A purification method for Tirzepatide

Abstract

This invention discloses a purification method for Tirzepatide. The method includes: dissolving crude Tirzepatide peptide in ammonia or sodium bicarbonate solution, filtering, and purifying by reversed-phase chromatography to obtain a purified sample; eluting the purified sample using reversed-phase high-performance liquid chromatography (RP-HPLC) with a sodium or potassium buffer solution as mobile phase A3 and one or a mixture of acetonitrile, methanol, and isopropanol in any proportion as mobile phase B, preparing a sodium or potassium salt fraction; cooling the sodium or potassium salt fraction to -20 to 10°C and maintaining the temperature; then adding an organic solvent to the fraction, controlling the temperature at -20 to 10°C to precipitate the sample; after complete precipitation, solid-liquid separation is performed, the solid is collected, dissolved in water, filtered, and freeze-dried. This invention improves the concentration speed, ensures product purity, and strictly controls impurities.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide drug synthesis technology, specifically relating to a purification method for Tirzepatide. Background Technology

[0002] People with type 2 diabetes mellitus (T2DM) frequently suffer from various comorbidities, including cognitive problems such as cognitive decline, cognitive impairment, or dementia. People with diabetes are 1.5 to 2 times more likely to experience cognitive decline, mild cognitive impairment, or dementia than those without the condition. This relationship is independent of other risk factors for cognitive impairment and accounts for a 13% prevalence in people with diabetes aged 65–74 years and a 24% prevalence in people aged 75 years or older. No single cause has been identified for the high risk of cognitive impairment in people with diabetes. The potential benefits of certain diabetes therapies, including glucagon-like peptide-1 (GLP-1) receptor agonists, for cognitive function have been proposed and are under investigation.

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

[0004] Tirzepatide is a dual GIP and GLP-1 agonist. Its efficacy is demonstrated by improving β-cell function and increasing insulin sensitivity. Tirzepatide showed dual improvements in efficacy and tolerability in patients starting at lower doses with smaller subsequent dose escalations. After 8 weeks of treatment with Tirzepatide, Japanese patients with type 2 diabetes showed significant reductions in AIC and weight. Tirzepatide also improved markers of non-alcoholic steatohepatitis (NASH, liver inflammation and cellular damage caused by hepatic fat) in patients with type 2 diabetes. These new Tirzepatide data build upon positive results from previous studies in patients with type 2 diabetes, providing additional evidence that Tirzepatide can significantly reduce AIC and weight levels in patients with type 2 diabetes, and that the drug can also treat other metabolic diseases.

[0005] CN112661815A discloses a purification method for Tirzepatide using C8 as the stationary phase. The first purification step involves a mobile phase A1 of 0.1% ammonia aqueous solution, adjusted to pH 2.0 with perchloric acid; and a mobile phase B of 100% acetonitrile. The second purification step involves a mobile phase A1 of 10 mmol / L sodium chloride aqueous solution, adjusted to pH 7.5 with sodium hydroxide; and a mobile phase B of 100% acetonitrile. The final fraction is concentrated by rotary evaporation and then freeze-dried to obtain a solid. However, the rotary evaporation process can easily lead to an increase in the size of high molecular weight impurities, potentially increasing toxicity.

[0006] CN113330024 A discloses a purification method for Tirzepatide using a C8 packed column. The first purification step uses mobile phases A (90% water, 0.1% TFA, and 10% ACN) and B (10% water, 0.1% TFA). The second purification step uses buffer C (90% NH4OAC aqueous solution, pH 8.0, 10% ACN) and buffer D (10% NH4OAC aqueous solution, pH 8.0, 90% ACN), followed by ion exchange conversion, and finally precipitation with methyl ether and isopropanol, followed by drying to obtain purified Tirzepatide. This method uses methyl ether and isopropanol to precipitate the peptide, and the residual solvent after drying can easily exceed the limit.

[0007] CN110903355 A discloses a method for preparing Tirzepatide. The method uses 10 μm reversed-phase C18 chromatographic packing material and two alternating mobile phase systems for purification. 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 (HPLC) is used for salt exchange, with a mobile phase system of 1% acetic acid / water solution-acetonitrile. The purification chromatographic packing material is 10 μm reversed-phase C18. The main peak of the salt exchange is collected and its purity is determined by analytical liquid chromatography. The salt exchange peak solutions are combined, concentrated under reduced pressure to obtain an aqueous solution of Tirzepatide in acetic acid, and then freeze-dried to obtain pure Tirzepatide. The process also requires rotary evaporation for concentration, which can easily lead to an increase in the size of high molecular weight impurities and increase toxicity. Summary of the Invention

[0008] The purpose of this invention is to address the problems of large fraction volume, slow concentration speed and long concentration time, and increased impurities in existing purification processes, which are detrimental to industrial production. This invention provides a purification method for Tirzepatide. This method improves the concentration speed, ensures product purity, strictly controls impurities, and facilitates large-scale industrial production.

