Purification method of tilpotide

Abstract

The invention aims to provide a purification method of tilpoitide. The purification method mainly solves the technical problem that an existing purification method is poor in removal effect on single impurities such as specific deletion peptide Ser11 and deletion peptide Ile12. The method comprises the following steps: 1) a dissolving step: dissolving a tilpotide raw material by using a carbonate buffer solution, filtering and collecting filtrate for later use; and 2) carrying out gradient elution by taking silica gel as a stationary phase, a carbonate buffer solution as a phase A and a polar organic solvent as a phase B, and collecting components with the purity of more than 93% and the individual impurity of less than 1%. The preparation method comprises the following steps of (1) purifying the components in the first step, (2) performing chromatographic purification on the components purified in the first step, and collecting the components with the purity of more than 98% and the single impurity of less than 0.1%, (4) performing functional silica gel chromatographic purification on the components purified in the second step, and collecting the components with the purity of more than 99.5% and the single impurity of less than 0.1%, and (5) concentrating the components purified in the third step, adjusting the pH value, and drying to obtain the tilpotide.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide synthesis and purification, and specifically relates to a purification method for tirzepatide. Background Art

[0002] Tirzepatide is a dual agonist of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors, which can activate two incretin receptors, GIP and GLP-1, involved in blood glucose control through a dual mechanism of action. Clinical studies have shown that tirzepatide can significantly improve the blood glucose level of type 2 diabetes patients and also reduce the patient's weight. However, it is difficult to remove and separate impurities in the existing solid-liquid mixing method, which affects the yield and purity of tirzepatide. Patent CN112661815A discloses a purification method for Tirzepatide, in which the crude peptide of Tirzepatide is dissolved in purified water, and the pH is adjusted to 8.0 with ammonia water until completely dissolved. After obtaining the crude peptide solution, Tirzepatide is obtained through two-step purification. The yield of the inventive method is about 70%, the purity of the obtained Tirzepatide is above 99.0%, and the single impurity is below 0.15%. However, the invention cannot completely control the single impurities such as the specific deletion peptide Ser11 and deletion peptide Ile12 to below 0.1%, and the removal effect is poor. Summary of the Invention

[0003] The purpose of the present invention is to provide a purification method for tirzepatide with good purification effect, mainly solving the technical problem that the existing purification methods have poor removal effects on single impurities such as the specific deletion peptide Ser11 and deletion peptide Ile12.

[0004] The technical solution of the present invention is as follows: A purification method for tirzepatide, comprising the following steps: 1) Dissolve the tirzepatide raw material with a carbonate buffer solution, filter and collect the filtrate for standby; 2) Perform the first-step purification on the filtrate obtained in step 1), using silica gel as the stationary phase, carbonate buffer solution as phase A, and polar organic solvent as phase B, for gradient elution, and collect the components with a purity greater than 93% and a single impurity less than 1%; 3) Perform the second-step purification on the components obtained in step 2), using a reversed-phase packing as the stationary phase, acetic acid solution as phase A, and polar organic solvent as phase B, for gradient elution, and collect the components with a purity greater than 98% and a single impurity less than 0.1%; 4) Perform the third-step purification on the components obtained in step 3), using silica gel as the stationary phase, carbonate buffer solution as phase A, and polar organic solvent as phase B, for gradient elution, and collect the components with a purity greater than 99.5% and a single impurity less than 0.1%; 5) Concentrate, adjust the pH, and dry the components obtained in step 4) to obtain tirzepatide.

[0005] In terms of volume percentage, the gradient elution in step 2) is as follows: A%: 70% - 50%, B%: 30% - 50%, with linear gradient elution for 80 - 90 min.

[0006] In terms of volume percentage, the gradient elution in step 3) is as follows: A%: 70% - 60%, B%: 30% - 40%, with linear gradient elution for 80 - 90 min.

[0007] In terms of volume percentage, the gradient elution in step 4) is as follows: A%: 65% - 55%, B%: 35% - 45%, with linear gradient elution for 60 - 70 min.

[0008] The polar organic solvent in steps 2) - 4) is selected from one of methanol, ethanol, acetonitrile or isopropanol.

[0009] The reverse-phase packing material in step 3) is selected from one of tetraalkylsilane-bonded silica gel packing material, octylsilane-bonded silica gel packing material or octadecylsilane-bonded silica gel packing material.

