Reversed-phase liquid chromatography detection method for impurities in tilpotide and application of reversed-phase liquid chromatography detection method

Abstract

The invention relates to a reversed-phase liquid chromatography detection method for impurities in tilpotide, and belongs to the technical field of medicinal chemistry. The invention provides a reversed-phase liquid chromatography detection method for impurities in tilpotide, which comprises the following steps: taking an acidic aqueous solution as a mobile phase A, taking a mixed solution of an organic reagent, acid and water as a mobile phase B, taking a single reversed-phase chromatographic column as a stationary phase, taking a detector as an ultraviolet detector, and carrying out gradient elution on tilpotide by adopting high performance liquid chromatography. The invention further provides application of the reversed-phase liquid chromatography detection method to detection and identification of impurities generated in the production or storage process of the tilpotide. According to the method disclosed by the invention, the reversed-phase liquid chromatography impurity detection of the telpotide impurities can be completed only by using one chromatographic column, the number of the detected telpotide impurities is large, and the separation degree exceeds 1.5.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a reverse phase liquid chromatography detection method for telpotide impurities and an application thereof. Background Art

[0002] During the synthesis, storage and transportation of peptide drugs, various types of related structural impurities will be generated. The related structural impurities present in peptide drugs will affect their biological functions and efficacy. For example, the impurities of synthetic peptide drugs have a complex impurity spectrum, including impurities introduced by the starting materials, process-related impurities, and impurities generated by degradation. It also includes peptide synthesis impurities such as missing peptides, inserted peptides, broken peptides, differential peptides, staggered peptides, oxidized peptides, polymers, side chain groups are modified, disulfide bonds are modified, deamidation impurities, amino acetylation and other impurities. The presence of these impurities affects the quality of the product and increases the risk of patients using peptide drugs. Therefore, during the drug development stage and clinical use, the analysis and control of related structural impurities is one of the important factors that need to be considered. It is necessary to establish a simple, fast, feasible, stable and reliable detection method to detect and evaluate the quality of peptide drug products.

[0003] Tirzepatide is the world's first GIP / GLP-1R dual-target agonist developed by Eli Lilly and Company. It was approved by the FDA in May 2022 for the treatment of type 2 diabetes in adults. It has shown extremely significant blood sugar lowering and weight loss effects on patients with type 2 diabetes, becoming a new mainstream treatment option for patients with type 2 diabetes. Tirzepatide's treatment field has expanded to endocrine and metabolic diseases, respiratory diseases, etc. In addition to blood sugar lowering and weight loss, tirzepatide was newly launched in December 2024 for obstructive sleep apnea (OSA), officially entering the field of respiratory diseases. It is the first drug approved by the FDA for the direct treatment of OSA, breaking the long-term dilemma of no clear indication for the disease, marking a new era for OSA treatment.

[0004] At present, the public technology for detecting impurities of telpotide, such as CN117890508A, discloses a high-performance liquid chromatography method for telpotide, which uses unbonded ethylene bridge hybrid particles as the stationary phase (HILIC), is a normal phase chromatography, suitable for the separation of special impurities, and therefore has limited ability to separate impurities. The method used in the patent CN117890508A is only a supplement to the reverse phase chromatography (such as C18 or C8 filler), and is mainly developed for the special telpotide impurities that are difficult to separate, while many other types of polypeptide impurities also need to be detected by appropriate means in order to more accurately evaluate the quality of polypeptide products. Summary of the invention

[0005] The purpose of the present invention is to provide a reverse phase liquid chromatography detection method for telpotide impurities in view of the problems existing in the above-mentioned prior art. The method provides a detection method with good separation effect, strong operability and low requirements on instruments and chromatographic columns based on the structural characteristics of telpotide impurities.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The invention discloses a reverse phase liquid chromatography detection method for tepote impurities, which is characterized by: using an acidic aqueous solution as a mobile phase A, using a mixed solution of an organic reagent, an acid and water as a mobile phase B, using a single reverse phase chromatographic column as a stationary phase, using an ultraviolet detector as a detector, and using high performance reverse phase liquid chromatography gradient elution to tepote; the gradient range of the mobile phase B is 10% to 90%; the column temperature during the gradient elution is 25°C to 65°C; the flow rate during the gradient elution is 0.05-1.5 mL / min; the impurities are selected from β-Asp 15 、D-[Ile] 12 , D-[Ala] 18 , Endo–[Gly] 30 、Des-[Thr] 7 、Fragment-[Pyr 19 -39]、Des-[Gln] 19 .