[0009] This invention provides a purification method for Tirzepatide. First, the crude product is dissolved and filtered, then purified by reversed-phase chromatography to obtain a purified sample. Next, the sample is converted to a sodium or potassium salt fraction. The fraction is then cooled and held at that temperature for a period of time. An organic solvent is slowly added to the fraction while maintaining the temperature, allowing the sample to precipitate. Solid-liquid separation is performed, the solvent is discarded, and the solid is retained. Water is added until the solid dissolves, and then the solid is freeze-dried.

[0010] The specific technical solution of the present invention is as follows:

[0011] This invention provides a method for purifying Tirzepatide, comprising the following steps:

[0012] (1) Dissolve crude Tirzepatide peptide in ammonia or sodium bicarbonate solution, filter, and purify the crude peptide solution by reverse phase chromatography to obtain a purified sample.

[0013] (2) The purified sample obtained in step (1) is subjected to reversed-phase high-performance liquid chromatography to change the salt. The buffer salt solution containing sodium or potassium is used as the mobile phase A3, and any one of acetonitrile, methanol, and isopropanol or a mixture of any proportions is used as the mobile phase B. The purified sample obtained in step (1) is eluted with mobile phase A3, water and mobile phase B to prepare the sodium or potassium salt fraction.

[0014] (3) Cool the obtained sodium or potassium salt fraction to -20 to 10°C and then keep it warm; then add organic solvent to the fraction and control the temperature to -20 to 10°C to precipitate the sample; after the sample has completely precipitated, separate the solid and liquid, collect the solid, add water to dissolve the solid, filter and freeze dry.

[0015] Furthermore, the filtration in step (1) uses a 0.45 μm filter membrane;

[0016] Preferably, the reversed-phase chromatography in step (1) is a C18 column, a C4 column, or a C8 column;

[0017] Preferably, the purity of the purified sample in step (1) is ≥99%, and the single impurity is ≤0.1%;

[0018] Preferably, the reversed-phase chromatography purification step in step (1) includes primary purification and secondary purification, specifically including:

[0019] 1) The obtained crude peptide solution was purified once by reversed-phase chromatography using a gradient elution with a mixed solution of mobile phase A1 and mobile phase B. The fraction with a purity ≥95.0% was collected to obtain the first purified fraction.

[0020] 2) The obtained primary purified fraction is subjected to secondary purification by reversed-phase chromatography, using a gradient elution with a mixed solution of mobile phase A2 and mobile phase B, and the fraction with a purity ≥99.0% and a single impurity ≤0.10% is collected, which is the purified sample.

[0021] The mobile phase A1 is a 0.05% to 0.20% TFA aqueous solution by volume, with the pH value adjusted by alkaline solution, and the pH value of the mobile phase A1 is 7.0 to 7.2;

[0022] Preferably, the pH of the mobile phase A1 is adjusted with NaOH;

[0023] The mobile phase A2 is a 50-100 mM ammonium acetate aqueous solution, and the pH value of the mobile phase A2 is 8.0-8.2;

[0024] Preferably, the pH of the mobile phase A2 is adjusted with ammonia.

[0025] The mobile phase B is any one of acetonitrile, methanol, and isopropanol, or a mixture thereof in any proportion.

[0026] Further, in step 1), the mobile phase B is acetonitrile, the elution gradient is (36-40)% to (56-60)% mobile phase B, and the elution time is 100 to 120 min;

[0027] Alternatively, the mobile phase B mentioned in step 1) is methanol, the elution gradient is 60% to 90% mobile phase B, and the elution time is 60 to 80 min;

[0028] Preferably, in step 1), the fractions with a purity of 60.0% ≤ 95.0% after gradient elution are combined, and then purified by reversed-phase chromatography using a mixed solution of mobile phase A1 and mobile phase B, collecting the fraction with a purity of ≥ 95.0%.

[0029] Further, in step 2), the mobile phase B is acetonitrile, the elution gradient is (34-38)% to (54-58)% mobile phase B, and the elution time is 100-120 min;

[0030] Alternatively, the mobile phase B mentioned in step 2) is methanol, the elution gradient is 60% to 90% mobile phase B, and the elution time is 80 to 100 min;

[0031] Alternatively, in step 2), the mobile phase B is isopropanol, the elution gradient is 34%–54% mobile phase B, and the elution time is 100–120 min.

[0032] Further, the reversed-phase high-performance liquid chromatography in step (2) is a C18 column, a C4 column, or a C8 column;

[0033] The sodium-containing buffer salt mentioned in step (2) is selected from any one or any mixture of sodium acetate, sodium trifluoroacetate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, sodium sulfate, sodium bicarbonate, sodium carbonate, and sodium citrate.