[0010] The carbonate buffer solution in each step is selected from one of ammonium bicarbonate, sodium bicarbonate, sodium carbonate, potassium carbonate or ammonium carbonate solution, and its concentration is 15 - 25 mmol / L.

[0011] The beneficial effects of the present invention are as follows: 1. Dissolving the telotristat ethyl raw material with carbonate buffer solution can effectively improve the solubility and separation effect.

[0012] 2. The method has strong applicability and good removal effect on impurities with similar polarities.

[0013] 3. Even under the condition that the purity of the crude peptide in the existing solid-phase synthesis is poor, the method can still achieve a purity of more than 99.5%, a single impurity control of less than 0.1%, and a total purification yield as high as more than 70%. Description of the Drawings

[0014] Figure 1 It is the high-performance liquid chromatography detection chart of the final product in Example 1.

[0015] Figure 2 It is the high-performance liquid chromatography detection chart of the final product in Example 2.

[0016] Figure 3 It is the high-performance liquid chromatography detection chart of the final product in Example 3. Detailed Embodiments Example 1

[0017] 1) Weigh the telotristat ethyl raw material and dissolve it with sodium carbonate buffer solution at a concentration of 30 g / L, filter and collect the filtrate for standby; 2) The filtrate obtained in step 1) is subjected to the first purification. Using tetraalkylsilane-bonded silica gel packing as the stationary phase, 15 mmol / L ammonium carbonate solution as phase A, and ethanol as phase B, gradient elution is carried out. The method of gradient elution is: A%: 70% - 50%, B%: 30% - 50%, linear gradient elution for 80 - 90 min. Collect the components with a purity greater than 93% and a single impurity less than 1%; 3) The components obtained in step 2) are subjected to the second purification. Using octadecylsilane-bonded silica gel packing as the stationary phase, 2% acetic acid aqueous solution with a mass percentage concentration as phase A, and methanol as phase B, gradient elution is carried out. The method of gradient elution is: A%: 70% - 60%, B%: 30% - 40%, linear gradient elution for 80 - 90 min. Collect the components with a purity greater than 98% and a single impurity less than 0.1%; 4) The components obtained in step 3) are subjected to the third purification. Using octylsilane-bonded silica gel packing as the stationary phase, 20 mmol / L ammonium bicarbonate solution as phase A, and acetonitrile as phase B, gradient elution is carried out. The method of gradient elution is: A%: 65% - 55%, B%: 35% - 45%, linear gradient elution for 60 - 70 min. Collect the components with a purity greater than 99.5% and a single impurity less than 0.1%; 5) The components obtained in step 4 are concentrated, pH adjusted, and dried to obtain tirzepatide. After weighing, the recovery rate is calculated to be 72.6%. The HPLC chromatogram of the product is shown in Figure 1 。 Example 2

[0018] 1) Weigh the tirzepatide raw material and dissolve it with ammonium bicarbonate buffer salt at a concentration of 25 g / L, filter and collect the filtrate for standby; 2) The filtrate obtained in step 1 is subjected to the first purification. Using tetraalkylsilane-bonded silica gel packing as the stationary phase, 15 mmol / L ammonium carbonate solution as phase A, and methanol as phase B, gradient elution is carried out. The method of gradient elution is: A%: 70% - 50%, B%: 30% - 50%, linear gradient elution for 80 - 90 min. Collect the components with a purity greater than 93% and a single impurity less than 1%; 3) The components obtained in step 2) are subjected to the second purification. Using octadecylsilane-bonded silica gel packing as the stationary phase, 3% acetic acid aqueous solution with a mass percentage concentration as phase A, and methanol as phase B, gradient elution is carried out. The method of gradient elution is: A%: 70% - 60%, B%: 30% - 40%, linear gradient elution for 80 - 90 min. Collect the components with a purity greater than 98% and a single impurity less than 0.1%; 4) Perform the third purification on the components obtained in step 3). Use octadecylsilyl silica gel packing as the stationary phase, 25 mmol / L sodium bicarbonate solution as phase A, and acetonitrile as phase B for gradient elution. The method of gradient elution is: A%: 65% - 55%, B%: 35% - 45%, with linear gradient elution for 60 - 70 min. Collect the components with a purity greater than 99.5% and a single impurity less than 0.1%; 5) Concentrate, adjust the pH, and dry the components obtained in step 4) to obtain tirzepatide. After weighing, the recovery rate is calculated to be 73.4%. See the HPLC chromatogram of the product Figure 2 . Example 3

