Polypeptide aggregation trend prediction method based on dynamic light scattering

Abstract

The invention belongs to the field of stability evaluation of polypeptide bulk drugs, discloses a method for predicting the aggregation trend of tilpoitide based on a dynamic light scattering technology, and aims at solving the problems that an existing polypeptide aggregation detection technology is complex in pretreatment, cannot monitor in real time or is low in detection flux. The method comprises the following steps: dispersing polypeptide in a buffer solution with the pH value of 6.5-8.5, adding diethylene glycol-3-amino propyl ether and N '-(4-methoxy-2, 3, 6-trimethylbenzenesulfonyl)-L-arginine as cosolvents, and performing oscillation on an accelerated shaking table for 2-6 hours to prepare a polypeptide solution; determining aggregation when the particle size is greater than the theoretical size of the polypeptide monomer through dynamic light scattering detection; the buffer solution comprises one of a disodium hydrogen phosphate solution, a sodium chloride solution and a tris (2-chloroethyl) phosphate solution. The method disclosed by the invention can be used for rapidly and nondestructively monitoring the aggregation trend of the polypeptide in real time, further replaces a traditional detection method and intuitively represents the aggregation property of the polypeptide.

Description

Technical Field

[0001] This invention belongs to the field of stability evaluation of peptide raw materials, and specifically relates to a method for predicting peptide aggregation trends based on dynamic light scattering. Background Technology

[0002] The aggregation of peptides refers to the spontaneous aggregation of peptide molecules under certain conditions through non-covalent interactions, forming aggregates with specific structures and functions, such as hydrogen bonds, hydrophobic interactions, and electrostatic interactions. Telpotritide is a GLP-1 / GIP dual receptor agonist used to treat type II diabetes and obesity. However, as a peptide drug, it is prone to molecular aggregation due to factors such as concentration, pH, light exposure, and excipients. Aggregation not only reduces efficacy but may also trigger immunogenic reactions. Understanding peptide aggregation trends is crucial for optimizing its application in the biomedical field.

[0003] Currently, various techniques are used to study peptide aggregation behavior, such as scanning electron microscopy (SEM), ELISA reader fluorescence intensity, and insoluble particle detectors. However, these techniques have certain limitations. For example, SEM and ELISA reader fluorescence intensity require complex sample pretreatment and can only provide static structural information, not real-time monitoring of peptide aggregation. While insoluble particle detectors can detect samples at the nanoscale, they require large sample volumes and have low throughput.

[0004] Dynamic light scattering (DLS) is a rapid, non-destructive, and real-time monitoring analytical technique that has been widely used in the characterization of nanomaterials. DLS measures the fluctuations in the intensity of scattered light caused by the Brownian motion of particles in a solution, thereby obtaining information such as particle size distribution and diffusion coefficient.

[0005] Therefore, there is an urgent need for a rapid, accurate, and reproducible method for predicting the aggregation trend of telpoide. Applying dynamic light scattering technology to the study of peptide aggregation trends can monitor the self-assembly and aggregation process of peptides in solution in real time, providing a powerful means to gain a deeper understanding of the aggregation mechanism of peptides. Summary of the Invention

[0006] The purpose of this invention is to provide a method for predicting peptide aggregation trends based on dynamic light scattering, in order to overcome the limitations of existing peptide aggregation detection technologies, such as scanning electron microscopy and ELISA reader fluorescence intensity detection, which require complex sample pretreatment and can only obtain static structural information and cannot monitor in real time; and insoluble particulate detectors require large sample volumes and have low detection throughput, making it difficult to efficiently analyze the peptide aggregation process.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows: A method for predicting peptide aggregation trends, comprising, The peptides were dispersed in a buffer solution and shaken on a shaker for 2-6 hours to prepare a peptide solution. Aggregation is determined when the particle size of the peptide solution is larger than the theoretical size of the peptide monomer by dynamic light scattering detection.

[0008] Preferably, the buffer solution comprises one of the following: a disodium hydrogen phosphate solution with pH 6.5-8.5, a 0.9 wt% sodium chloride solution with pH 6.5-8.5, and a tri(2-chloroethyl) phosphate solution with pH 7.0-8.0; Preferably, the polypeptide includes telpoeptide.

