Method for detecting impurities in polypeptide medicine and application of method

Abstract

The invention provides a method for detecting impurities in a polypeptide drug and application thereof, and the detection method comprises the following steps: detecting a test solution and an impurity reference solution of the polypeptide drug by liquid chromatography, and calculating the impurity content according to the detection result; mobile phases adopted by the liquid chromatography comprise a mobile phase A and a mobile phase B; the mobile phase A and the mobile phase B are both phosphate solution-acetonitrile mixed solutions; the volume ratio of the phosphate solution in the mobile phase A is 70%-80%; the volume ratio of acetonitrile in the mobile phase B is 70%-80%; the polypeptide drug is P-017, the amino acid sequence is shown as SEQ ID NO: 1, and lysine at the 42nd site contains cholesterol succinic acid monoester group modification. The detection method can realize quantitative analysis of the impurities in the polypeptide, has the advantages of good impurity specificity, good repeatability, high precision and sensitivity, is accurate and reliable, and can be widely applied to detection of impurity residues in the polypeptide and preparation products thereof.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, specifically relating to a method for detecting impurities in polypeptide drugs and its application. Background Technology

[0002] P-017 is a broad-spectrum, highly effective coronavirus membrane fusion inhibitor with antiviral activity. It is a peptide drug used for the prevention and treatment of COVID-19. This product is a lipopeptide compound composed of 43 amino acids. P-017 interacts with the heptapeptide repeat region 1 (HR1) of the S2 subunit of the SARS-CoV-2 spike protein to form a heterohexahelical bundle (6-HB), thereby inhibiting the formation of homologous 6-HB between the HR1 and HR2 domains of the virus itself, blocking the fusion process between the virus and the host cell, and thus achieving antiviral effects.

[0003] P-017 is a white or off-white powder with the molecular formula C. 17 H 25 N3O2, peptide sequence is: Ac-Ser-Val-Val-Asn-Ile-Gln-Lys-Glu-Ile- 10 Asp-Arg-Leu-Asn-Glu-Val-Ala-Lys-Asn-Leu- 20 Asn-Glu-Ser-Leu-Ile-Asp-Leu-Gln-Glu-Leu- 30 Gly-Lys-Tyr-Glu-Gln-Tyr-Ile-Lys-Glu-Ala-Ala- 40 Ala-Lys-Lys(Chol)-NH2.

[0004] The synthesis of P-017 is complex, and its large molecular weight generates a series of impurities with similar properties, including deletion peptides, insertion peptides, mislinked peptides, transpeptides, and Asp / Asn-related impurities. These impurities may affect the efficacy and safety of the drug, and even pose a risk of genotoxicity. Furthermore, these impurities are very similar in structure and properties to P-017, making the separation of P-017 from these impurities challenging. No methods for the separation and detection of impurities in P-017 or other similarly structured peptides have been found.

[0005] Therefore, how to provide a method that can effectively detect impurities in P-017 has become an urgent problem to be solved. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for detecting impurities in polypeptide drugs and its application.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for detecting impurities in a polypeptide drug, the method comprising the following steps: using liquid chromatography to detect a test solution of the polypeptide drug and an impurity reference solution, and calculating the impurity content based on the detection results; the mobile phase used in the liquid chromatography includes mobile phase A and mobile phase B; both mobile phase A and mobile phase B are phosphate solution-acetonitrile mixed solutions; wherein, the volume percentage of phosphate solution in mobile phase A is 70%-80% (e.g., 70%, 72%, 74%, 75%, 76%, 78%, or 80%, preferably 75%); the volume percentage of acetonitrile in mobile phase B is 70%-80% (e.g., 70%, 72%, 74%, 75%, 76%, 78%, or 80%, preferably 78%). The polypeptide drug is P-017, and the amino acid sequence of P-017 is shown in SEQ ID NO:1, wherein the lysine residue at position 42 contains a cholesterol succinate monoester group (Chol) modified.

[0008] The peptide sequence of P-017 is as follows: Ac-Ser-Val-Val-Asn-Ile-Gln-Lys-Glu-Ile- 10 Asp-Arg-Leu-Asn-Glu-Val-Ala-Lys-Asn-Leu- 20 Asn-Glu-Ser-Leu-Ile-Asp-Leu-Gln-Glu-Leu- 30 Gly-Lys-Tyr-Glu-Gln-Tyr-Ile-Lys-Glu-Ala- 40 Ala-Ala-Lys-Lys(Chol)-NH2.

[0009] SEQ ID NO:1 SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKEAAKK.

[0010] The detection method in this invention can simultaneously and accurately detect multiple impurities in P-017, with high sensitivity, high accuracy, high reproducibility, and good durability, making it convenient for accurate detection and quality monitoring of impurities in P-017.

[0011] In this invention, the sample to be tested is mixed with a diluent to obtain a test solution; an impurity standard is mixed with a diluent to obtain an impurity reference solution. The diluent is a phosphoric acid-water solution, wherein the volume ratio of phosphoric acid to water is (0.1-0.4):100, preferably 0.2:100.

[0012] Preferably, the phosphate solution is an aqueous solution containing 6-12 mmol / L potassium dihydrogen phosphate and 40-45 mmol / L anhydrous disodium hydrogen phosphate.

[0013] Among them, 6-12 mmol / L potassium dihydrogen phosphate can be, for example, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, or 12 mmol / L, etc. 40-45 mmol / L anhydrous disodium hydrogen phosphate can be, for example, 40 mmol / L, 41 mmol / L, 42 mmol / L, 43 mmol / L, 44 mmol / L, or 45 mmol / L, etc.