[0008] A reverse phase liquid chromatography detection method for telpotide impurities described in the present invention has a further preferred technical solution that: the reverse phase chromatography column is preferably a C18 reverse phase chromatography column or a C8 reverse phase chromatography column.

[0009] A reverse phase liquid chromatography detection method for telpotide impurities described in the present invention has a further preferred technical solution that: the filling particle size of the chromatographic column is 1.5 to 10 um, preferably 1.7 um.

[0010] The reverse phase liquid chromatography detection method of telpotide impurities of the present invention has a further preferred technical solution that: the acidic aqueous solution comprises at least one of trifluoroacetic acid, acetic acid, phosphoric acid, hydrochloric acid and formic acid, preferably trifluoroacetic acid;

[0011] The reverse phase liquid chromatography detection method of telpotide impurities of the present invention has a further preferred technical solution as follows: the content of trifluoroacetic acid is in the range of 0.01% to 5.0%, preferably 0.1%;

[0012] A reverse phase liquid chromatography detection method for telpotide impurities according to the present invention, a further preferred technical solution is: the organic reagent is at least one of acetonitrile, methanol, ethanol and isopropanol, preferably acetonitrile;

[0013] The reverse phase liquid chromatography detection method of telpotide impurities described in the present invention has a further preferred technical solution that the volume ratio of the organic reagent to water in the mixed solution of the organic reagent, acid and water is 5:5 to 9:1, and preferably the volume ratio of the organic reagent to water is 9:1.

[0014] The reverse phase liquid chromatography detection method of telpotide impurities described in the present invention has a further preferred technical solution: the column temperature in the gradient elution process is preferably 40°C;

[0015] A reverse phase liquid chromatography detection method for telpotide impurities described in the present invention has a further preferred technical solution that the flow rate in the gradient elution process is preferably 0.1 mL / min.

[0016] A further preferred technical solution of the reverse phase liquid chromatography detection method for telpotide impurities described in the present invention is that the wavelength of the ultraviolet detector is 215 nm.

[0017] The reverse phase liquid chromatography detection method of telpotide impurities is used in detecting and identifying telpotide impurities generated during drug production or storage. In the application, the impurity is selected from β-Asp 15 、D-[Ile] 12 , D-[Ala] 18 , Endo–[Gly] 30 、Des-[Thr] 7 、Fragment-[Pyr 19 -39]、Des-[Gln] 19 .

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The method of the present invention adopts a reverse phase chromatography (C18 or C8 filler) impurity separation method for telpotide impurities, which only requires one chromatographic column and can be used to separate process impurities and degradation impurities in the synthesis and storage process of telpotide. In addition, the types and numbers of telpotide impurities detected are not only large, but also the separation degree exceeds 1.5. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the chromatogram of the telportopeptide sample detected by the present method in Example 3.

[0021] Figure 2 For example 3, the present method was applied to detect the presence of β-Asp 15 、D-[Ile] 12 , D-[Ala] 18, Endo–[Gly] 30 、Des-[Thr] 7 、Fragment-[Pyr 19 -39]、Des-[Gln] 19 Separation chromatograms of 7 impurities.

[0022] Figure 3 and Figure 4 Des-[Gln] in Example 3 19 Mass spectrum for impurity structure confirmation. DETAILED DESCRIPTION

[0023] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation modes of the present invention are described in detail below, but the contents of the present invention are not limited thereto.

[0024] β-Asp 15 、D-[Ile] 12 , D-[Ala] 18 , Endo–[Gly] 30 、Des-[Thr] 7 、Fragment-[Pyr 19 -39]、Des-[Gln] 19 7 impurities, all obtained by our company's directional synthesis, among which the impurity Des-[Gln] 19 The mass spectrum for structure confirmation is shown in Figure 3 and Figure 4 The other impurities were confirmed and labeled in the same way. The details are shown in Table 2.