[0009] Preferably, the mass-to-volume ratio of the polypeptide to the buffer solution is 0.1-50 mg: 1 mL.

[0010] Preferably, the theoretical monomer size of telpoeptide is 0.1-10 nm.

[0011] Preferably, when the detection particle size of telpolide is 10-1000 nm, it is determined that a polymer has formed; when the detection particle size of telpolide is greater than 1000 nm, it is determined that a flocculent aggregate has formed.

[0012] Preferably, the shaking temperature is 2-8℃ and the shaking speed is 10-300 rpm.

[0013] Preferably, the scattering angle of dynamic light scattering is 20-130°, and the detection temperature of dynamic light scattering is 23-27℃.

[0014] Preferably, the polypeptide solution includes a co-solvent.

[0015] Preferably, the co-solvent comprises diethylene glycol-3-aminopropyl ether and N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine.

[0016] Preferably, the mass-to-volume ratio of diethylene glycol-3-aminopropyl ether to the buffer solution is 1-5 mg:1 mL.

[0017] Preferably, the mass-to-volume ratio of N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine to the buffer solution is 1-10 mg:1 mL.

[0018] Diethylene glycol-3-aminopropyl ether and N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine, as a composite co-solvent, effectively reduce the probability of direct collisions and contacts between peptide molecules due to motion, significantly improve the solvent's solubility compatibility with telpoeptide, enhance the interaction between the solvent and peptide molecules, and prevent peptide aggregation and precipitation due to limited solubility; it also lowers the aggregation energy barrier of peptide molecules in solution, reduces local aggregation caused by differences in interfacial interactions, and ensures that the peptide remains in a stable dissolved state in the buffer solution.

[0019] Application of peptide aggregation trend prediction methods in stability evaluation of peptide active pharmaceutical ingredients.

[0020] Preferably, the polypeptide active pharmaceutical ingredient includes telpolide.

[0021] Preferably, the stability evaluation includes assessing the storage conditions or process parameters of the peptide active pharmaceutical ingredient.

[0022] More preferably, the cosolvent includes 2-(2-(2-morpholinoethoxy)ethoxy)ethanol, with a mass-to-volume ratio of 2-(2-(2-morpholinoethoxy)ethoxy)ethanol to the buffer solution of 0.5-3 mg:1 mL. When 2-(2-(2-morpholinoethoxy)ethoxy)ethanol is used in combination with diethylene glycol-3-aminopropyl ether and N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine as a cosolvent, they jointly construct a denser intermolecular interaction network, increasing the solubility of telpoeptide, thereby significantly weakening the intermolecular aggregation driving force and significantly improving the short-term dispersion stability and long-term storage stability of the peptide solution.

[0023] This invention also provides a method for preparing a polypeptide solution, comprising: Disperse the peptide in buffer solution and shake on a shaker for 2-6 hours to obtain a peptide solution.

[0024] Preferably, the polypeptide includes telpoeptide.

[0025] Preferably, the buffer solution comprises one of the following: a disodium hydrogen phosphate solution with pH 6.5-8.5, a 0.9 wt% sodium chloride solution with pH 6.5-8.5, and a tri(2-chloroethyl) phosphate solution with pH 7.0-8.0.

[0026] Preferably, the shaking temperature is 2-8℃.

[0027] Preferably, the rotational speed of the shaking table is 10-300 rpm.

[0028] More preferably, the shaking speed of the shaker is 50-60 rpm.

[0029] Preferably, the mass-to-volume ratio of the polypeptide to the buffer solution is 0.1-50 mg: 1 mL.

[0030] More preferably, a solubilizer may be added to the polypeptide solution.

[0031] More preferably, the co-solvent includes diethylene glycol-3-aminopropyl ether and N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine.

[0032] More preferably, the mass-to-volume ratio of diethylene glycol-3-aminopropyl ether to the buffer solution is 1-5 mg:1 mL.

[0033] More preferably, the mass-to-volume ratio of N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine to the buffer solution is 1-10 mg:1 mL.