[0014] Preferably, the pH value of the phosphate solution is in the range of 1.8-2.2, for example, it can be 1.8, 1.9, 2.0, 2.1 or 2.2, etc., preferably 2.0.

[0015] Preferably, the liquid chromatography employs gradient elution, and the gradient elution procedure is as follows: At min 0, the volume fraction of mobile phase A is 95%-95.5% (for example, it could be 95%, 95.1%, 95.2%, 95.3%, 95.4%, or 95.5%, etc.), and the remainder is mobile phase B; From 0 to 10 min, the volume fraction of mobile phase A changes uniformly from 95%-95.5% (e.g., 95%, 95.1%, 95.2%, 95.3%, 95.4%, or 95.5%) to 55%-55.5% (e.g., 55%, 55.1%, 55.2%, 55.3%, 55.4%, or 55.5%), with the remainder being mobile phase B. Between 10 and 40 minutes, the volume fraction of mobile phase A is uniformly changed from 55%-55.5% (e.g., it could be 55%, 55.1%, 55.2%, 55.3%, 55.4%, or 55.5%) to 30%-30.5% (e.g., it could be 30%, 30.1%, 30.2%, 30.3%, 30.4%, or 30.5%), with the remainder being mobile phase B. Between 40 and 55 minutes, the volume fraction of mobile phase A changes uniformly from 30%-30.5% (e.g., 30%, 30.1%, 30.2%, 30.3%, 30.4%, or 30.5%) to 0%-0.5% (e.g., 0%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%), with the remainder being mobile phase B. From 55 to 55.1 min, the volume fraction of mobile phase A changes uniformly from 0%-0.5% (e.g., 0%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%) to 95%-95.5% (e.g., 95%, 95.1%, 95.2%, 95.3%, 95.4%, or 95.5%), with the remainder being mobile phase B. From 55.1 to 65 min, the volume fraction of mobile phase A is 95%-95.5% (e.g., it can be 95%, 95.1%, 95.2%, 95.3%, 95.4%, or 95.5%), with the remainder being mobile phase B.

[0016] In this invention, the gradient elution procedure described above yields a spectrum with a good peak shape for the main peak P-017, a tailing factor of 1.7, and a peak-to-valley ratio greater than 1.00 between P-017 and adjacent impurities. This enables the separation and detection of five known impurities in P-017.

[0017] Preferably, the chromatographic column used in the liquid chromatography is an octadecylsilane-bonded chromatographic column.

[0018] In this invention, the main peak P-017 in the spectrum obtained by using an octadecylsilane-bonded chromatographic column has a good peak shape, and the tailing factors of each impurity are all between 0.9 and 1.4, with the tailing factor of P-017 being 1.7. All impurities can be well separated from P-017, and the peak-to-valley ratio of P-017 and each impurity is greater than 1.00, which can achieve the separation and detection of five known impurities in P-017.

[0019] Preferably, the chromatographic column used in the liquid chromatography has a particle size of 1.5-5 μm (e.g., 1.5 μm, 2 μm, 3 μm, 4 μm or 5 μm, etc.), a column length of 100-300 mm (e.g., 100 mm, 200 mm or 300 mm, etc.), and an inner diameter of 2-4.6 mm (e.g., 2 mm, 3 mm, 4 mm or 4.6 mm, etc.).

[0020] Preferably, the column temperature of the liquid chromatography column is 45-55℃ (e.g., 45℃, 46℃, 48℃, 50℃, 52℃, 54℃ or 55℃, etc.), preferably 48-52℃, and more preferably 50℃.

[0021] Preferably, the flow rate of the mobile phase in the liquid chromatography is 0.5-1.0 mL / min (e.g., 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min or 1.0 mL / min, etc.), more preferably 0.6-0.8 mL / min, and even more preferably 0.7 mL / min.

[0022] Preferably, the injection volume of the liquid chromatograph is 5-15 μL (e.g., 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL or 15 μL, etc.), and more preferably 10 μL.

[0023] Preferably, the detector used in the liquid chromatography is an ultraviolet detector.

[0024] Preferably, the impurity is selected from any one or a combination of at least two of impurity 19, impurity 34, impurity 35, impurity 51 or impurity 53; The name of impurity 19 is impurity [Lys] 43 [(Dihchol)]P-017, the amino acid sequence is shown in SEQ ID NO:1; wherein, the lysine at position 43 is modified with a hydrogenated cholesterol succinate monoester group (Dihchol); The peptide sequence of impurity 19 is as follows: Ac-Ser-Val-Val-Asn-Ile-Gln-Lys-Glu-Ile- 10 Asp-Arg-Leu-Asn-Glu-Val-Ala-Lys-Asn-Leu- 20 Asn-Glu-Ser-Leu-Ile-Asp-Leu-Gln-Glu-Leu- 30 Gly-Lys-Tyr-Glu-Gln-Tyr-Ile-Lys-Glu-Ala- 40 Ala-Ala-Lys-Lys(Dihchol)-NH2.

