[0043] Chromatographic column: Sepax Bio-C8(2), particle size 10 μm, pore size Specifications: 4.6×250mm.

[0044] (1) Treatment of a pure sample: The crude semaglutide peptide was dissolved in 10% acetonitrile water, and the pH was adjusted to 8-9 with 10% ammonia water. The dissolved concentration was 10 mg / mL, and the sample was filtered through a 0.22 μm organic filter membrane before use.

[0045] (2) A pure mobile phase: Phase A is prepared with acetic acid into a 3% acetic acid-water solution, and phase B is prepared with acetonitrile to which 0.15% ammonia water is added.

[0046] (3) One-step purification:

[0047] Equilibration: Equilibrate the column with 90% phase A.

[0048] Sample loading: The treated samples were loaded at a crude loading of 15 mg / mL.

[0049] Post-equilibration: Equilibrate the column with 90% phase A.

[0050] Elution: Phase A and phase B are used as mobile phases for elution, and the proportion of phase B is gradually increased for gradient elution. The elution gradient is as follows:

[0051] Time (min) Mobile phase B (%) Mobile phase A (%) 0 10 90 10 15 85 135 40 60 135.5 80 20 145 80 20

[0052] (4) Treatment of second pure samples: The first pure eluate was combined, the pH was adjusted to 8-9 with 10% ammonia water, and then diluted to half with purified water, and filtered with a 0.22 μm organic filter membrane for later use.

[0053] (5) Two pure mobile phases: Phase A is 0.15% ammonia solution, and the pH is adjusted to 7.5 with phosphoric acid. Phase B is acetonitrile with 0.1% phosphoric acid added.

[0054] (6) Secondary purification step:

[0055] Equilibration: Equilibrate the column with 90% phase A.

[0056] Loading: Load the processed samples.

[0057] Post-equilibration: Equilibrate the column with 90% phase A.

[0058] Elution: Phase A and phase B are used as mobile phases for elution, and the proportion of phase B is gradually increased for gradient elution. The elution gradient is as follows:

[0059] Time (min) Mobile phase B (%) Mobile phase A (%) 0 10 90 10 15 85 135 40 60 135.01 80 20 145 80 20

[0060] Example 1 For the purification of crude semaglutide peptide, the results are shown in Table 1:

[0061] Table 1. Purification results of crude semaglutide peptide

[0062] sample purity Recovery rate Qian Za Post-Miscellaneous Loading 65.45％ - 3.72％ 2.77％ A pure elution 98.32％ 84.56％ 0.31％ 0.25％ Second pure elution 99.72％ 81.36％ 0.06％ 0.04％

[0063] As shown in Table 1, after the first and second purification elutions, the difficult-to-remove front impurities in the crude semaglutide peptide were significantly controlled. The first purification was able to remove nearly 92% of the front impurities and 91% of the back impurities, and the second purification was able to remove nearly 81% of the front impurities and 84% of the back impurities. The first purification improved the purification effect by nearly 96% compared with Comparative Example 1, and the removal effect of the back impurities was close.

[0064] Example 2

[0065] Sample type: Crude semaglutide peptide. Semaglutide obtained by capturing, enzymatically cleaving, and side chain modification of semaglutide fusion protein expressed in E. coli fermentation supernatant.

[0066] Chromatographic column: Sepax BR-C18(2), particle size 8 μm, pore size Specifications: 4.6×250mm.

[0067] (1) Sample treatment: The synthesized crude semaglutide peptide was dissolved in 10% acetonitrile water, and the pH was adjusted to 8-9 with 10% ammonia water. The dissolved concentration was 10 mg / mL, and the solution was filtered through a 0.22 μm organic filter membrane before use.

[0068] (2) Mobile phase: Phase A is prepared with acetic acid in a 2% acetic acid-water solution, and phase B is prepared with acetonitrile to which 0.15% ammonia water is added.

[0069] (3) Purification step:

[0070] Equilibration: Equilibrate the column with 90% phase A.

[0071] Sample loading: The treated samples were loaded at a crude loading of 15 mg / mL.

[0072] Post-equilibration: Equilibrate the column with 90% phase A.

[0073] Elution: Phase A and phase B are used as mobile phases for elution, and the proportion of phase B is gradually increased for gradient elution. The elution gradient is as follows:

[0074] Time (min) Mobile phase B (%) Mobile phase A (%) 0 10 90 10 20 80 210 40 60

[0075] Example 2 For the purification of crude semaglutide peptide, the results are shown in Table 2:

[0076] Table 2. Purification results of crude semaglutide peptide

[0077] sample purity Recovery rate Close to the main peak Close to the main peak Loading 67.30％ - 2.53％ 1.35％ Elution 98.93％ 91.44％ 0.12％ 0.13％

[0078] As shown in Table 2, after a pure elution, the front and back impurities that are difficult to remove in the crude semaglutide peptide are also significantly removed, removing about 95% of the front impurities and about 90% of the back impurities. Compared with Comparative Example 2, the purification effect of the front impurities is improved by nearly 45%, and the purification effect of the back impurities is improved by 17%. This may be due to the addition of a small amount of pH regulator causing a more delicate change in pH during the linear elution process, resulting in an increase in the relative retention time difference between impurities and target substances, thereby improving the resolution.