[0034] The potassium-containing buffer salt mentioned in step (2) is selected from any one or any mixture of potassium acetate, potassium trifluoroacetate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium chloride, potassium sulfate, potassium bicarbonate, potassium carbonate, and potassium citrate.

[0035] Preferably, the pH value of the sodium- or potassium-containing buffer salt solution is 6.0 to 9.0, more preferably 7.0 to 8.0;

[0036] Preferably, the concentration of the sodium or potassium buffer solution is 10–200 mM, more preferably 30–100 mM, and even more preferably 20–80 mM.

[0037] Further, the method for preparing the purified sample obtained in step (1) into a sodium or potassium salt fraction in step (2) includes: after loading the purified sample obtained in step (1), washing it with 90% mobile phase A3 + 10% mobile phase B for 15-25 min, then washing it with 90% water + 10% mobile phase B for 10-20 min, and then washing it with (20-45)% water + (55-80)% mobile phase B, collecting the main peak, and obtaining the sodium or potassium salt fraction.

[0038] Furthermore, in step (3), the sodium or potassium salt fraction is cooled to -20 to 10°C and then kept at that temperature for 0.5 to 5 hours.

[0039] Preferably, in step (3), the obtained sodium or potassium salt fraction is cooled to -10 to 5°C.

[0040] Furthermore, in step (3), an organic solvent is added to the distillate, the temperature is controlled at -20 to 10°C, and the holding time is 0.5 to 5 hours;

[0041] Preferably, in step (3), an organic solvent is added to the distillate, and the temperature is controlled at -10 to 5°C.

[0042] Furthermore, the organic solvent mentioned in step (3) is any one of acetonitrile, methanol, and isopropanol or a mixture thereof in any proportion;

[0043] Preferably, the volume percentage of the organic solvent to the total volume of the organic solvent and the distillate is 75% to 95%, more preferably 80% to 90%.

[0044] Further, in step (3), the concentration of the solid after dissolution is 10-200 g / L, preferably 20-120 g / L;

[0045] Preferably, the filtration in step (3) is a 0.22-micron filtration.

[0046] The beneficial effects of this invention are as follows:

[0047] The purification method for Tirzepatide provided by this invention first involves dissolving and filtering the crude product, then purifying it using reversed-phase chromatography to obtain a purified sample. The sample is then converted to sodium or potassium salt fractions. The fractions are then cooled and held at that temperature for a period of time. An organic solvent is slowly added to the fractions while maintaining the temperature under controlled conditions to precipitate the sample. Solid-liquid separation is performed, the solvent is discarded, and the solid is retained. Water is added until the solid dissolves, and the sample is freeze-dried to obtain Tirzepatide peptides with high purity, low impurities, low high molecular weight impurities, and low residual solvent. This method avoids the problems of decreased purity and increased degradation and high molecular weight impurities caused by prolonged rotary evaporation concentration, as well as the high solvent residue caused by direct drying after precipitation. This method can obtain acetate samples with a purity ≥99.0% and single impurity ≤0.10%, with high single sample loading, short production cycle, and low waste volume, which is environmentally friendly. This method improves concentration speed, ensures product purity, strictly controls impurities, and is conducive to industrial-scale production. Attached Figure Description

[0048] Figure 1 The UPLC chromatogram of Example 1;

[0049] Figure 2The UPLC chromatogram for Example 2;

[0050] Figure 3 The UPLC chromatogram for Example 3;

[0051] Figure 4 The UPLC chromatogram for Example 4;

[0052] Figure 5 The UPLC chromatogram for Example 5;

[0053] Figure 6 The UPLC chromatogram of Example 6;

[0054] Figure 7 The UPLC chromatogram for Comparative Example 1;

[0055] Figure 8 This is the UPLC chromatogram of Comparative Example 2;

[0056] Figure 9 The UPLC chromatogram for Comparative Example 3;

[0057] Figure 10 This is the UPLC chromatogram of Comparative Example 4. Detailed Implementation

[0058] To better understand the present invention, it is now further described with reference to the following embodiments and accompanying drawings. The embodiments are for illustrative purposes only and do not limit the invention in any way. In the embodiments, all original reagents and materials are commercially available, and experimental methods not specifically specified are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0059] Example 1:

[0060] (1) Reverse-phase purification

[0061] Weigh 50.0 g of crude Tirzepatide (containing 32.5 g of Tirzepatide), dissolve in 5% sodium bicarbonate solution to a volume of 2 L, and filter through a 0.45 μm filter membrane. The preparative column was a 10 cm DAC packed with C18 packing material. Mobile phase A1 was a 0.1% TFA aqueous solution, adjusted to pH 7.2 with sodium hydroxide, and mobile phase B was acetonitrile. The mixture was equilibrated with 90% A1 and 10% B for 10 min at a flow rate of 240 mL / min and a wavelength of 280 nm. After loading the sample, gradient elution was performed at a gradient of 40% B - 60% B / (100 min), and the fraction with a purity ≥95.0% was collected. Fractions with a purity ≤60.0% and <95.0% were combined, loaded, and then purified again by gradient elution with A1 and B, collecting the fraction with a purity ≥95.0%.

