Method for purifying Tirzeptide

Abstract

The invention provides a purification method of Tirzeptide, which comprises the following steps: (1) dissolving crude peptide with ammonia water, filtering, and loading a sample into a preparative column; (2) carrying out one-step purification by using a mobile phase A1 and a mobile phase B to obtain a sample with the purity of more than 95%; wherein the pH of the mobile phase A1 is adjusted by alkali liquor; (3) carrying out two-step purification by using a mobile phase A2 and a mobile phase B to obtain a sample of which the purity is greater than 99% and the single impurity is less than 0.10%; wherein the pH of the mobile phase A2 is adjusted by alkali liquor; and (4) converting the mobile phase A3 and the mobile phase B into ammonium salt, and carrying out spray drying treatment to obtain solid Tirzepine. According to the purification method provided by the invention, the refined Tirzeptide refined peptide is obtained through reversed-phase purification 1, reversed-phase purification 2, salt conversion purification and spray drying. According to the scheme, an acetate sample with the purity being greater than or equal to 99.0% and the single impurity being less than or equal to 0.10% can be The single-time sample loading amount is high, the production period is short, the waste liquid amount is small, and environmental protection is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a method for purifying Tirzepatide. Background Art

[0002] Patients with type 2 diabetes mellitus (T2DM) frequently suffer from a variety of comorbidities, including cognitive problems such as cognitive decline, cognitive impairment, or dementia. Patients with diabetes are 1.5 to 2 times more likely to experience cognitive decline, mild cognitive impairment, or dementia than those without the disease. This relationship is independent of other risk factors for cognitive dysfunction and accounts for a prevalence of 13% in patients with diabetes aged 65 to 74 years and 24% in patients aged 75 years or older. No single cause for the increased risk of cognitive dysfunction in patients with diabetes has been identified. The possibility that certain diabetes therapies, including glucagon-like peptide-1 (GLP-1) receptor agonists, may have a beneficial effect on cognitive function has been proposed and is being studied.

[0003] Patients with type 2 diabetes (T2DM) are at risk for developing heart failure with preserved ejection fraction (HFpEF) as well as heart failure with reduced ejection fraction (HFrEF). See, e.g., J. Ho et al., Predictors of New-Onset Heart Failure; Circulation: Heart Failure 6:279-286 (2013). Current treatments for HFpEF may include lifestyle changes to induce weight loss and administration of symptom-relieving agents for comorbid conditions. Bariatric surgery has shown some benefits for patients with HFpEF. See, e.g., Mikhalkova et al., Obesity (2018). Despite the increasing incidence of HFpEF, effective treatment options have been largely unsuccessful.

[0004] Tirzepatide is a dual agonist of GIP and GLP-1. Tirzepatide has demonstrated efficacy by improving beta cell function and insulin sensitivity. Tirzepatide has shown dual improvements in efficacy and tolerability in patients who initially use a lower dose and have smaller subsequent dose increments. After 8 weeks of treatment with Tirzepatide, Japanese patients with type 2 diabetes showed significant reductions in AIC and body weight. Tirzepatide can improve markers of non-alcoholic steatohepatitis (NASH, liver inflammation and cell damage caused by liver fat) in patients with type 2 diabetes. "These new Tirzepatide data build on the positive results of studies conducted to date in patients with type 2 diabetes, providing additional evidence that Tirzepatide can bring meaningful reductions in AIC and body weight levels in patients with type 2 diabetes, and that the drug can also treat other metabolic diseases.

[0005] The existing synthesis process has the problems of low purity, difficulty in controlling impurities below 0.10%, low yield, complicated operation steps, and being unfavorable for industrial production. Therefore, it is very necessary to provide a new purification method of Tirzepatide to improve the purity of refined peptides, simplify the purification steps, and facilitate industrial scale-up production. Summary of the invention