[0079] Embodiment 3:

[0080] Sample type: Crude telportin peptide, derived from solid phase synthesis.

[0081] Chromatographic column: Sepax Bio-C8(2), particle size 8 μm, pore size Specifications: 4.6×250mm.

[0082] (1) Treatment of pure samples: The synthesized crude telportin peptide was dissolved in 10% acetonitrile water, the pH was adjusted to 8-9 with 10% ammonia water, heated to 45° C. in a water bath and incubated for 2 h. The dissolved concentration was 10 mg / mL, and filtered through a 0.22 μm organic filter membrane for later use.

[0083] (2) A pure mobile phase: Phase A is made of acetic acid prepared with 0.15% ammonia-phosphoric acid and adjusted to pH 6.0, and phase B is made of acetonitrile to which 0.15% ammonia is added.

[0084] (3) One-step purification:

[0085] Equilibration: Equilibrate the column with 90% phase A.

[0086] Sample loading: Load the treated samples at a crude loading of 10 mg / mL.

[0087] Post-equilibration: Equilibrate the column with 90% phase A.

[0088] Elution: Phase A and phase B are used as mobile phases for elution, and the proportion of phase B is gradually increased for gradient elution. The elution gradient is as follows:

[0089] Time (min) Mobile phase B (%) Mobile phase A (%) 0 10 90 5 34 66 6 34 66 88 39 61

[0090] (4) Treatment of second pure samples: The first pure eluate was combined and diluted to half with purified water, and then filtered through a 0.22 μm organic filter membrane for later use.

[0091] (5) Two pure mobile phases: Phase A is 2% acetic acid aqueous solution, and phase B is acetonitrile with 1% acetic acid added.

[0092] (6) Secondary purification step:

[0093] Equilibration: Equilibrate the column with 90% phase A.

[0094] Loading: Load the processed samples.

[0095] Post-equilibration: Equilibrate the column with 90% phase A.

[0096] Elution: Phase A and phase B are used as mobile phases for elution, and the proportion of phase B is gradually increased for gradient elution. The elution gradient is as follows:

[0097] Time (min) Mobile phase B (%) Mobile phase A (%) 0 10 90 5 10 90 6 38 62 66 44 56

[0098] Example 3 For the purification of crude telportin peptide, the results are shown in Table 3:

[0099] Table 3. Purification results of crude semaglutide peptide

[0100] sample purity Recovery rate Qian Za Post-Miscellaneous Loading 78.73％ - 1.02％ 1.24％ A pure elution 98.83％ 78.76％ 0.46％ 0.15％ Second pure elution 99.75％ 85.10％ 0.06％ 0.04％

[0101] As shown in Table 3, after the first step of purification, the front and back impurities which are difficult to remove in the crude tepoxetine peptide are also significantly removed, and after two steps of purification, the single impurity can be controlled within 0.1%.

[0102] Comparative Example 1

[0103] Compared with Example 1, the phase B in the mobile phase was changed.

[0104] Sample type: Crude semaglutide peptide. Semaglutide obtained by capturing, enzymatically cleaving, and side chain modification of semaglutide fusion protein expressed in E. coli fermentation supernatant.

[0105] Chromatographic column: Sepax Bio-C8(2), particle size 10 μm, pore size Specifications: 4.6×250mm.

[0106] (1) Sample treatment: The crude semaglutide peptide after side chain modification was dissolved in 10% acetonitrile water, and the pH was adjusted to 8-9 with 10% ammonia water. The dissolved concentration was 10 mg / mL, and filtered through a 0.22 μm organic filter membrane before use.

[0107] (2) Mobile phase: Phase A is acetic acid prepared into a 3% acetic acid-water solution, and phase B is pure acetonitrile.

[0108] (3) Purification step:

[0109] Equilibration: Equilibrate the column with 90% phase A.

[0110] Sample loading: The treated samples were loaded at a crude loading of 15 mg / mL.

[0111] Post-equilibration: Equilibrate the column with 90% phase A.

[0112] Elution: Phase A and phase B are used as mobile phases for elution, and the proportion of phase B is gradually increased for gradient elution. The elution gradient is as follows:

[0113] Time (min) Mobile phase B (%) Mobile phase A (%) 0 10 90 10 15 85 135 40 60 135.01 80 20 145 80 20

[0114] The purification results of Comparative Example 1 are shown in Table 3:

[0115] Table 3, purification results of comparative example 1

[0116]

[0117]

[0118] As shown in Table 3, under this purification system, the separation effect of impurities with smaller polarity difference close to the main peak is poor, and 47% of the front impurities and 91% of the back impurities can be removed.

[0119] Comparative Example 2

[0120] Compared with Example 2, the phase B in the mobile phase was changed.