[0025] The impurity 34 is named impurity [Lys] 43 [Chol(O)]P-017, the amino acid sequence is shown in SEQ ID NO:1; wherein, the lysine at position 43 is modified with an oxidized cholesterol succinate monoester group (Chol(O)); The peptide sequence of impurity 34 is as follows: Ac-Ser-Val-Val-Asn-Ile-Gln-Lys-Glu-Ile- 10 Asp-Arg-Leu-Asn-Glu-Val-Ala-Lys-Asn-Leu- 20 Asn-Glu-Ser-Leu-Ile-Asp-Leu-Gln-Glu-Leu- 30 Gly-Lys-Tyr-Glu-Gln-Tyr-Ile-Lys-Glu-Ala-40 Ala-Ala-Lys-Lys[Chol(O)]-NH2.

[0026] The name of the impurity 35 is impurity [Endo-Ser] 1a P-017, the amino acid sequence is shown in SEQ ID NO:2, wherein the lysine at position 43 is modified with a cholesterol succinate monoester group (Chol); The peptide sequence of impurity 35 is as follows: Ac-Ser-Ser-Val-Val-Asn-Ile-Gln-Lys-Glu-Ile- 10 Asp-Arg-Leu-Asn-Glu-Val-Ala-Lys-Asn-Leu- 20 Asn-Glu-Ser-Leu-Ile-Asp-Leu-Gln-Glu-Leu- 30 Gly-Lys-Tyr-Glu-Gln-Tyr-Ile-Lys-Glu-Ala- 40 Ala-Ala-Lys-Lys(Chol)-NH2.

[0027] SEQ ID NO:2 SSVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKEAAKK.

[0028] The impurity 51 is named impurity [Asm] 10-11 P-017, the amino acid sequence is shown in SEQ ID NO:3, wherein the lysine at position 41 is modified with a cholesterol succinate monoester group (Chol); The peptide sequence of impurity 51 is as follows: Ac-Ser-Val-Val-Asn-Ile-Gln-Lys-Glu-Ile- 10 Asm-Leu-Asn-Glu-Val-Ala-Lys-Asn-Leu- 20 Asn-Glu-Ser-Leu-Ile-Asp-Leu-Gln-Glu-Leu- 30 Gly-Lys-Tyr-Glu-Gln-Tyr-Ile-Lys-Glu-Ala- 40 Ala-Ala-Lys-Lys(Chol)-NH2.

[0029] SEQ ID NO:3 SVVNIQKEIXLNEVAKNLNESLIDLQELGKYEQYIKEAAKK. X represents aspartic acid succinimide.

[0030] The name of the impurity 53 is impurity [Asp]. 13 P-017, the amino acid sequence is shown in SEQ ID NO:4, wherein the lysine at position 43 is modified with a cholesterol succinate monoester group (Chol); The peptide sequence of impurity 53 is as follows: Ac-Ser-Val-Val-Asn-Ile-Gln-Lys-Glu-Ile- 10 Asp-Arg-Leu-Asp-Glu-Val-Ala-Lys-Asn-Leu- 20 Asn-Glu-Ser-Leu-Ile-Asp-Leu-Gln-Glu-Leu- 30 Gly-Lys-Tyr-Glu-Gln-Tyr-Ile-Lys-Glu-Ala- 40 Ala-Ala-Lys-Lys(Chol)-NH2.

[0031] SEQ ID NO:4 SVVNIQKEIDRLDNEVAKNLNESLIDLQELGKYEQYIKEAAKK.

[0032] Preferably, the impurity includes impurity 19, and also includes any one or a combination of at least two of impurities 53, 51, 34, and 35.

[0033] In this invention, the use of the specific parameters and reagent materials described above can effectively improve the sensitivity and accuracy of detection.

[0034] As a preferred embodiment of the present invention, the method for detecting impurities in the polypeptide drug includes the following steps: (1) Mix the sample to be tested with the diluent to obtain the test solution; (2) Mix the impurity standard with the diluent to obtain the impurity reference solution; (3) Perform liquid chromatography detection on the test solution and the impurity reference solution, and calculate the impurity content based on the detection results; The mobile phase used in the liquid chromatography includes mobile phase A and mobile phase B; Both mobile phase A and mobile phase B are phosphate solution-acetonitrile mixed solutions; The mobile phase A is a phosphate solution-acetonitrile mixture with a high proportion of phosphate solution, and the volume proportion of phosphate solution is 70%-80%. The mobile phase B is a phosphate solution-acetonitrile mixture with a high acetonitrile content, where the volume percentage of acetonitrile is 70%-80%. The phosphate solution is an aqueous solution containing 6-12 mmol / L potassium dihydrogen phosphate and 40-45 mmol / L anhydrous disodium hydrogen phosphate; the pH range of the phosphate solution is 1.8-2.2. The liquid chromatography employs gradient elution, and the gradient elution procedure is as follows: At min 0, the volume fraction of mobile phase A is 95%-95.5%, with the remainder being mobile phase B; From 0 to 10 min, the volume fraction of mobile phase A changes uniformly from 95%-95.5% to 55%-55.5%, with the remainder being mobile phase B; Between 10 and 40 minutes, the volume fraction of mobile phase A changes uniformly from 55%-55.5% to 30%-30.5%, with the remainder being mobile phase B. Between 40 and 55 minutes, the volume fraction of mobile phase A changes uniformly from 30% to 30.5% to 0% to 0.5%, with the remainder being mobile phase B. From 55 to 55.1 min, the volume fraction of mobile phase A changed uniformly from 0%-0.5% to 95%-95.5%, with the remainder being mobile phase B; From 55.1 to 65 min, the volume fraction of mobile phase A was 95%-95.5%, with the remainder being mobile phase B.