[0034] More preferably, the cosolvent includes 2-(2-(2-morpholinoethoxy)ethoxy)ethanol.

[0035] More preferably, the mass-to-volume ratio of 2-(2-(2-morpholinoethoxy)ethoxy)ethanol to the buffer solution is 0.5-3 mg:1 mL.

[0036] This invention also provides a method for detecting clustering trends, comprising: At 23-27℃, dynamic light scattering was used to detect peptide solutions. When the detected particle size was larger than the theoretical size of the monomer, it was determined that the peptide had aggregated.

[0037] Preferably, the scattering angle of dynamic light scattering is 20-130°.

[0038] Preferably, the theoretical size of the monomer is 0.1-10 nm.

[0039] This invention employs dynamic light scattering technology as its core detection method. It utilizes diethylene glycol-3-aminopropyl ether and N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine as co-solvents to construct a buffer system with a pH of 6.5-8.5. The peptide solution is prepared by accelerated shaking at 2-8°C. Dynamic light scattering enables precise detection of peptide aggregation trends, thus offering the following advantages: rapid, non-destructive, and real-time monitoring of the peptide aggregation process; clear analysis of the effects of concentration, pH, light, and co-solvents on aggregation; effective assistance in optimizing storage conditions and process parameters for peptide raw materials; ensuring the stability and batch-to-batch uniformity of the preparation process; and strong support for in-depth research on peptide aggregation mechanisms. Therefore, this invention is a highly efficient, accurate, and reproducible method for predicting peptide aggregation trends in the field of peptide raw material stability evaluation. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the test results for peptide aggregation trend in Example 7. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0043] Example 1: Preparation of peptide solution: The peptide was dispersed in buffer solution and shaken on a shaker at 6℃ and 60 rpm for 4 h to obtain the peptide solution. The peptide was telpoeptide, and the buffer solution was a 10 mmol / L tri(2-chloroethyl) phosphate solution at pH 7.4. The mass-to-volume ratio of peptide to buffer solution was 1 mg:1 mL.

[0044] Aggregation trend detection: The peptide solution was detected using dynamic light scattering at 25°C. The scattering angle of the dynamic light scattering was 90°.

[0045] Example 2: The only difference between this example and Example 1 is the preparation of the polypeptide solution.

[0046] Preparation of peptide solution: The peptide was dispersed in buffer solution and shaken on a shaker at 6℃ and 60 rpm for 4 h to obtain the peptide solution. The peptide was telpoeptide, and the buffer solution was a 10 mmol / L tri(2-chloroethyl) phosphate solution at pH 7.4. The mass-to-volume ratio of peptide to buffer solution was 5 mg: 1 mL.

[0047] Example 3: The only difference between this example and Example 1 is the preparation of the polypeptide solution.

[0048] Preparation of peptide solution: The peptide was dispersed in buffer solution and shaken on a shaker at 6℃ and 60 rpm for 4 h to obtain the peptide solution. The peptide was telpoeptide, and the buffer solution was a 10 mmol / L tri(2-chloroethyl) phosphate solution at pH 7.4. The mass-to-volume ratio of peptide to buffer solution was 10 mg: 1 mL.

[0049] Example 4: The only difference between this example and Example 1 is the preparation of the polypeptide solution.

[0050] Preparation of peptide solution: The peptide was dispersed in buffer solution and shaken on a shaker at 6℃ and 60 rpm for 4 h to obtain the peptide solution. The peptide was telpoeptide, and the buffer solution was a 10 mmol / L tri(2-chloroethyl) phosphate solution at pH 7.4. The mass-to-volume ratio of peptide to buffer solution was 25 mg: 1 mL.

[0051] Example 5: The only difference between this example and Example 1 is the preparation of the polypeptide solution.

[0052] Preparation of peptide solution: The peptide was dispersed in buffer solution and shaken on a shaker at 6℃ and 60 rpm for 4 h to obtain the peptide solution. The peptide was telpoeptide, and the buffer solution was a 10 mmol / L tri(2-chloroethyl) phosphate solution at pH 7.4. The mass-to-volume ratio of peptide to buffer solution was 50 mg: 1 mL.