[0062] All qualified fractions purified in the first step (i.e., fractions with a purity greater than 95% obtained in the first step) were collected and diluted with an equal volume of water for a second step purification. Mobile phase A2 was a 100 mM ammonium acetate aqueous solution, with the pH adjusted to 8.0 by ammonia. Mobile phase B was acetonitrile. The mixture was equilibrated at 90% A2 + 10% B for 10 min at a flow rate of 240 mL / min. The sample with a purity greater than 95% obtained in the first step was loaded onto the sample, followed by gradient elution at a gradient of 38% B - 58% B / (100 min). Fractions with a purity ≥99.0% and a single impurity ≤0.10% were collected.

[0063] (2) Salt conversion and purification

[0064] The chromatographic column used was the same as that used in step (1) for reverse-phase purification. Mobile phase A3 was a 50 mM sodium acetate solution, with pH adjusted to 8.0 using sodium hydroxide. Mobile phase B was acetonitrile. The solution was equilibrated with 90% A3 + 10% B for 10 min at a flow rate of 240 mL / min. After loading the sample, the solution was washed with 90% A3 + 10% B for 15 min, then with 90% water + 10% B for 10 min, and finally with 40% water + 60% B. The main peak was collected to obtain the sodium salt fraction. The fraction volume was 1 L, and the Tirzepatide concentration was 23 g / L.

[0065] (3) Freeze drying

[0066] The sodium salt fraction was cooled to 10°C and maintained for 0.5 hours. 5 L of acetonitrile (90% acetonitrile ratio) was slowly added to the fraction, and the mixture was stirred for 0.5 hours while maintaining the temperature at 10°C. After the sample precipitated, it was allowed to stand for 1 hour, filtered, and the solid was collected; the filtrate was discarded. The collected solid was dissolved in water to a concentration of 57.5 g / L in 0.4 L of water, filtered through a 0.22 μm filter, and then freeze-dried to obtain 24.2 g of Tirzepatide.

[0067] The purification recovery rate was 74.5%, the purity was 99.66%, the maximum single impurity was 0.06%, the high molecular weight impurity was 0.02%, and the acetonitrile residue was 10 ppm.

[0068] Example 2:

[0069] (1) Reverse-phase purification

[0070] Weigh 50.0 g of crude Tirzepatide (containing 32.5 g of Tirzepatide), dissolve in 5% sodium bicarbonate solution to a volume of 2 L, and filter through a 0.45 μm filter membrane. The preparative column was a 10 cm DAC packed with C4 packing material. Mobile phase A1 was a 0.1% TFA aqueous solution, with the pH adjusted to 7.1 using sodium hydroxide. Mobile phase B was methanol. The mixture was equilibrated with 90% A1 and 10% B for 15 min at a flow rate of 200 mL / min and a wavelength of 280 nm. After loading the sample, gradient elution was performed with an elution gradient of 60% B - 90% B / (60 min). Fractions with a purity ≥ 95.0% were collected. Fractions with a purity ≤ 60.0% and < 95.0% were combined, loaded, and then purified again by gradient elution with A1 and B, collecting fractions with a purity ≥ 95.0%.

[0071] All qualified fractions purified in the first step (i.e., fractions with a purity greater than 95% obtained in the first step) were collected and diluted with an equal volume of water for a second step purification. Mobile phase A2 was a 100 mM ammonium acetate aqueous solution, with the pH adjusted to 8.0 by ammonia. Mobile phase B was methanol. The mixture was equilibrated at 90% A2 + 10% B for 15 min at a flow rate of 200 mL / min. The sample with a purity greater than 95% obtained in the first step was loaded onto the sample, followed by gradient elution at a gradient of 60% B - 90% B / (90 min). Fractions with a purity ≥99.0% and a single impurity ≤0.10% were collected.

[0072] (2) Salt conversion and purification

[0073] The chromatographic column used was the same as that used in step (1) for reverse-phase purification. Mobile phase A3 was a 100 mM sodium chloride solution, with pH adjusted to 8.0 using sodium hydroxide. Mobile phase B was methanol. The solution was equilibrated with 90% A3 + 10% B for 15 min at a flow rate of 200 mL / min. After loading the sample, the solution was washed with 90% A3 + 10% B for 20 min, then with 90% water + 10% B for 15 min, and finally with 20% water + 80% B. The main peak was collected to obtain the sodium salt fraction. The fraction volume was 2.2 L, and the Tirzepatide concentration was 10.2 g / L.