[0025] Example 1, a reverse phase liquid chromatography detection method for impurities of tepote

[0026] The method uses an acidic aqueous solution as mobile phase A, a mixed solution of an organic reagent, an acid and water as mobile phase B, a single chromatographic column as the stationary phase, an ultraviolet detector as the detector, and high-performance reversed-phase liquid chromatography gradient elution to remove telpotide impurities.

[0027] The chromatographic column is a C18 reverse phase chromatographic column;

[0028] The filling particle size of the chromatographic column is 3um;

[0029] The acidic aqueous solution is a phosphoric acid aqueous solution, the phosphoric acid content is 0.1%, the volume ratio of the organic reagent to water in the mixed solution of the organic reagent, acid and water is 9:1, the acid is trifluoroacetic acid, and the trifluoroacetic acid content is 0.1%;

[0030] The gradient range of the mobile phase B is 10% to 90%;

[0031] During the gradient elution, the column temperature was 25°C and the flow rate was 1 mL / min;

[0032] The wavelength of the ultraviolet detector is 215 nm.

[0033] The reverse phase liquid chromatography detection method of telpotide impurities is used in detecting and identifying telpotide impurities generated during drug production or storage. The impurities are selected from β-Asp 15 、D-[Ile] 12 , D-[Ala] 18 , Endo–[Gly] 30 、Des-[Thr] 7 、Fragment-[Pyr 19 -39]、Des-[Gln] 19 , the separation degree can be greater than 1.5.

[0034] In the above embodiments, when the particle sizes of the chromatographic columns are 1.5um, 1.7um, 3um, 5um, 7um, and 10um respectively, the other conditions remain unchanged, and the method of the present invention is used to detect and identify the impurities of tepote produced during the production or storage of drugs, and the impurities are selected from β-Asp 15 、D-[Ile] 12 , D-[Ala] 18 , Endo–[Gly] 30 、Des-[Thr] 7 、Fragment-[Pyr 19 -39]、Des-[Gln] 19 , the separation degree can be greater than 1.5.

[0035] Example 2, a reverse phase liquid chromatography detection method for impurities of tepote

[0036] The method uses an acidic aqueous solution as mobile phase A, a mixed solution of an organic reagent and water as mobile phase B, a reversed-phase chromatographic column as a stationary phase, an ultraviolet detector as a detector, and high-performance reversed-phase liquid chromatography gradient elution to remove telpotide impurities.

[0037] The chromatographic column is a C8 chromatographic column;

[0038] The filling particle size of the chromatographic column is 10um;

[0039] The acidic aqueous solution is a formic acid aqueous solution with a formic acid content of 5.0%, and the organic reagent is isopropanol;

[0040] The volume ratio of the organic reagent to water in the mixed solution of the organic reagent and water is 9:1;

[0041] The gradient range of the mobile phase B is 20-80%;

[0042] During the gradient elution, the column temperature was 45°C and the flow rate was 1.5 mL / min;

[0043] The wavelength of the ultraviolet detector is 215 nm.

[0044] The reverse phase liquid chromatography detection method of telpotide impurities is used in detecting and identifying telpotide impurities generated during drug production or storage. The impurities are selected from β-Asp 15 、D-[Ile] 12 , D-[Ala] 18 , Endo–[Gly] 30 、Des-[Thr] 7 、Fragment-[Pyr 19 -39]、Des-[Gln] 19 , the separation degree can be greater than 1.5.

[0045] In the above embodiment, when the gradient elution column temperature is 25°C, 45°C, and 65°C respectively, and the other conditions remain unchanged, the method of the present invention is used to detect and identify the impurities of tepote produced during the production or storage of drugs, and the impurities are selected from β-Asp 15 、D-[Ile] 12 , D-[Ala] 18 , Endo–[Gly] 30 、Des-[Thr] 7 、Fragment-[Pyr 19 -39]、Des-[Gln] 19 , the separation degree can be greater than 1.5.

[0046] Example 3, detection experiment

[0047] Solution preparation:

[0048] (1) Telpotide sample solution: Weigh an appropriate amount of telpotide, dissolve it in water and dilute it to a sample solution with a concentration of 1 mg / ml.