[0035] The liquid chromatography uses an octadecylsilane-bonded column; the column particle size is 1.5-5 μm, the column length is 100-300 mm, and the column inner diameter is 2-4.6 mm; the column temperature is 45-55℃; and the mobile phase flow rate is 0.5-1.0 mL / min.

[0036] Secondly, the present invention provides the application of the method for detecting impurities in polypeptide drugs described in the first aspect in the quality testing of polypeptide P-017 and its formulation products.

[0037] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0038] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention provides a method for simultaneously separating and detecting multiple impurities in polypeptide P-017. The peak-to-valley ratio between P-017 and its adjacent impurity peaks is the lowest at 1.68. The peak-to-valley ratios between impurities 19, 34, 35, 51, and 53 and their adjacent impurity peaks are also the lowest at 1.68, significantly better than the peak-to-valley ratio standard (>1.00). The peak shapes are distinct, enabling the separation of P-017 from the five impurities with good peak-to-valley ratios. No method for detecting impurities in P-017 is described in the prior art. The method of this invention achieves effective separation and detection of impurities in P-017, which is beneficial for the quality control of P-017.

[0039] (2) This invention improves the peak shape of P-017 and its impurities by optimizing the conditions, thereby further enhancing the sensitivity of each component detection. The method of this invention uses a UV detector (DAD) for detection. Based on the characteristics of the detector itself, the main component self-comparison method is used to quantitatively calculate the impurities in P-017. Through recovery rate tests, the accuracy of this method is verified to be good, and it also has high reproducibility and robustness, which facilitates accurate detection and quality monitoring of impurities in peptide P-017. Attached Figure Description

[0040] Figure 1 This is a chromatogram of a mixed impurity control solution (system suitability).

[0041] Figure 2 This is the chromatogram of the test sample solution.

[0042] Figure 3 This is a chromatogram of a mixed impurity control solution at a flow rate of 0.6 mL / min.

[0043] Figure 4 This is a chromatogram of a mixed impurity control solution at a flow rate of 0.8 mL / min.

[0044] Figure 5 This is a chromatogram of a mixed impurity control solution at a column temperature of 48°C.

[0045] Figure 6 This is a chromatogram of a mixed impurity control solution at a column temperature of 52°C. Detailed Implementation

[0046] Explanation of abbreviations in this invention.

[0047] Ac-: Acetyl group, a modifying group at the N-terminus of a polypeptide.

[0048] -NH2: amino group, a C-terminal modification group of the polypeptide.

[0049] Serine: Serine.

[0050] Val: Valine.

[0051] Asn: Asparagine.

[0052] Ile: Isoleucine.

[0053] Gln: Glutamine.

[0054] Lys: Lysine.

[0055] Glu: Glutamic acid.

[0056] Asp: Aspartic acid.

[0057] Arg: Arginine.

[0058] Leu: Leucine; Ala: Alanine.

[0059] Gly: Glycine.

[0060] Tyr: Tyrosine.

[0061] Chol: Cholesterol succinate monoester group, a modifying group for Lys residues.

[0062] Dihchol (Diol): Hydrogenated cholesterol succinate monoester group, which is a modifying group of Lys43 residue in impurity 19.

[0063] Chol(O): Oxidized cholesterol succinate monoester group, which is a modifying group of Lys43 residue in impurity 34.

[0064] Asm: Aspartic acid succinimide, which is the amino acid-related group at positions 10-11 in impurity 51.

[0065] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0066] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0067] Example 1 This embodiment provides a method for detecting related substances in polypeptides, as detailed below: 1. Solution preparation.

[0068] Diluent: Phosphoric acid-water (0.2:100) (v:v).

[0069] Impurity 19 positioning solution: Take an appropriate amount of impurity 19 reference standard, accurately weigh it, add diluent to dissolve it and quantitatively dilute it to prepare a solution containing about 2.5 μg of impurity 19 per 1 mL.

[0070] Impurity 34 positioning solution: Take an appropriate amount of impurity 34 reference standard, accurately weigh it, add diluent to dissolve it and quantitatively dilute it to prepare a solution containing about 2.5 μg of impurity 34 per 1 mL.

[0071] Impurity 35 positioning solution: Take an appropriate amount of impurity 35 reference standard, accurately weigh it, add diluent to dissolve it and quantitatively dilute it to prepare a solution containing about 2.5 μg of impurity 35 per 1 mL.

[0072] Impurity 51 positioning solution: Take an appropriate amount of impurity 51 reference standard, accurately weigh it, add diluent to dissolve it and quantitatively dilute it to prepare a solution containing about 2.5 μg of impurity 51 per 1 mL.

[0073] Impurity 53 positioning solution: Take an appropriate amount of impurity 53 reference standard, accurately weigh it, add diluent to dissolve it and quantitatively dilute it to prepare a solution containing about 2.5 μg of impurity 53 per 1 mL.

[0074] Mixed impurity reference solution (system suitability solution): Take appropriate amounts of P-017 reference standard, impurity 19, impurity 34, impurity 35, impurity 51 and impurity 53 reference standards, accurately weigh them, dissolve them in diluent and quantitatively dilute them to prepare a solution containing approximately 0.5 mg of P-017 and approximately 2.5 μg each of impurity 19, impurity 34, impurity 35, impurity 51 and impurity 53 per mL.

[0075] Test solution: Take an appropriate amount of P-017, accurately weigh it, add diluent to dissolve it and quantitatively dilute it to prepare a solution containing about 0.5 mg of P-017 per 1 mL.