[0006] The above technologies have disadvantages such as unsatisfactory purification effect, poor control of racemic impurities, or complicated purification steps. In view of this, the present invention provides a solution to solve the above problems, and obtains refined Tirzepatide peptide by reverse phase purification 1, reverse phase purification 2, salt conversion purification and spray drying. This scheme can obtain Tirzepatide peptide with a purity of ≥99.0% and a single impurity of ≤0.10%. In addition, the single sample loading is high, the production cycle is short, the amount of waste liquid is small, and it is beneficial to environmental protection.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a method for purifying Tirzepatide, comprising the following steps: (1) dissolving a crude peptide with ammonia water and filtering, and then loading the sample onto a preparation column; (2) performing one-step purification with mobile phase A1 and mobile phase B to obtain a sample with a purity of more than 95%; wherein the mobile phase A1 is conditioned by alkaline solution to adjust the pH; (3) performing two-step purification with mobile phase A2 and mobile phase B to obtain a sample with a purity of more than 99% and a single impurity of less than 0.10%; wherein the mobile phase A2 is conditioned by alkaline solution to adjust the pH; (4) converting the peptide into ammonium salt with mobile phase A3 and mobile phase B, and performing spray drying to obtain solid Tirzepatide. It should be noted that in step (4), the qualified fraction after salt conversion does not need to be concentrated, and can be directly subjected to spray drying to obtain fine Tirzepatide.

[0009] Furthermore, the internal filler of the preparation column is any one of reverse phase C18, C8 or C4.

[0010] Furthermore, the particle size of the filler in the preparation column is 5 μm to 10 μm, and the pore size of the filler is 5 nm to 30 nm. The pore size of the filler is 5 nm to 30 nm, that is,

[0011] Furthermore, the loading amount is 0.5% to 5.0% of the filler mass; preferably, the loading amount is 1.0% to 3.0% of the filler weight.

[0012] Furthermore, in steps (2) to (4), the mobile phase B is one or more of acetonitrile, methanol, and isopropanol.

[0013] Furthermore, in step (2), the mobile phase A1 is a TFA (trifluoroacetic acid) salt buffer, and the concentration of TFA used is 0.01% to 2%; the alkali solution for adjusting the pH is any one or more of sodium hydroxide, potassium hydroxide, and ammonia water, and the pH is adjusted in the range of 7.0 to 9.0.

[0014] Furthermore, in step (3), the mobile phase A2 is an acetate buffer, and the concentration of the acetic acid used is 0.01% to 2%; the alkaline solution for adjusting the pH is any one or more of sodium hydroxide, potassium hydroxide, and ammonia water, and the pH is adjusted in the range of 7.0 to 9.0.

[0015] Furthermore, in step (4), the mobile phase A3 is aqueous ammonia, and the concentration of the aqueous ammonia used is 0.01% to 2%.

[0016] Compared with the prior art, the technical solution provided by the present invention has at least the following advantages:

[0017] The invention provides a novel purification method of Tirzepatide. The purification method can control a single impurity within 0.10% through two-step purification on reverse phase chromatography, and convert it into an ammonium salt on reverse phase chromatography without the need for an additional ion exchange chromatography column. The solvent used is simple, and purification is performed within a range higher than the isoelectric point of Tirzepatide, so that Tirzepatide will not be precipitated due to crossing the isoelectric point. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] One or more embodiments are exemplarily described by pictures in the corresponding drawings, and these exemplified descriptions do not constitute limitations on the embodiments. Unless otherwise stated, the pictures in the drawings do not constitute proportional limitations.

[0019] Figure 1 is the UPLC chromatogram of the refined peptide of Example 1 of the present invention;

[0020] Figure 2 is the UPLC chromatogram of the refined peptide of Example 2 of the present invention;

[0021] Figure 3 is the UPLC chromatogram of the refined peptide of Example 3 of the present invention;

[0022] Figure 4 is the UPLC chromatogram of the refined peptide of Example 4 of the present invention;

[0023] Figure 5 is the UPLC chromatogram of the refined peptide of Example 5 of the present invention;

[0024] Figure 6 This is the UPLC chromatogram of the refined peptide of Example 6 of the present invention;

[0025] Figure 7 This is the UPLC chromatogram of the refined peptide of Comparative Example 1 of the present invention;

[0026] Figure 8 This is the UPLC chromatogram of the refined peptide of Comparative Example 2 of the present invention;

[0027] Fig. 9 It is the UPLC chromatogram of the refined peptide of Comparative Example 3 of the present invention. DETAILED DESCRIPTION

[0028] The inventors found that CN112661815A in the prior art provides a method for purifying Tirzepatide, using C8 as the stationary phase. In the first step of purification, the mobile phase A1 is 0.1% ammonia aqueous solution, and the pH value is adjusted to 2.0 with perchloric acid; the phase B is 100% acetonitrile. In the second step of purification, the mobile phase A1 is 10mmol / L sodium chloride aqueous solution, and the pH value is adjusted to 7.5 with sodium hydroxide; the phase B is 100% acetonitrile. Oxidizing perchloric acid is used in the purification. Since the isoelectric point of Tirzepatide is 4.3, the two-step purification uses acidic and alkaline buffer salts respectively, which will cause sample precipitation in the process of crossing the isoelectric point, which is not good for the physical stability of the sample.