[0053] Example 6: The only difference between this example and Example 1 is the preparation of the polypeptide solution.

[0054] Preparation of peptide solution: The peptide was dispersed in buffer solution and shaken on a shaker at 6℃ and 60 rpm for 4 h to obtain the peptide solution. The peptide was telpoide, and the buffer solution was a 5 mmol / L disodium hydrogen phosphate solution at pH 6.8. The mass-to-volume ratio of peptide to buffer solution was 10 mg: 1 mL.

[0055] Example 7: The only difference between this example and Example 1 is the preparation of the polypeptide solution.

[0056] Preparation of peptide solution: The peptide was dispersed in buffer solution and shaken on a shaker at 6℃ and 60 rpm for 4 h to obtain the peptide solution. The peptide was telpoide, and the buffer solution was a 5 mmol / L disodium hydrogen phosphate solution at pH 7.0. The mass-to-volume ratio of peptide to buffer solution was 10 mg: 1 mL.

[0057] Example 8: The only difference between this example and Example 5 is the preparation of the polypeptide solution.

[0058] Preparation of peptide solution: The peptide was dispersed in buffer solution and shaken on a shaker at 6℃ and 60 rpm for 4 h to obtain the peptide solution. The peptide was telpoide, and the buffer solution was a 5 mmol / L disodium hydrogen phosphate solution at pH 8.0. The mass-to-volume ratio of peptide to buffer solution was 10 mg: 1 mL.

[0059] Example 9: The only difference between this example and Example 5 is the preparation of the polypeptide solution.

[0060] Preparation of the peptide solution: The peptide, diethylene glycol-3-aminopropyl ether, and N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine were dispersed in a buffer solution and shaken at 6°C and 60 rpm for 4 h to obtain the peptide solution. The peptide was telpoeptide, the buffer solution was a 10 mmol / L tri(2-chloroethyl) phosphate solution at pH 7.4, the mass-to-volume ratio of peptide to buffer was 50 mg:1 mL, the mass-to-volume ratio of diethylene glycol-3-aminopropyl ether to buffer was 2 mg:1 mL, and the mass-to-volume ratio of N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine to buffer was 2 mg:1 mL.

[0061] Example 10: The only difference between this example and Example 5 is the preparation of the polypeptide solution.

[0062] Preparation of the peptide solution: The peptide, diethylene glycol-3-aminopropyl ether, and N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine were dispersed in a buffer solution and shaken at 6°C and 60 rpm for 4 h to obtain the peptide solution. The peptide was telpoeptide, the buffer solution was a 10 mmol / L tri(2-chloroethyl) phosphate solution at pH 7.4, the mass-to-volume ratio of peptide to buffer was 50 mg:1 mL, the mass-to-volume ratio of diethylene glycol-3-aminopropyl ether to buffer was 2 mg:1 mL, and the mass-to-volume ratio of N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine to buffer was 4 mg:1 mL.

[0063] Example 11: The only difference between this example and Example 5 is the preparation of the polypeptide solution.

[0064] Preparation of the peptide solution: The peptide, diethylene glycol-3-aminopropyl ether, N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine, and 2-(2-(2-morpholinoethoxy)ethoxy)ethanol were dispersed in a buffer solution and shaken at 6°C and 60 rpm for 4 h to obtain the peptide solution. The peptide was telpoeptide, the buffer solution was a 10 mmol / L tri(2-chloroethyl) phosphate solution at pH 7.4, the mass-to-volume ratio of peptide to buffer was 50 mg:1 mL, the mass-to-volume ratio of diethylene glycol-3-aminopropyl ether to buffer was 2 mg:1 mL, the mass-to-volume ratio of N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine to buffer was 2 mg:1 mL, and the mass-to-volume ratio of 2-(2-(2-morpholinoethoxy)ethoxy)ethanol to buffer was 1 mg:1 mL.

[0065] Example 12: The only difference between this example and Example 5 is the preparation of the polypeptide solution.