[0076] Self-control solution: Accurately measure 1 mL of the test solution into a 100 mL volumetric flask, dilute to the mark with diluent, and shake well.

[0077] The test solution and the solutions for each impurity are subjected to liquid chromatography for detection, and the types and contents of impurities are determined based on the detection results.

[0078] 2. The test conditions are as follows.

[0079] Instrument: High performance liquid chromatograph with DAD detector.

[0080] Column: Phenomenex Kinetex C18 (4.6 mm × 150 mm, 2.6 μm); C18 (4.6 mm × 50 mm) impurity trapping column and C18 pre-column were used.

[0081] Mobile phase A: A phosphate solution-acetonitrile mixture with a high phosphate solution content; the phosphate solution accounts for 75%.

[0082] Mobile phase B: A phosphate solution-acetonitrile mixture with a high acetonitrile content; the acetonitrile content is 78%.

[0083] The phosphate solution is an aqueous solution containing 9 mmol / L potassium dihydrogen phosphate and 45 mmol / L disodium hydrogen phosphate, with the pH adjusted to 2.0 by phosphoric acid.

[0084] Column temperature: 50℃. Injection volume: 10 μL. Detector: UV detector. Flow rate: 0.7 mL / min. Sample tray temperature: 5℃. Elution method: Gradient elution (Table 1). Elution time: 65 min.

[0085] Table 1 3. Exclusivity.

[0086] (1) Accurately measure 10 μL each of the above-prepared blank solvent, each impurity positioning solution and the mixed impurity reference solution, and inject them into the high performance liquid chromatograph. Record the chromatograms and results. The results are shown in Table 2 below.

[0087] Table 2 Note: If the relative deviation of retention time RD < 1%, it is considered to be a characteristic peak of the same substance.

[0088] (2) Conclusion.

[0089] The above samples were separated and detected using high-performance liquid chromatography (HPLC). The experimental results show that: Specific localization tests were performed using blank solvent. No extra characteristic peaks were generated in the chromatogram of the blank solvent, indicating that the blank solvent does not interfere with the determination of the main peak and various impurities.

[0090] System suitability tests were conducted using a mixed impurity reference solution, and the results are as follows: Figure 1 The results showed that the peak-to-valley ratios of impurities 53, 51, 34, 35, and 19 with the main peak of P-017 were all greater than 1.00, and the peak shapes were obvious, which enabled the separation of the main peak of P-017 from the four impurities.

[0091] The results of the test solution and impurity localization solution showed that the retention times of P-017, impurity 19, impurity 34, impurity 35, impurity 51, and impurity 53 were 36.655 min, 26.009 min, 32.597 min, 36.655 min, and 37.198 min, respectively. The chromatogram of the test solution is as follows: Figure 2 .

[0092] 4. Linearity and range.

[0093] (1) Solution preparation.

[0094] Diluent: Phosphoric acid-water (0.2:100) (v:v).

[0095] Linear solutions: Accurately weigh appropriate amounts of each impurity reference standard, add diluent to prepare each stock solution, accurately measure appropriate amounts, dilute with diluent to prepare a series of linear solutions, shake well, and the solution is obtained.

[0096] (2) Experimental steps and conclusions.

[0097] Accurately measure 10 µL of each of the above linear solutions and inject them into the high performance liquid chromatograph. Record the results, which are shown in Table 3 below.

[0098] Table 3 (3) Conclusion.

[0099] Impurity 34, within the concentration range of 0.156 μg / mL to 4.334 μg / mL, showed a linear equation of y = 5.8831x - 0.7578, with r = 0.998 > 0.990, indicating a significant linear regression and a good linear relationship.

[0100] For impurity 35, within the concentration range of 0.118 μg / mL to 4.711 μg / mL, the linear equation was y = 4.6352x -0.5834, r = 0.998 > 0.990, indicating a significant linear regression and a good linear relationship.

[0101] For impurity 51, within the concentration range of 0.159 μg / mL to 4.403 g / mL, the linear equation was y = 4.9846x - 0.7428, r = 0.998 > 0.990, indicating a significant linear regression and a good linear relationship.

[0102] Impurity 53, within the concentration range of 0.170 μg / mL to 4.732 μg / mL, showed a linear equation of y = 4.5802x - 0.6087, with r = 0.998 > 0.990, indicating a significant linear regression and a good linear relationship.

[0103] For impurity 19, within the concentration range of 0.109 μg / mL to 4.372 μg / mL, the linear equation was y = 4.9318x - 0.6476, r = 0.997 > 0.990, indicating a significant linear regression and a good linear relationship.

[0104] 5. Precision.

[0105] (1) Solution preparation.

[0106] Diluent: Phosphoric acid-water (0.2:100) (v:v).

[0107] Test solution: Take an appropriate amount of P-017, accurately weigh it, add diluent to dissolve it and quantitatively dilute it to prepare a solution containing about 0.5 mg of P-017 per mL. Prepare 6 parallel solutions.

[0108] (2) Experimental steps and conclusions.

[0109] Accurately measure 10 µL of the above test solution and inject it into the high performance liquid chromatograph. Record the results, which are shown in Table 4 below.