[0074] (3) Freeze drying

[0075] The sodium salt fraction was cooled to 0°C and held for 0.5 hours. 8 L of methanol (95% methanol content) was slowly added to the fraction, and the temperature was lowered to -5°C. The mixture was stirred for 2 hours. After the sample precipitated, it was allowed to stand for 2 hours, filtered, and the solid was collected. The filtrate was discarded. The collected solid was dissolved in water to a concentration of 56.1 g / L in 0.4 L of water. After filtration through a 0.22 μm filter, the solution was freeze-dried to obtain 23.5 g of Tirzepatide.

[0076] The purification recovery rate was 72.3%, the purity was 99.55%, the maximum single impurity was 0.06%, the high molecular weight impurity was 0.03%, and the methanol residue was undetectable.

[0077] Example 3:

[0078] (1) Reverse-phase purification

[0079] Weigh 50.0 g of crude Tirzepatide (containing 32.5 g of Tirzepatide), dissolve in 5% sodium bicarbonate solution to a volume of 2 L, and filter through a 0.45 μm filter membrane. The preparative column was a 10 cm DAC packed with C8 packing material. Mobile phase A1 was a 0.1% TFA aqueous solution, adjusted to pH 7.0 with sodium hydroxide, and mobile phase B was acetonitrile. The mixture was equilibrated with 90% A1 + 10% B for 10 min at a flow rate of 240 mL / min and a wavelength of 280 nm. After loading the sample, gradient elution was performed with an elution gradient of 38% B - 58% B / (100 min). Fractions with a purity ≥ 95.0% were collected. Fractions with a purity ≤ 60.0% and < 95.0% were combined, loaded, and then purified again by gradient elution with A1 and B, collecting fractions with a purity ≥ 95.0%.

[0080] All qualified fractions purified in the first step (i.e., fractions with a purity greater than 95% obtained in the first step) were collected and diluted with an equal volume of water for a second step purification. Mobile phase A2 was a 100 mM ammonium acetate aqueous solution, with the pH adjusted to 8.2 by ammonia. Mobile phase B was isopropanol. The mixture was equilibrated with 90% A2 + 10% B for 10 min at a flow rate of 240 mL / min. The sample with a purity greater than 95% obtained in the first step was loaded onto the sample, followed by gradient elution. The elution gradient was 34% B - 54% B / (120 min). Fractions with a purity ≥ 99.0% and a single impurity ≤ 0.10% were collected.

[0081] (2) Salt conversion and purification

[0082] The chromatographic column used was the same as that used in step (1) for reverse-phase purification. Mobile phase A3 was a 30 mM sodium bicarbonate solution, without pH adjustment. Mobile phase B was isopropanol. After equilibration with 90% A3 + 10% B for 20 min at a flow rate of 150 mL / min, the sample was loaded and washed with 90% A3 + 10% B for 25 min, then washed with 90% water + 10% B for 20 min, and finally washed with 40% water + 60% B. The main peak was collected to obtain the sodium salt fraction. The fraction volume was 2.5 L, and the Tirzepatide concentration was 9.2 g / L.

[0083] (3) Freeze drying

[0084] The sodium salt fraction was cooled to -20°C and maintained for 1 hour. 7.5 L of isopropanol (90% isopropanol ratio) was slowly added to the fraction, and the temperature was lowered to -20°C while stirring for 1 hour. After the sample precipitated, it was allowed to stand for 1 hour, filtered, and the solid was collected; the filtrate was discarded. The collected solid was dissolved in water to a concentration of 57.5 g / L (0.4 L), filtered through a 0.22 μm filter, and then freeze-dried to obtain 23.9 g of Tirzepatide.

[0085] The purification recovery rate was 73.6%, the purity was 99.65%, the maximum single impurity was 0.05%, the high molecular weight impurity was 0.02%, and the isopropanol residue was 10 ppm.

[0086] Example 4:

[0087] (1) Reverse-phase purification

[0088] Weigh 50.0 g of crude Tirzepatide (containing 32.5 g of Tirzepatide), dissolve in 5% sodium bicarbonate solution to a volume of 2 L, and filter through a 0.45 μm filter membrane. The preparative column was a 10 cm DAC packed with C18 packing material. Mobile phase A1 was a 0.1% TFA aqueous solution, adjusted to pH 7.2 with sodium hydroxide, and mobile phase B was acetonitrile. The mixture was equilibrated with 90% A1 + 10% B for 10 min at a flow rate of 240 mL / min and a wavelength of 280 nm. After loading the sample, gradient elution was performed with an elution gradient of 40% B - 60% B / (100 min). Fractions with a purity ≥ 95.0% were collected. Fractions with a purity ≤ 60.0% and < 95.0% were combined, loaded, and then purified again by gradient elution with A1 and B, collecting fractions with a purity ≥ 95.0%.