[0066] Preparation of the peptide solution: The peptide, diethylene glycol-3-aminopropyl ether, N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine, and 2-(2-(2-morpholinoethoxy)ethoxy)ethanol were dispersed in a buffer solution and shaken at 6°C and 60 rpm for 4 h to obtain the peptide solution. The peptide was telpoeptide, the buffer solution was a 10 mmol / L tri(2-chloroethyl) phosphate solution at pH 7.4, the mass-to-volume ratio of peptide to buffer was 50 mg:1 mL, the mass-to-volume ratio of diethylene glycol-3-aminopropyl ether to buffer was 2 mg:1 mL, the mass-to-volume ratio of N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine to buffer was 2 mg:1 mL, and the mass-to-volume ratio of 2-(2-(2-morpholinoethoxy)ethoxy)ethanol to buffer was 2 mg:1 mL.

[0067] Comparative Example 1: The only difference between this comparative example and Example 1 is the preparation of the polypeptide solution.

[0068] Preparation of peptide solution: The peptide was dispersed in buffer solution and shaken on a shaker at 6℃ and 60 rpm for 4 h to obtain the peptide solution. The peptide was telpoide, and the buffer solution was a 5 mmol / L disodium hydrogen phosphate solution at pH 5.5. The mass-to-volume ratio of peptide to buffer solution was 10 mg: 1 mL.

[0069] Comparative Example 2: The only difference between this comparative example and Example 1 is the preparation of the polypeptide solution.

[0070] Preparation of peptide solution: The peptide was dispersed in buffer solution and shaken on a shaker at 6℃ and 60 rpm for 4 h to obtain the peptide solution. The peptide was telpoide, and the buffer solution was a 5 mmol / L disodium hydrogen phosphate solution at pH 9.0. The mass-to-volume ratio of peptide to buffer solution was 10 mg: 1 mL.

[0071] Comparative Example 3: The only difference between this comparative example and Example 1 is the preparation of the polypeptide solution.

[0072] Preparation of peptide solution: The peptide was dispersed in buffer solution while wrapped in aluminum foil and shaken on a shaker at 6℃ and 60 rpm for 4 h to obtain the peptide solution. The peptide was telpoide, and the buffer solution was a 5 mmol / L disodium hydrogen phosphate solution at pH 7.0. The mass-to-volume ratio of peptide to buffer solution was 10 mg: 1 mL.

[0073] Comparative Example 4: The only difference between this comparative example and Example 1 is the preparation of the polypeptide solution.

[0074] Preparation of peptide solution: The peptide was dispersed in buffer solution and subjected to visible light (4500 Lux) and ultraviolet irradiance (100 μW / cm²). 2 The peptide solution was obtained by irradiation under light for 2 hours and shaking on a shaker at 6℃ and 60 rpm for 4 hours. The peptide was telpoeptide, and the buffer solution was a 5 mmol / L disodium hydrogen phosphate solution at pH 7.0. The mass-to-volume ratio of peptide to buffer solution was 10 mg: 1 mL.

[0075] Comparative Example 5: The only difference between this comparative example and Example 1 is the preparation of the polypeptide solution.

[0076] Preparation of peptide solution: The peptide was dispersed in buffer solution and subjected to visible light (4500 Lux) and ultraviolet irradiance (100 μW / cm²). 2 The peptide solution was obtained by irradiation under light for 4 hours and shaking on a shaker at 6℃ and 60 rpm for 4 hours. The peptide was telpoeptide, and the buffer solution was a 5 mmol / L disodium hydrogen phosphate solution at pH 7.0. The mass-to-volume ratio of peptide to buffer solution was 10 mg: 1 mL.

[0077] Comparative Example 6: The only difference between this comparative example and Example 1 is the preparation of the polypeptide solution.

[0078] Preparation of peptide solution: The peptide was dispersed in buffer solution and subjected to visible light (4500 Lux) and ultraviolet irradiance (100 μW / cm²). 2 The peptide solution was obtained by irradiation under light for 6 hours and shaking on a shaker at 6°C and 60 rpm for 4 hours. The peptide was telpoide, and the buffer solution was a 5 mmol / L disodium hydrogen phosphate solution at pH 7.0. The mass-to-volume ratio of peptide to buffer solution was 10 mg:1 mL. Comparative Example 7: This comparative example differs from Example 9 only in the preparation of the peptide solution.