[0049] (2) Telpotide resolution solution: Weigh appropriate amounts of telpotide and impurities, add water to dissolve and dilute to a resolution solution containing 1 mg / ml telpotide and 10 μg / ml impurities;

[0050] Chromatographic conditions:

[0051] Instrument: Thermo Fisher Vanquish High Performance Reversed Phase Liquid Chromatography Column: PeptideCSH TMC18130A (2.1mm*150mm, 1.7μm);

[0052] Detection wavelength: 215nm;

[0053] Flow rate: 0.1ml / min;

[0054] Column temperature: 40°C;

[0055] Injection volume: 20 μl;

[0056] Mobile phase A: water: trifluoroacetic acid = 100: 0.1 (v / v);

[0057] Mobile phase B: acetonitrile: water: trifluoroacetic acid = 90:10:0.1 (v / v / v);

[0058] Collection time: 75 minutes

[0059] Elution conditions: as shown in Table 1

[0060] Table 1 Gradient elution

[0061]

[0062] Separation effect: Figure 2 As shown in Table 3, the separation degrees of the seven impurities and the main component are all greater than 1.5, and the separation effect is good.

[0063] Table 2 Information on tepote and its impurities

[0064]

[0065]

[0066]

[0067] Table 3 Telpotide separation solution test results

[0068]

[0069] From the separation solution test results in Table 3, the separation degrees of all impurities and tepoxetine are greater than 1.5, and the separation effect is good.

Claims

1. A reverse phase liquid chromatography detection method for telpotide impurities, characterized in that: An acidic aqueous solution is used as mobile phase A, a mixed solution of an organic reagent, an acid and water is used as mobile phase B, a single reverse phase chromatographic column is used as the stationary phase, the detector is an ultraviolet detector, and high performance reverse phase liquid chromatography gradient elution is used for telportide; the gradient range of the mobile phase B is 10% to 90%; the column temperature during the gradient elution process is 25°C to 65°C; the flow rate during the gradient elution process is 0.05-1.5 mL / min; the impurity is selected from β-Asp 15 、D-[Ile] 12 , D-[Ala] 18 , Endo–[Gly] 30 、Des-[Thr] 7 、Fragment-[Pyr 19 -39]、Des-[Gln] 19 .

2. The reverse phase liquid chromatography detection method of telpotide impurities according to claim 1, characterized in that: The reverse phase chromatographic column is selected from a C18 reverse phase chromatographic column or a C8 reverse phase chromatographic column, and the filling particle size of the chromatographic column is 1.5 to 10 um.

3. The reverse phase liquid chromatography detection method of telpotide impurities according to claim 2, characterized in that: The filling particle size of the chromatographic column is 1.7 um.

4. The reverse phase liquid chromatography detection method of telpotide impurities according to claim 1, characterized in that: The acidic aqueous solution is selected from at least one of trifluoroacetic acid, acetic acid, phosphoric acid, hydrochloric acid and formic acid; the organic reagent is at least one of acetonitrile, methanol, ethanol and isopropanol.

5. The reverse phase liquid chromatography detection method of telpotide impurities according to claim 1, characterized in that: The trifluoroacetic acid content of the acidic aqueous solution is 0.01% to 5.0%, preferably 0.1%.

6. The reverse phase liquid chromatography detection method of telpotide impurities according to claim 1, characterized in that: The volume ratio of the organic reagent to water in the mixed solution of the organic reagent, acid and water is 5:5 to 9:

1.

7. The reverse phase liquid chromatography detection method of telpotide impurities according to claim 1, characterized in that: The volume ratio of organic reagent to water is 9:1; The column temperature during the gradient elution was 40°C; The flow rate during the gradient elution was 0.1 mL / min; The wavelength of the ultraviolet detector is 215 nm.

8. The use of the reverse phase liquid chromatography detection method of telpotide impurities according to any one of claims 1 to 7, characterized in that: The reverse phase liquid chromatography detection method is applied to detect and identify the impurities of telpotide produced during the production or storage of drugs, wherein the impurities are selected from β-Asp 15 、D-[Ile] 12 , D-[Ala] 18 , Endo–[Gly] 30 、Des-[Thr] 7 、Fragment-[Pyr 19 -39]、Des-[Gln] 19 .

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