[0110] Table 4 Conclusions: Repeatability test: The contents of impurities 34 and 51 are both <0.1%, with a maximum RSD of 22.4 <25%. The contents of other impurities are all between 0.2% and 0.5%, with a maximum RSD of 9.1% <10%. Intermediate precision test: The contents of impurities 34 and 51 are both <0.1%, with a maximum RSD of 13.8 <25%. The content of impurity 53 is <0.2%, with an RSD of 4.5% <20%. The contents of other impurities are all between 0.2% and 0.5%, with a maximum RSD of 4.1% <10%. Precision test: The contents of impurities 34 and 51 are both <0.1%, with a maximum RSD of 24.9 <25%. The maximum RSD of other impurities is 9.5% <15%, and the impurity profiles are consistent, indicating that the method has good precision.

[0111] 6. Recovery rate.

[0112] (1) Solution preparation.

[0113] Diluent: Phosphoric acid-water (0.2:100) (v:v).

[0114] Linear stock solution with mixed impurities: Take appropriate amounts of impurity 19, impurity 34, impurity 35, impurity 51 and impurity 53 reference standards, weigh them accurately, dissolve them in diluent and dilute them quantitatively to prepare a solution containing approximately 10 μg each of impurity 19, impurity 34, impurity 35, impurity 51 and impurity 53 per mL.

[0115] Impurity reference solution: Accurately measure 2.5 mL of the mixed impurity linear stock solution into a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0116] Background solution: Take an appropriate amount of P-017, accurately weigh it, add diluent to dissolve it and quantitatively dilute it to prepare a solution containing approximately 2 mg of P-017 per 1 mL.

[0117] Preparation of R1-50% solution: Accurately measure 2.5 mL of the background solution into a 10 mL volumetric flask, accurately add 1.25 mL of the mixed impurity linear stock solution, dilute to the mark with diluent, and shake well. Prepare 3 parallel aliquots.

[0118] Preparation of R2-100% solution: Accurately measure 2.5 mL of the background solution into a 10 mL volumetric flask, accurately add 2.5 mL of the mixed impurity linear stock solution, dilute to the mark with diluent, and shake well. Prepare 3 parallel aliquots.

[0119] Preparation of R3-150% solution: Accurately measure 2.5 mL of the background solution into a 10 mL volumetric flask, accurately add 3.75 mL of the mixed impurity linear stock solution, dilute to the mark with diluent, and shake well. Prepare 3 parallel aliquots.

[0120] Preparation of R4-200% solution: Accurately measure 2.5 mL of the background solution into a 10 mL volumetric flask, accurately add 5 mL of the mixed impurity linear stock solution, dilute to the mark with diluent, and shake well. Prepare 3 parallel batches.

[0121] Control solution: Accurately measure 0.25 mL of the background solution into a 100 mL volumetric flask, dilute to the mark with diluent, and mix well. Prepare 3 parallel aliquots.

[0122] (2) Experimental steps and conclusions.

[0123] Accurately measure 10 µL of the above diluent, impurity reference solution, self-reference solution and spiked solution, and inject them into the high performance liquid chromatograph. Record the chromatograms and results, and calculate the recovery rate according to the external standard method. The results are shown in Table 5 below.

[0124] Table 5 Conclusion: At the three concentration levels of 50%, 100%, and 150%, the recoveries calculated using the external standard method were all between 90% and 110%. The maximum value was 108.7%, and the minimum value was 91.2%. The maximum RSD was 6.9%, and the minimum value was 2.0%, all less than 10.0%, indicating that the method has good accuracy.

[0125] 7. Durability.

[0126] (1) Solution preparation.

[0127] Diluent: Phosphoric acid-water (0.2:100) (v:v).

[0128] Test solution: Accurately weigh 10 mg of this product, place it in a 20 mL volumetric flask, dissolve and dilute to the mark with diluent, and shake well to obtain the test solution.

[0129] Self-control solution: Accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0130] Mixed impurity reference solution (system suitability solution): Take appropriate amounts of P-017 reference standard, impurity 19, impurity 34, impurity 35, impurity 51 and impurity 53 reference standards, accurately weigh them, dissolve them in diluent and quantitatively dilute them to prepare a solution containing approximately 0.5 mg of P-017 and approximately 2.5 μg each of impurity 19, impurity 34, impurity 35, impurity 51 and impurity 53 per mL.

[0131] (2) Experimental steps and conclusions.

[0132] Accurately measure 10 μL of each of the above solutions and inject them into the liquid chromatograph, recording the chromatograms. The results are shown in Table 6 (System Applicability Results) and Table 7 (Impurity Content in the Test Solution). Figures 3-6 .

[0133] Table 6 Table 7 Conclusion: Under the chromatographic conditions, with column temperature adjusted within ±2℃, the RSD of the main peak area in the system suitability solution was less than 2.0%, and the peak-to-valley ratio of P-017 to adjacent impurities was greater than 1.00, indicating a good system. When the phosphate solution pH was 1.8, the RSD of the main peak area in the system suitability solution was less than 2.0%, and the peak-to-valley ratio of P-017 to adjacent impurities was greater than 1.00. When the phosphate solution pH was 2.2, the RSD of the main peak area in the system suitability solution was less than 2.0%, and the peak-to-valley ratio of P-017 to adjacent impurities was 1.68, indicating a good system. At a flow rate of 0.6 mL / min, the RSD of the main peak area in the system suitability solution was less than 2.0%, and the peak-to-valley ratio of P-017 to adjacent impurities was greater than 1.00. At a flow rate of 0.8 mL / min, the RSD of the main peak area in the system suitability solution was less than 2.0%, and the peak-to-valley ratio of P-017 to adjacent impurities was greater than 1.00, indicating a good system.