[0079] Preparation of peptide solution: The peptide and diethylene glycol-3-aminopropyl ether were dispersed in buffer solution and shaken on a shaker at 6℃ and 60 rpm for 4 h to obtain the peptide solution. The peptide was telpoeptide, the buffer solution was a 10 mmol / L tri(2-chloroethyl) phosphate solution at pH 7.4, the mass-to-volume ratio of peptide to buffer solution was 50 mg:1 mL, and the mass-to-volume ratio of diethylene glycol-3-aminopropyl ether to buffer solution was 2 mg:1 mL.

[0080] Comparative Example 8: This comparative example differs from Example 9 only in the preparation of the polypeptide solution.

[0081] Preparation of peptide solution: The peptide and N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine were dispersed in buffer solution and shaken at 6℃ and 60 rpm for 4 h to obtain the peptide solution. The peptide was telpoeptide, the buffer solution was a 10 mmol / L tri(2-chloroethyl) phosphate solution at pH 7.4, the mass-to-volume ratio of peptide to buffer solution was 50 mg:1 mL, and the mass-to-volume ratio of N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine to buffer solution was 2 mg:1 mL.

[0082] Comparative Example 9: The only difference between this comparative example and Example 1 is the preparation of the polypeptide solution.

[0083] Preparation of peptide solution: The peptide and L-arginine were dispersed in buffer solution and shaken on a shaker at 6℃ and 60 rpm for 4 h to obtain the peptide solution. The peptide was telpoeptide, the buffer solution was a 10 mmol / L tri(2-chloroethyl) phosphate solution at pH 7.4, the mass-to-volume ratio of peptide to buffer solution was 50 mg:1 mL, and the mass-to-volume ratio of L-arginine to buffer solution was 2 mg:1 mL.

[0084] Comparative Example 10: This comparative example differs from Example 1 only in the preparation of the polypeptide solution.

[0085] Preparation of peptide solution: The peptide was dispersed in buffer solution and allowed to stand at 6°C for 4 hours to obtain the peptide solution. The peptide was telpoeptide, and the buffer solution was a 10 mmol / L tri(2-chloroethyl) phosphate solution at pH 7.4. The mass-to-volume ratio of peptide to buffer solution was 1 mg:1 mL.

[0086] Comparative Example 11: This comparative example differs from Example 1 only in the preparation of the polypeptide solution.

[0087] Preparation of peptide solution: The peptide was dispersed in buffer solution and allowed to stand at 6°C for 4 hours to obtain the peptide solution. The peptide was telpoeptide, and the buffer solution was a 10 mmol / L tri(2-chloroethyl) phosphate solution at pH 7.4. The mass-to-volume ratio of peptide to buffer solution was 5 mg: 1 mL.

[0088] Comparative Example 12: This comparative example differs from Example 1 only in the preparation of the polypeptide solution.

[0089] Preparation of peptide solution: The peptide was dispersed in buffer solution and allowed to stand at 6°C for 4 hours to obtain the peptide solution. The peptide was telpoeptide, and the buffer solution was a 10 mmol / L tri(2-chloroethyl) phosphate solution at pH 7.4. The mass-to-volume ratio of peptide to buffer solution was 10 mg: 1 mL.

[0090] Comparative Example 13: The only difference between this comparative example and Example 1 is the preparation of the polypeptide solution.

[0091] Preparation of peptide solution: The peptide was dispersed in buffer solution and allowed to stand at 6°C for 4 hours to obtain the peptide solution. The peptide was telpoeptide, and the buffer solution was a 10 mmol / L tri(2-chloroethyl) phosphate solution at pH 7.4. The mass-to-volume ratio of peptide to buffer solution was 25 mg: 1 mL.

[0092] Comparative Example 14: This comparative example differs from Example 1 only in the preparation of the polypeptide solution.