[0029] CN 113330024A provides a method for purifying Tirzepatide, using C8 as a filling column. In the first step of purification, A (90% water, 0.1% TFA and 10% ACN) and B (10% water, 0.1% TFA) are used as mobile phases. In the second step of purification, buffer C (90% NH4OAC aqueous solution pH 8.0, 10% ACN) and buffer D (10% NH4OAC aqueous solution pH 8.0, 90% ACN) are used, and then ion exchange is used for salt conversion, and finally tertiary methyl ether and isopropanol are used for precipitation, and dried to obtain refined Tirzepatide. This method requires the use of two stationary phases, which is relatively costly, and tertiary methyl ether and isopropanol are used to precipitate the refined peptide in the later stage, which is large in amount and is not conducive to environmental protection.

[0030] CN 110903355A discloses a method for purifying Tirzepatide. A chromatographic filler of 10 μm reverse phase C18 is used for alternating purification using two mobile phase systems, the first mobile phase system is 0.1% TFA / water solution-0.1% TFA / acetonitrile solution, and the second mobile phase system is 50 mmol ammonium acetate / water solution-acetonitrile. High performance liquid chromatography is used for salt exchange, the mobile phase system is 1% acetic acid / water solution-acetonitrile, and the chromatographic filler for purification is 10 μm reverse phase C18. The main peak of salt exchange is collected and the purity is detected by analytical liquid phase. The main peak solution of salt exchange is combined, concentrated under reduced pressure, and Tirzepatide acetic acid aqueous solution is obtained, and freeze-dried to obtain pure Tirzepatide. The pH of the ammonium acetate used is not clear, and it is finally prepared as acetate. Since the isoelectric point of Tirzepatide is 4.3, acetate is not conducive to its redissolution and storage stability.

[0031] In view of this, the present invention provides a method for purifying Tirzepatide, comprising the following steps:

[0032] First, the crude product was dissolved and filtered;

[0033] Using a certain concentration of TFA (adjusting the pH to 7.0-9.0 with alkaline solution) as mobile phase A1, a sample with a purity of ≥95.0% was purified in one step;

[0034] Then, a certain concentration of hydrochloric acid (adjusting the pH to 7.0-9.0 with alkaline solution) was used as mobile phase A2, and a qualified sample with a purity of ≥99.0% and a single impurity of ≤0.10% was obtained through two-step purification;

[0035] Then, a certain concentration of ammonia water is used as the mobile phase A3 to convert it into ammonium salt. The qualified fractions of salt conversion do not need to be concentrated, and solid Tirzepatide is directly obtained by spray drying.

[0036] The present invention can control a single impurity within 0.10% by two-step purification on reverse phase chromatography, and convert it into ammonium salt on reverse phase chromatography without the need for an additional ion exchange chromatography column. The solvent used is simple, and the purification is performed within a range higher than the isoelectric point of Tirzepatide, and Tirzepatide will not be precipitated due to crossing the isoelectric point. The whole process is simple to operate, the production cycle is short, and it is conducive to large-scale industrial production.

[0037] The present invention is described in detail below in conjunction with specific implementation modes.

[0038] Embodiment 1:

[0039] Weigh 32.00 g crude Tirzepatide (containing 18.50 g Tirzepatide), dissolve it in 10% ammonia water to 1 L, and filter it through 0.45 μm. The preparation column is 10 cm DAC, filled with 1.2 kg C18 filler. Mobile phase A1 was 0.1% TFA aqueous solution, pH was adjusted to 7.2 with ammonia water, and mobile phase B was acetonitrile. 90% A1+10% B was balanced for 10 min, flow rate was 300 mL / min, wavelength was 280 nm, gradient elution was performed after sample loading, the elution gradient was 40% B-60% B / (100 min), fractions with purity ≥95.0% were collected, fractions with purity 60.0%≤<95.0% were combined, and gradient elution with A1 and B was performed after sample loading for re-purification.

[0040] All qualified fractions from the first step purification were collected, diluted with an equal volume of water, and then subjected to the second step purification. Mobile phase A2 was 0.1% acetic acid aqueous solution, pH was adjusted to 7.5 with ammonia water, mobile phase B was acetonitrile, 90% A2 + 10% B was balanced for 10 min, flow rate was 300 mL / min, the sample with a purity greater than 95% obtained from the first step purification was loaded, and then gradient elution was performed, the elution gradient was 40% B-60% B / (100 min), and fractions with a purity ≥ 99.0% and fractions with single impurities ≤ 0.10% were collected.