[0134] After adjusting the column temperature, phosphate solution pH, and flow rate, the results for each impurity showed no significant difference compared to those under normal conditions. In summary, the column temperature, phosphate solution pH, and flow rate exhibited good robustness in the chromatographic conditions.

[0135] Example 2 Based on the chromatographic conditions of Example 1, the chromatographic column was changed to a C8 packed column, while other chromatographic conditions remained unchanged. P-017 and its five known impurities were detected. 10 μL of the mixed impurity reference solution prepared in Example 1 was accurately measured and injected into the high-performance liquid chromatograph. The results are shown in Table 8 below.

[0136] Table 8 Conclusion: High-performance liquid chromatography (HPLC) analysis of P-017 and its impurities using the two different chromatographic columns described above showed that the chromatographic column with C18 packing material from Example 1 produced a more accurate chromatogram. Figure 1 In the chromatographic column of Method 1, the main peak P-017 has a good peak shape, and the tailing factors of each impurity are between 0.9 and 1.4, with P-017 having a tailing factor of 1.7. All impurities can be well separated from P-017, and the peak-to-valley ratio of P-017 and each impurity is greater than 1.00, enabling the separation and detection of the five known impurities in P-017. In contrast, when using the column of Method 1, the main peak P-017 in the obtained spectrum has a poor peak shape and severe broadening. The peak-to-valley ratio between the main peak P-017 and the adjacent impurities (impurities 35 and 53) is less than 1.00, and the impurity peaks may be wrapped by the main peak, making it impossible to separate and detect the main peak and the five impurities.

[0137] Example 3 Selection of mobile phase solvent.

[0138] Based on the chromatographic conditions of Example 1, the mobile phase solvent was changed while other chromatographic conditions remained unchanged, and P-017 and its five known impurities were detected. 10 μL of the mixed impurity reference solution prepared in Example 1 was accurately measured and injected into the high-performance liquid chromatograph. The results are shown in Table 9 below.

[0139] Table 9 Conclusion: High-performance liquid chromatography (HPLC) analysis of P-017 and its impurities using the mobile phases of the three different solvents described above revealed that using phosphoric acid-water (0.2:100) as the solvent in Example 1 yielded the best chromatogram. Figure 1 In the results, the main peak P-017 showed a good peak shape, and the tailing factors of each impurity ranged from 0.9 to 1.4, with P-017 having a tailing factor of 1.7. All impurities could be well separated from P-017, with the peak-to-valley ratio of P-017 and adjacent impurities greater than 1.00, enabling the separation and detection of the five known impurities in P-017. In contrast, when using the dissolution and mobile phases of methods 1 and 2, the main peak P-017 in the obtained spectra showed a poor peak shape and severe broadening. The peak-to-valley ratio between the main peak P-017 and adjacent impurities was less than 1.00, and impurity peaks might be encased by the main peak, making it impossible to separate and detect the main peak and the five impurities.

[0140] Example 4 Selection of the amount of phosphate added in the mobile phase.

[0141] Based on the chromatographic conditions of Example 1, the amount of ion-pairing reagent (potassium dihydrogen phosphate-disodium hydrogen phosphate) added to the mobile phase was changed, while other chromatographic conditions remained unchanged. P-017 and its five known impurities were detected. 10 μL of the mixed impurity reference solution prepared in Example 1 was accurately measured and injected into the high-performance liquid chromatograph, and the results were recorded.

[0142] The mixed impurity reference solutions prepared in Example 1 were injected into a high-performance liquid chromatograph, and the results were recorded (as shown in Table 10).

[0143] Table 10 Conclusion: High-performance liquid chromatography (HPLC) analysis of P-017 and its impurities using mobile phases with different amounts of the above-mentioned ion-pairing reagents revealed that when using Method 1 with an addition of 4.5 mmol / L potassium dihydrogen phosphate and 22.5 mmol / L disodium hydrogen phosphate, the resulting spectrum showed poor main peak shape, a tailing factor of 3.0, and a peak-to-valley ratio of less than 1.00 for P-017 and adjacent impurities. However, the graph clearly showed problems with peak tail elution, with the chromatographic peaks of impurities 35, 53, and 19 being encapsulated. When using Method 2 with 18 mmol / L potassium dihydrogen phosphate and 90 mmol / L disodium hydrogen phosphate, P-017 was indistinguishable from impurities 53 and 19 in the resulting spectrum. When using the 9 mmol / L potassium dihydrogen phosphate and 45 mmol / L disodium hydrogen phosphate concentration from Example 1, the resulting spectrum (…) Figure 1 The main peak P-017 showed good peak shape, with a tailing factor of 1.7. The peak-to-valley ratio between P-017 and adjacent impurities was greater than 1.00, enabling the separation and detection of five known impurities in P-017. Considering the influence of ion-pairing reagents on the peak-to-valley ratio, instrument, and chromatographic column, the optimal dosage of ion-pairing reagent was selected as 9 mmol / L potassium dihydrogen phosphate - 45 mmol / L disodium hydrogen phosphate.

[0144] Example 5 Selection of elution gradient.

[0145] Based on the chromatographic conditions of Example 1, the elution gradient was changed while other chromatographic conditions remained unchanged, and P-017 and its five known impurities were detected. 10 μL of the mixed impurity reference solution prepared in Example 1 was accurately measured and injected into the high-performance liquid chromatograph, and the results were recorded (Table 11).