[0121] Sample type: Crude semaglutide peptide. Semaglutide obtained by capturing, enzymatically cleaving, and side chain modification of semaglutide fusion protein expressed in E. coli fermentation supernatant.

[0122] Chromatographic column: Sepax BR-C18(2), particle size 8 μm, pore size Specifications: 4.6×250mm.

[0123] (1) Sample treatment: The synthesized crude semaglutide peptide was dissolved in 10% acetonitrile water, and the pH was adjusted to 8-9 with 10% ammonia water. The dissolved concentration was 10 mg / mL, and the solution was filtered through a 0.22 μm organic filter membrane before use.

[0124] (2) Mobile phase: Phase A is acetic acid prepared into a 2% acetic acid-water solution, and phase B is pure acetonitrile.

[0125] (3) Purification step:

[0126] Equilibration: Equilibrate the column with 90% phase A.

[0127] Sample loading: The treated samples were loaded at a crude loading of 15 mg / mL.

[0128] Post-equilibration: Equilibrate the column with 90% phase A.

[0129] Elution: Phase A and phase B are used as mobile phases for elution, and the proportion of phase B is gradually increased for gradient elution. The elution gradient is as follows:

[0130] Time (min) Mobile phase B (%) Mobile phase A (%) 0 10 90 10 20 80 210 40 60

[0131] The purification results of Comparative Example 2 are shown in Table 4:

[0132] Table 4, Purification results of Comparative Example 4

[0133] sample purity Recovery rate Close to the main peak Close to the main peak Loading 67.30％ - 2.53％ 1.35％ Elution 96.09％ 92.54％ 0.78％ 0.31％

[0134] As shown in Table 4, under this purification system, the separation effect of impurities with smaller polarity differences close to the main peak is poor, and 69% of the front impurities and 77% of the back impurities can be removed.

[0135] Therefore, the purification method of the crude GLP-1 analog peptide of the present invention reduces the difficulty of purification by targeting the impurities that are difficult to remove near the main peak, thereby solving the problems of high purification difficulty, low recovery rate and high cost in the prior art.

Claims

1. A method for purifying a crude GLP-1 analog peptide, characterized in that: The following steps are involved: (1) Pure sample treatment: Dissolve the crude GLP-1 analog peptide in acetonitrile and water and adjust the pH, then filter and set aside; (2) a pure mobile phase: phase A is an acidic solution including TFA, acetic acid or phosphoric acid, and phase B is an organic solvent with a pH adjuster added, including acetonitrile or methanol. The pH adjuster includes TFA, acetic acid, phosphoric acid, ammonia water or triethylamine; (3) One-step purification process: equilibration, using 80-95% phase A to equilibrate the chromatographic column; loading, loading the treated sample; post-equilibration, using 80-95% phase A to equilibrate the chromatographic column; elution, using phase A and phase B as mobile phases, gradually increasing the proportion of phase B for gradient elution, and purification is completed.

2. A purification method according to claim 1, characterized in that: After the primary pure gradient elution, a primary pure eluate is taken and combined with a sample for secondary pure purification, wherein the secondary pure purification comprises the following steps: (4) Treatment of the second pure sample: Take the first pure eluate and mix the sample, adjust the pH, dilute with water, and filter for later use; (5) Two pure mobile phases: Phase A is a weak alkaline solution, including ammonium salt or sodium salt, and phase B is an organic solvent with a pH adjuster added, including acetonitrile or methanol. The pH adjuster includes TFA, acetic acid, phosphoric acid, ammonia or triethylamine; (6) Secondary purification: balance, using 80-95% A phase to balance the chromatographic column; loading, loading the treated sample; post-balance, using 80-95% A phase to balance the chromatographic column; elution, using A phase and B phase as mobile phases, gradually increasing the proportion of B phase for gradient elution, and purification is completed.

3. The purification method according to claim 1 or 2, characterized in that The crude GLP-1 analog peptide includes semaglutide or telpotide.

4. The purification method according to claim 1 or 2, characterized in that The purification method uses a chromatographic column of Sepax Bio-C8 or Sepax BR-C18, a filler particle size of 8 μm or 10 μm, and a pore size of or 5. The purification method according to claim 1, characterized in that In step (1), a pure sample is processed, specifically, a crude GLP-1 analog peptide is taken, dissolved in 10%-20% acetonitrile water, adjusted to pH 8-9 with 10%-20% ammonia water, and the concentration is 5-15 mg / mL, and filtered with an organic filter membrane for standby use.

6. The purification method according to claim 1, characterized in that In step (2), the volume fraction of the pH adjuster in the phase B in the organic solvent is 0.1% to 1%.

7. The purification method according to claim 2, characterized in that In step (4), the two pure samples are processed, specifically, taking a pure eluent sample, adjusting the pH to 8-9 with 10%-20% ammonia water, diluting it by half with purified water, filtering it with an organic filter membrane and setting it aside.

8. The purification method according to claim 2, characterized in that In step (5), the volume fraction of the pH adjuster in the phase B in the organic solvent is 0.1% to 1%.

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