[0041] The mobile phase A3 is 0.01% ammonia water, the mobile phase B is acetonitrile, 90% A3+10% B is balanced for 10 minutes, the flow rate is 300mL / min, after loading, it is rinsed with 90% A3+10% B for 20 minutes, and then rinsed with 40% A3+60% B, and the main peak is collected. The qualified salt conversion fraction does not need to be concentrated, and is directly spray-dried to obtain 7.88g Tirzepatide, the purification recovery rate is 42.59%, the purity is 99.84%, and the maximum single impurity is 0.07%. The UPLC chromatogram of the Tirzepatide sample prepared in Example 1 is shown as follows: Figure 1 shown.

[0042] Embodiment 2:

[0043] Weigh 34.00 g crude Tirzepatide (containing 19.66 g Tirzepatide), dissolve it in 10% ammonia water to 1 L, and filter it through 0.45 μm. The preparation column is 10 cm DAC, filled with 1.2 kg C8 filler. Mobile phase A1 was 0.15% TFA aqueous solution, pH was adjusted to 7.5 with ammonia water, and mobile phase B was 90% acetonitrile + 10% isopropanol. 90% A1 + 10% B was balanced for 10 min, flow rate was 300 mL / min, wavelength was 280 nm, gradient elution was performed after sample loading, the elution gradient was 38% B-58% B / (100 min), fractions with purity ≥ 95.0% were collected, fractions with purity 60.0% ≤ < 95.0% were combined, and gradient elution with A1 and B was performed after sample loading for re-purification.

[0044] All qualified fractions from the first step purification were collected, diluted with an equal volume of water, and then subjected to the second step purification. Mobile phase A2 was 0.15% acetic acid aqueous solution, pH adjusted to 7.8 with sodium hydroxide, mobile phase B was 90% acetonitrile + 10% isopropanol, 90% A2 + 10% B was balanced for 10 min, flow rate was 300 mL / min, the sample with a purity greater than 95% obtained from the first step purification was loaded, and then gradient elution was performed, the elution gradient was 38% B-58% B / (100 min), and fractions with a purity ≥ 99.0% and fractions with a single impurity ≤ 0.10% were collected.

[0045] The mobile phase A3 was 0.015% ammonia water, the mobile phase B was acetonitrile, 90% A3+10% B was balanced for 10 min, the flow rate was 300 mL / min, after loading, it was rinsed with 90% A3+10% B for 20 min, and then rinsed with 40% A3+60% B, and the main peak was collected. The qualified salt conversion fraction was directly spray-dried without concentration to obtain 8.45 g of Tirzepatide, the purification recovery rate was 42.98%, the purity was 99.85%, and the maximum single impurity was 0.05%. The UPLC chromatogram of the Tirzepatide sample prepared in Example 2 is shown in FIG. Figure 2 shown.

[0046] Embodiment 3:

[0047] Weigh 30.00 g crude Tirzepatide (containing 17.34 g Tirzepatide), dissolve it in 10% ammonia water to 1 L, and filter it through 0.45 μm. The preparation column is 10 cm DAC, filled with 1.2 kg C4 filler. Mobile phase A1 was 0.2% TFA aqueous solution, pH was adjusted to 7.8 with ammonia water, and mobile phase B was 90% acetonitrile + 10% methanol. 90% A1 + 10% B was balanced for 10 min, flow rate was 300 mL / min, wavelength was 280 nm, gradient elution was performed after sample loading, and the elution gradient was 32% B-58% B / (100 min), fractions with purity ≥ 95.0% were collected, fractions with purity 60.0% ≤ < 95.0% were combined, and gradient elution with A1 and B was performed after sample loading for re-purification.

[0048] All qualified fractions from the first step purification were collected, diluted with an equal volume of water, and then subjected to the second step purification. Mobile phase A2 was 0.2% acetic acid aqueous solution, pH was adjusted to 8.2 with potassium hydroxide, mobile phase B was 90% acetonitrile + 10% methanol, 90% A2 + 10% B was balanced for 10 min, flow rate was 300 mL / min, the sample with a purity greater than 95% obtained from the first step purification was loaded, and then gradient elution was performed, the elution gradient was 32% B-58% B / (100 min), and fractions with a purity ≥ 99.0% and fractions with a single impurity ≤ 0.10% were collected.