[0019] 1) Weigh the tirzepatide raw material and dissolve it with potassium carbonate buffer salt at a concentration of 30 g / L. Filter and collect the filtrate for standby; 2) Perform the first purification on the filtrate obtained in step 1). Use tetraalkylsilyl silica gel packing as the stationary phase, 15 mmol / L potassium bicarbonate solution as phase A, and methanol as phase B for gradient elution. The method of gradient elution is: A%: 70% - 50%, B%: 30% - 50%, with linear gradient elution for 80 - 90 min. Collect the components with a purity greater than 93% and a single impurity less than 1%; 3) Perform the second purification on the components obtained in step 2). Use octadecylsilyl silica gel packing as the stationary phase, 2% acetic acid aqueous solution with mass percentage concentration as phase A, and isopropanol as phase B for gradient elution. The method of gradient elution is: A%: 70% - 60%, B%: 30% - 40%, with linear gradient elution for 80 - 90 min. Collect the components with a purity greater than 98% and a single impurity less than 0.1%; 4) Perform the third purification on the components obtained in step 3). Use octadecylsilyl silica gel packing as the stationary phase, 20 mmol / L sodium carbonate solution as phase A, and acetonitrile as phase B for gradient elution. The method of gradient elution is: A%: 65% - 55%, B%: 35% - 45%, with linear gradient elution for 60 - 70 min. Collect the components with a purity greater than 99.5% and a single impurity less than 0.1%; 5) Concentrate, adjust the pH, and dry the components obtained in step 4) to obtain tirzepatide. After weighing, the recovery rate is calculated to be 71.8%. See the HPLC chromatogram of the product Figure 3 .

Claims

1. A method for purifying tirzepatide, characterized in that, It includes the following steps: 1) Dissolve the telotristat raw material with a carbonate buffer solution, filter and collect the filtrate for standby; 2) Perform the first purification on the filtrate obtained in step 1). Using silica gel as the stationary phase, carbonate buffer solution as phase A, and polar organic solvent as phase B, perform gradient elution, and collect the components with a purity greater than 93% and a single impurity less than 1%; 3) Perform the second purification on the components obtained in step 2). Using a reversed-phase packing as the stationary phase, acetic acid solution as phase A, and polar organic solvent as phase B, perform gradient elution, and collect the components with a purity greater than 98% and a single impurity less than 0.1%; 4) Perform the third purification on the components obtained in step 3). Using silica gel as the stationary phase, carbonate buffer solution as phase A, and polar organic solvent as phase B, perform gradient elution, and collect the components with a purity greater than 99.5% and a single impurity less than 0.1%; 5) Concentrate, adjust the pH, and dry the components obtained in step 4) to obtain telotristat.

2. The purification method according to claim 1, wherein, Calculated by volume percentage, the method of gradient elution in step 2) is: A%: 70% - 50%, B%: 30% - 50%, linear gradient elution for 80 - 90 min.

3. The purification method according to claim 1, wherein Calculated by volume percentage, the method of gradient elution in step 3) is: A%: 70% - 60%, B%: 30% - 40%, linear gradient elution for 80 - 90 min.

4. The purification method according to claim 1, characterized in that, Calculated by volume percentage, the method of gradient elution in step 4) is: A%: 65% - 55%, B%: 35% - 45%, linear gradient elution for 60 - 70 min.

5. The purification method according to claim 1, characterized in that, The polar organic solvent in steps 2) - 4) is selected from one of methanol, ethanol, acetonitrile or isopropanol.

6. The purification method according to claim 1, wherein The reversed-phase packing in step 3) is selected from one of tetraalkylsilane-bonded silica gel packing, octylsilane-bonded silica gel packing or octadecylsilane-bonded silica gel packing.

7. The purification method according to claim 1, wherein, The carbonate buffer solution in each step is selected from one of ammonium bicarbonate solution, sodium bicarbonate solution, sodium carbonate solution, potassium carbonate solution or ammonium carbonate solution, and its concentration is 15 - 25 mmol / L.

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