[0089] All qualified fractions purified in the first step (i.e., fractions with a purity greater than 95% obtained in the first step) were collected and diluted with an equal volume of water for a second step purification. Mobile phase A2 was a 100 mM ammonium acetate aqueous solution, with the pH adjusted to 8.0 by ammonia. Mobile phase B was acetonitrile. The mixture was equilibrated at 90% A2 + 10% B for 10 min at a flow rate of 240 mL / min. The sample with a purity greater than 95% obtained in the first step was loaded onto the sample, followed by gradient elution at a gradient of 38% B - 58% B / (100 min). Fractions with a purity ≥99.0% and a single impurity ≤0.10% were collected.

[0090] (2) Salt conversion and purification

[0091] The chromatographic column used was the same as that used in step (1) for reverse-phase purification. Mobile phase A3 was a 100 mM potassium dihydrogen phosphate solution, with pH adjusted to 8.0 using potassium hydroxide. Mobile phase B was acetonitrile. The solution was equilibrated with 90% A3 + 10% B for 10 min at a flow rate of 240 mL / min. After loading the sample, the solution was washed with 90% A3 + 10% B for 15 min, then with 90% water + 10% B for 10 min, and finally with 40% water + 60% B. The main peak was collected to obtain the potassium salt fraction. The fraction volume was 1 L, and the Tirzepatide concentration was 23.2 g / L.

[0092] (3) Freeze drying

[0093] The potassium salt fraction was cooled to 10°C and maintained for 0.5 hours. 5 L of acetonitrile (90% acetonitrile ratio) was slowly added to the fraction, and the mixture was stirred for 0.5 hours while maintaining the temperature at 10°C. After the sample precipitated, it was allowed to stand for 1 hour, filtered, and the solid was collected; the filtrate was discarded. The collected solid was dissolved in water to a concentration of 58 g / L (0.4 L), filtered through a 0.22 μm filter, and then freeze-dried to obtain 24.4 g of Tirzepatide.

[0094] The purification recovery rate was 75.1%, the purity was 99.76%, the maximum single impurity was 0.07%, the high molecular weight impurity was 0.02%, and the acetonitrile residue was 10 ppm.

[0095] Example 5:

[0096] (1) Reverse-phase purification

[0097] Weigh 15000.0 g of crude Tirzepatide (containing 9750.0 g of Tirzepatide), dissolve in 5% sodium bicarbonate solution to a volume of 500 L, and filter through a 0.45 μm filter membrane. The preparative column was a 60 cm DAC packed with C8 packing material. Mobile phase A1 was a 0.1% TFA aqueous solution, with the pH adjusted to 7.1 using sodium hydroxide. Mobile phase B was acetonitrile. The mixture of 90% A1 and 10% B was equilibrated for 10 min at a flow rate of 8 L / min and a wavelength of 280 nm. After loading the sample, gradient elution was performed at a gradient of 36% B - 56% B / (120 min). Fractions with a purity ≥ 95.0% were collected. Fractions with a purity ≤ 60.0% and < 95.0% were combined, loaded, and then purified again by gradient elution with A1 and B, collecting fractions with a purity ≥ 95.0%.

[0098] All qualified fractions purified in the first step (i.e., fractions with a purity greater than 95% obtained in the first step) were collected and diluted with an equal volume of water for a second step purification. Mobile phase A2 was a 100 mM ammonium acetate aqueous solution, with the pH adjusted to 8.1 by ammonia. Mobile phase B was acetonitrile. The mixture was equilibrated at 90% A2 + 10% B for 10 min at a flow rate of 8 L / min. The sample with a purity greater than 95% obtained in the first step was loaded onto the sample and then subjected to gradient elution at a gradient of 34% B - 54% B / (120 min). Fractions with a purity ≥99.0% and a single impurity ≤0.10% were collected.

[0099] (2) Salt conversion and purification

[0100] The chromatographic column used was the same as that used in step (1) for reverse-phase purification. Mobile phase A3 was a 60 mM sodium acetate solution, with pH adjusted to 8.0 using sodium hydroxide. Mobile phase B was acetonitrile. The solution was equilibrated with 90% A3 + 10% B for 15 min at a flow rate of 8 L / min. After loading the sample, the solution was washed with 90% A3 + 10% B for 15 min, then with 90% water + 10% B for 10 min, and finally with 40% water + 60% B. The main peak was collected to obtain the sodium salt fraction. The fraction volume was 400 L, and the Tirzepatide concentration was 17.75 g / L.

[0101] (3) Freeze drying

[0102] The sodium salt fraction was cooled to 0°C and held for 1 hour. Then, 1800 L of acetonitrile (90% acetonitrile) was slowly added to the fraction, and the temperature was lowered to 0°C. The mixture was stirred for 5 hours. After the sample precipitated, it was allowed to stand for 2 hours, filtered, and the solid was collected. The filtrate was discarded. The collected solid was dissolved in water to a final volume of 120.0 L (59.2 g / L), filtered through a 0.22 μm filter, and then freeze-dried to obtain 7150.0 g of Tirzepatide.