[0049] The mobile phase A3 was 0.02% ammonia water, the mobile phase B was acetonitrile, 90% A3+10% B was balanced for 10 min, the flow rate was 300 mL / min, after loading, it was rinsed with 90% A3+10% B for 20 min, and then rinsed with 50% A3+50% B, and the main peak was collected. The qualified salt conversion fraction was directly spray-dried without concentration to obtain 7.55 g of Tirzepatide, the purification recovery rate was 43.54%, the purity was 99.81%, and the maximum single impurity was 0.08%. The UPLC chromatogram of the Tirzepatide sample prepared in Example 3 is shown in FIG. Figure 3 shown.

[0050] The inventors simultaneously carried out the following examples and comparative examples, and the preparation columns were all 10 cm DAC, filled with 1.2 kg C8 filler. The mobile phase B was acetonitrile. The differences from the above Examples 1, 2 and 3 are summarized in Table 1.

[0051] Table 1 Purification parameters of Tirzepatide in Examples 4 to 6 and Comparative Examples 1 to 3

[0052]

[0053]

[0054] The UPLC chromatogram of the Tirzepatide sample prepared in Example 4 is as follows: Figure 4 As shown, the UPLC chromatogram of the Tirzepatide sample prepared in Example 5 is as shown Figure 5 As shown, the UPLC chromatogram of the Tirzepatide sample prepared in Example 6 is as follows Figure 6 As shown; the UPLC chromatogram of the Tirzepatide sample prepared in Comparative Example 1 is as shown Figure 7 As shown, the UPLC chromatogram of the Tirzepatide sample prepared in Comparative Example 2 is as follows Figure 8 As shown, the UPLC chromatogram of the Tirzepatide sample prepared in Comparative Example 3 is as follows Fig. 9 shown.

[0055] Those skilled in the art can understand that the above-mentioned embodiments are specific examples for implementing the present application, and in practical applications, various changes can be made to them in form and detail without departing from the spirit and scope of the present application. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present application, so the scope of protection of the present application shall be based on the scope defined in the claims.

Claims

1. A method for purifying Tirzepatide, characterized in that: The following steps are involved: (1) Dissolve the crude peptide in aqueous ammonia and filter it, then load it onto a preparative column; (2) performing one-step purification using mobile phase A1 and mobile phase B to obtain a sample with a purity greater than 95%; wherein the pH of the mobile phase A1 is adjusted with alkaline solution; (3) performing two-step purification using mobile phase A2 and mobile phase B to obtain a sample with a purity of >99% and a single impurity of <0.10%; wherein the pH of the mobile phase A2 is adjusted with an alkaline solution; (4) The mobile phase A3 and the mobile phase B are converted into ammonium salts and spray-dried to obtain solid Tirzepatide.

2. The method for purifying Tirzepatide according to claim 1, characterized in that The internal filler of the preparation column is any one of reverse phase C18, C8 or C4.

3. The method for purifying Tirzepatide according to claim 1, characterized in that: The particle size of the internal filler of the preparation column is 5 μm to 10 μm, and the pore size of the filler is 5 nm to 30 nm.

4. The method for purifying Tirzepatide according to claim 2 or 3, characterized in that: The loading amount is 0.5% to 5.0% of the filler mass; preferably, the loading amount is 1.0% to 3.0% of the filler weight.

5. The method for purifying Tirzepatide according to claim 1, characterized in that: In steps (2) to (4), the mobile phase B is one or more of acetonitrile, methanol, and isopropanol.

6. The method for purifying Tirzepatide according to claim 1, characterized in that: In step (2), the mobile phase A1 is a TFA (trifluoroacetic acid) salt buffer, and the concentration of TFA used is 0.01% to 2%; The alkali solution for adjusting pH is any one or more of sodium hydroxide, potassium hydroxide and ammonia water, and the pH is adjusted in the range of 7.0 to 9.

0.

7. The method for purifying Tirzepatide according to claim 1, characterized in that: In step (3), the mobile phase A2 is an acetate buffer, and the concentration of acetic acid used is 0.01% to 2%; The alkali solution for adjusting pH is any one or more of sodium hydroxide, potassium hydroxide and ammonia water, and the pH is adjusted in the range of 7.0 to 9.

0.

8. The method for purifying Tirzepatide according to claim 1, characterized in that: In step (4), the mobile phase A3 is aqueous ammonia, and the concentration of the aqueous ammonia used is 0.01% to 2%.

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