[0146] Table 11 Conclusion: High-performance liquid chromatography (HPLC) analysis of P-017 and its impurities using different elution gradients revealed that with method 1, the peak-to-valley ratio of P-017 to adjacent impurities in the resulting spectrum was less than 1.00. The graph clearly shows that low proportions of organic ions resulted in poor elution of the main peak and impurities, with the main peak and impurities being densely concentrated and poorly separated. With method 2, the impurity separation was improved compared to method 1, but impurities 19 and 53 showed encapsulation, and the peak-to-valley ratio of the main peak P-017 to subsequent impurities was less than 1.00, requiring further optimization. The spectrum obtained using the elution gradient in Example 1 (…) Figure 1 The main peak P-017 has a good peak shape, a tailing factor of 1.7, and a peak-to-valley ratio greater than 1.00 between P-017 and adjacent impurities, enabling the separation and detection of five known impurities in P-017.

[0147] The applicant declares that this invention illustrates the method for detecting impurities in P-017 and its application through the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials in the product of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0148] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0149] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for detecting impurities in a polypeptide drug, characterized in that, The detection method includes the following steps: detecting the test solution and impurity reference solution of the polypeptide drug using liquid chromatography, and calculating the impurity content based on the detection results; the mobile phase used in the liquid chromatography includes mobile phase A and mobile phase B; both mobile phase A and mobile phase B are phosphate solution-acetonitrile mixed solutions; wherein, the volume percentage of phosphate solution in mobile phase A is 70%-80%; and the volume percentage of acetonitrile in mobile phase B is 70%-80%. The polypeptide drug is P-017, and the amino acid sequence of P-017 is shown in SEQ ID NO:1, wherein the lysine residue at position 42 is modified with a cholesterol succinate monoester group.

2. The method for detecting impurities in a polypeptide drug according to claim 1, characterized in that, The phosphate solution is an aqueous solution containing 6-12 mmol / L potassium dihydrogen phosphate and 40-50 mmol / L anhydrous disodium hydrogen phosphate; Preferably, the pH range of the phosphate solution is 1.8-2.

2.

3. The method for detecting impurities in a polypeptide drug according to claim 1 or 2, characterized in that, The liquid chromatography employs gradient elution, and the gradient elution procedure is as follows: At min 0, the volume fraction of mobile phase A is 95%-95.5%, with the remainder being mobile phase B; From 0 to 10 min, the volume fraction of mobile phase A changes uniformly from 95%-95.5% to 55%-55.5%, with the remainder being mobile phase B; Between 10 and 40 minutes, the volume fraction of mobile phase A changes uniformly from 55%-55.5% to 30%-30.5%, with the remainder being mobile phase B. Between 40 and 55 minutes, the volume fraction of mobile phase A changes uniformly from 30% to 30.5% to 0% to 0.5%, with the remainder being mobile phase B. From 55 to 55.1 min, the volume fraction of mobile phase A changed uniformly from 0%-0.5% to 95%-95.5%, with the remainder being mobile phase B; From 55.1 to 65 min, the volume fraction of mobile phase A was 95%-95.5%, with the remainder being mobile phase B.

4. The method for detecting impurities in a polypeptide drug according to any one of claims 1-3, characterized in that, The liquid chromatography column used is an octadecylsilane bonded column.

5. The method for detecting impurities in a polypeptide drug according to any one of claims 1-4, characterized in that, The liquid chromatography column used has a particle size of 1.5-5 μm, a column length of 100-300 mm, and an inner diameter of 2-4.6 mm.

6. The method for detecting impurities in a polypeptide drug according to any one of claims 1-5, characterized in that, The column temperature of the liquid chromatography column is 45-55℃, preferably 48-52℃, and more preferably 50℃.

7. The method for detecting impurities in a polypeptide drug according to any one of claims 1-6, characterized in that, The flow rate of the mobile phase in the liquid chromatography is 0.5-1.0 mL / min, preferably 0.6-0.8 mL / min.

8. The method for detecting impurities in a polypeptide drug according to any one of claims 1-7, characterized in that, The impurity is selected from any one or a combination of at least two of impurity 19, impurity 34, impurity 35, impurity 51 or impurity 53; The name of impurity 19 is impurity [Lys] 43 [(Dihchol)]P-017, the amino acid sequence is shown in SEQ ID NO:1, wherein the lysine at position 43 is modified with a hydrogenated cholesterol succinate monoester group; The impurity 34 is named impurity [Lys] 43 [Chol(O)]P-017, the amino acid sequence is shown in SEQ ID NO:1, wherein the lysine at position 43 is modified with an oxidized cholesterol succinate monoester group; The name of the impurity 35 is impurity [Endo-Ser] 1a P-017, the amino acid sequence is shown in SEQ ID NO:2, wherein the lysine at position 43 is modified with a cholesterol succinate monoester group; The impurity 51 is named impurity [Asm] 10-11 P-017, the amino acid sequence is shown in SEQ ID NO:3, wherein the lysine at position 41 is modified with a cholesterol succinate monoester group; The name of the impurity 53 is impurity [Asp]. 13 P-017, the amino acid sequence is shown in SEQ ID NO:4, wherein the lysine at position 43 is modified with a cholesterol succinate monoester group.

9. The method for detecting impurities in a polypeptide drug according to any one of claims 1-8, characterized in that, The impurities include impurity 19, and also include any one or a combination of at least two of impurities 53, 51, 34, and 35.

10. The method for detecting impurities in the polypeptide drug according to any one of claims 1-9 is used in the quality testing of polypeptide P-017 and its formulation products.

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