[0103] The purification recovery rate was 73.3%, the purity was 99.67%, the maximum single impurity was 0.08%, the high molecular weight impurity was 0.02%, and the acetonitrile residue was 15 ppm.

[0104] Example 6:

[0105] (1) Reverse-phase purification

[0106] Weigh 50.0 g of crude Tirzepatide (containing 32.5 g of Tirzepatide), dissolve in 5% sodium bicarbonate solution to a volume of 2 L, and filter through a 0.45 μm filter membrane. The preparative column was a 10 cm DAC packed with C18 packing material. Mobile phase A1 was a 0.1% TFA aqueous solution, adjusted to pH 7.2 with sodium hydroxide, and mobile phase B was acetonitrile. The mixture was equilibrated with 90% A1 and 10% B for 10 min at a flow rate of 240 mL / min and a wavelength of 280 nm. After loading the sample, gradient elution was performed at a gradient of 40% B - 60% B / (100 min), and the fraction with a purity ≥95.0% was collected. Fractions with a purity ≤60.0% and <95.0% were combined, loaded, and then purified again by gradient elution with A1 and B, collecting the fraction with a purity ≥95.0%.

[0107] All qualified fractions purified in the first step (i.e., fractions with a purity greater than 95% obtained in the first step) were collected and diluted with an equal volume of water for a second step purification. Mobile phase A2 was a 100 mM ammonium acetate aqueous solution, with the pH adjusted to 8.0 by ammonia. Mobile phase B was acetonitrile. The mixture was equilibrated at 90% A2 + 10% B for 10 min at a flow rate of 240 mL / min. The sample with a purity greater than 95% obtained in the first step was loaded onto the sample, followed by gradient elution at a gradient of 38% B - 58% B / (100 min). Fractions with a purity ≥99.0% and a single impurity ≤0.10% were collected.

[0108] (2) Salt conversion and purification

[0109] The chromatographic column used was the same as that used in step (1) for reverse-phase purification. Mobile phase A3 was 50 mM sodium dihydrogen phosphate solution, adjusted to pH 7.8 with sodium hydroxide. Mobile phase B was acetonitrile. The solution was equilibrated with 90% A3 + 10% B for 10 min at a flow rate of 240 mL / min. After loading the sample, the solution was washed with 90% A3 + 10% B for 15 min, then with 90% water + 10% B for 10 min, and finally with 40% water + 60% B. The main peak was collected to obtain the sodium salt fraction. The fraction volume was 1.05 L, and the Tirzepatide concentration was 22.4 g / L.

[0110] (3) Freeze drying

[0111] The sodium salt fraction was cooled to -20°C and maintained for 0.5 hours. 5 L of acetonitrile (90% acetonitrile ratio) was slowly added to the fraction, and the mixture was stirred for 0.5 hours while maintaining the temperature at -20°C. After the sample precipitated, it was allowed to stand for 1 hour, filtered, and the solid was collected; the filtrate was discarded. The collected solid was dissolved in water to a concentration of 58.8 g / L in 0.4 L of water, filtered through a 0.22 μm filter, and then freeze-dried to obtain 24.7 g of Tirzepatide.

[0112] The purification recovery rate was 76.0%, the purity was 99.66%, the maximum single impurity was 0.07%, the high molecular weight impurity was 0.02%, and the acetonitrile residue was 10 ppm.

[0113] The inventors also conducted the following comparative examples, all using 10cm DAC columns packed with C8 packing material. The differences from Embodiment 1 described above are summarized in the table below.

[0114]

[0115]

[0116] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method of purifying Tirzepatide, characterized by, Includes the following steps: (1) Dissolve crude Tirzepatide peptide in ammonia or sodium bicarbonate solution, filter, and purify the crude peptide solution by reverse phase chromatography to obtain a purified sample. (2) The purified sample obtained in step (1) is subjected to reversed-phase high-performance liquid chromatography to change the salt. The buffer salt solution containing sodium or potassium is used as the mobile phase A3, and any one of acetonitrile, methanol, and isopropanol or a mixture of any proportions is used as the mobile phase B. The purified sample obtained in step (1) is eluted with mobile phase A3, water and mobile phase B to prepare the sodium or potassium salt fraction. (3) Cool the obtained sodium or potassium salt fraction to -20 to 10°C and then keep it warm; then add organic solvent to the fraction and control the temperature to -20 to 10°C to precipitate the sample; after the sample has completely precipitated, separate the solid and liquid, collect the solid, add water to dissolve the solid, filter and freeze dry.

2. The purification method according to claim 1, characterized in that, The filtration in step (1) uses a 0.45 μm filter membrane; Preferably, the reversed-phase chromatography in step (1) is a C18 column, a C4 column, or a C8 column; Preferably, the purity of the purified sample in step (1) is ≥99%, and the single impurity is ≤0.1%; Preferably, the reversed-phase chromatography purification step in step (1) includes primary purification and secondary purification, specifically including: 1) The obtained crude peptide solution was purified once by reversed-phase chromatography using a gradient elution with a mixed solution of mobile phase A1 and mobile phase B. The fraction with a purity ≥95.0% was collected to obtain the first purified fraction. 2) The obtained primary purified fraction is subjected to secondary purification by reversed-phase chromatography, using a gradient elution with a mixed solution of mobile phase A2 and mobile phase B, and the fraction with a purity ≥99.0% and a single impurity ≤0.10% is collected, which is the purified sample. The mobile phase A1 is a TFA aqueous solution with a volume percentage of 0.05% to 0.20%, and the pH value is adjusted with an alkaline solution. The pH value of the mobile phase A1 is 7.0 to 7.

2. Preferably, the pH of the mobile phase A1 is adjusted with NaOH; The mobile phase A2 is a 50-100 mM ammonium acetate aqueous solution, and the pH value of the mobile phase A2 is 8.0-8.2; Preferably, the pH of the mobile phase A2 is adjusted with ammonia. The mobile phase B is any one of acetonitrile, methanol, and isopropanol, or a mixture thereof in any proportion.

3. The purification method of claim 2, wherein, The mobile phase B mentioned in step 1) is acetonitrile, the elution gradient is (36-40)% to (56-60)% mobile phase B, and the elution time is 100 to 120 min; Alternatively, the mobile phase B mentioned in step 1) is methanol, the elution gradient is 60% to 90% mobile phase B, and the elution time is 60 to 80 min; Preferably, in step 1), the fractions with a purity of 60.0% ≤ 95.0% after gradient elution are combined, and then purified by reversed-phase chromatography using a mixed solution of mobile phase A1 and mobile phase B, collecting the fraction with a purity of ≥ 95.0%.

4. The purification method of claim 2, wherein, In step 2), the mobile phase B is acetonitrile, the elution gradient is (34-38)% to (54-58)% mobile phase B, and the elution time is 100-120 min. Alternatively, the mobile phase B mentioned in step 2) is methanol, the elution gradient is 60% to 90% mobile phase B, and the elution time is 80 to 100 min; Alternatively, in step 2), the mobile phase B is isopropanol, the elution gradient is 34%–54% mobile phase B, and the elution time is 100–120 min.

5. The purification method of claim 1, wherein, The reversed-phase high-performance liquid chromatography described in step (2) uses a C18 column, a C4 column, or a C8 column; The sodium-containing buffer salt mentioned in step (2) is selected from any one or any mixture of sodium acetate, sodium trifluoroacetate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, sodium sulfate, sodium bicarbonate, sodium carbonate, and sodium citrate. The potassium-containing buffer salt mentioned in step (2) is selected from any one or any mixture of potassium acetate, potassium trifluoroacetate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium chloride, potassium sulfate, potassium bicarbonate, potassium carbonate, and potassium citrate. Preferably, the pH value of the sodium- or potassium-containing buffer salt solution is 6.0 to 9.0, more preferably 7.0 to 8.0; Preferably, the concentration of the sodium or potassium buffer solution is 10–200 mM, more preferably 30–100 mM, and even more preferably 20–80 mM.

6. The purification method of claim 1, wherein, The method for preparing sodium or potassium salt fractions from the purified sample obtained in step (1) in step (2) includes: loading the purified sample obtained in step (1) onto the sample, washing it with 90% mobile phase A3 + 10% mobile phase B for 15-25 min, then washing it with 90% water + 10% mobile phase B for 10-20 min, and then washing it with (20-45)% water + (55-80)% mobile phase B, collecting the main peak, and obtaining sodium or potassium salt fractions.

7. The purification method of claim 1, wherein, In step (3), the sodium or potassium salt fraction is cooled to -20 to 10°C and then kept at that temperature for 0.5 to 5 hours. Preferably, in step (3), the obtained sodium or potassium salt fraction is cooled to -10 to 5°C.

8. The purification method of claim 1, wherein, In step (3), an organic solvent is added to the distillate, the temperature is controlled at -20 to 10°C, and the holding time is 0.5 to 5 hours; Preferably, in step (3), an organic solvent is added to the distillate, and the temperature is controlled at -10 to 5°C.

9. The purification method of claim 1, wherein, The organic solvent mentioned in step (3) is any one of acetonitrile, methanol, and isopropanol or a mixture thereof in any proportion; Preferably, the volume percentage of the organic solvent to the total volume of the organic solvent and the distillate is 75% to 95%, more preferably 80% to 90%.

10. The purification method of claim 1, wherein, The concentration of the solid after dissolution in step (3) is 10-200 g / L, preferably 20-120 g / L; Preferably, the filtration in step (3) is a 0.22-micron filtration.

Images