[0093] Preparation of peptide solution: The peptide was dispersed in buffer solution and allowed to stand at 6°C for 4 hours to obtain the peptide solution. The peptide was telpoeptide, and the buffer solution was a 10 mmol / L tri(2-chloroethyl) phosphate solution at pH 7.4. The mass-to-volume ratio of peptide to buffer solution was 50 mg: 1 mL.

[0094] Experimental Example 1: Effect of concentration on peptide aggregation tendency test.

[0095] Test samples: Examples 1-5 for detecting clustering trends.

[0096] Test method: The particle size distribution and polydispersity index of the peptide solutions prepared according to Examples 1-5 were detected. Particles with a size less than 10 nm were monomers, particles with a size of 10-1000 nm were polymers, and particles with a size greater than 1000 nm were flocculent aggregates.

[0097] The results of the test on the effect of concentration on the aggregation trend of peptides are shown in Table 1.

[0098] Table 1. Results of the test on the effect of concentration on peptide aggregation tendency

[0099] In Examples 1-3, telpoeptide molecules were uniformly dispersed in the buffer solution with relatively large intermolecular distances, resulting in a low probability of contact and binding through non-covalent interactions. Consequently, the particle size was less than 10 nm and the polydispersity index was less than 0.18, maintaining a stable monomeric state. In Example 4, the concentration of telpoeptide was increased to 25 mg / mL. At this point, the peptide molecule density in the solution increased significantly, the average intermolecular distance shortened, and the strength and frequency of non-covalent interactions increased substantially. Some molecules began to spontaneously aggregate to form polymers, thus improving both the particle size and the polydispersity index. In Example 5, the concentration was further increased to 50 mg / mL. The probability of intermolecular contact between peptide molecules increased further, the aggregation network formed by non-covalent interactions became denser, and the polymers continued to grow and undergo secondary aggregation, eventually resulting in a small amount of flocculent precipitate with a significantly increased particle size. This indicates that telpoeptide begins to exhibit aggregation behavior at concentrations exceeding 10 mg / mL, and the higher the concentration, the more significant the aggregation trend.

[0100] Experimental Example 2: Test on the effect of pH on peptide aggregation tendency.

[0101] Test samples: Examples 6-8 and Comparative Examples 1-2 for detecting aggregation trends.

[0102] Test method: Based on the peptide solutions of Examples 6-8 and Comparative Examples 1-2, the particle size distribution and polydispersity index were detected. Particles with a size less than 10 nm were monomers, particles with a size of 10-1000 nm were polymers, and particles with a size greater than 1000 nm were flocculent aggregates.

[0103] The test results of the effect of pH on peptide aggregation tendency are shown in Table 2. The test results of peptide aggregation tendency in Example 7 are as follows. Figure 1 As shown.

[0104] Table 2. Results of the test on the effect of pH on peptide aggregation tendency

[0105] Comparative Example 1, under acidic conditions at pH 5.5, is close to the isoelectric point of telpoeptide, causing the net charge of telpoeptide molecules to approach zero. This significantly weakens the electrostatic repulsion between molecules, and the basic groups such as amino groups in telpoeptide molecules are prone to protonation. The interaction between hydrophilic groups and water molecules is weakened, the exposure of hydrophobic regions is significantly increased, and the binding ability between molecules through hydrophobic interactions is greatly enhanced, further promoting the formation of aggregate networks, ultimately resulting in a large number of flocculent aggregates. In Example 6, the weakly acidic environment reduces the proton concentration of the solution, significantly reducing the protonation of amino groups in telpoeptide molecules, decreasing the exposure of hydrophobic regions, and weakening the strength of non-covalent interactions between molecules. The aggregation tendency is significantly alleviated compared to Comparative Example 1. Although some molecules still undergo slight aggregation, no flocculent structure is formed. Examples 7 and 8 are neutral. In a weakly alkaline environment, the charge state of telpoeptide molecules tends to stabilize, with amino protonation and carboxyl deprotonation reaching equilibrium. The hydrophilic groups on the molecular surface can form stable hydrogen bonds with water molecules in the buffer solution, effectively shielding the hydrophobic regions. Non-covalent interactions between molecules are suppressed to the maximum extent, maintaining a stable monomeric state. In contrast, in a strongly alkaline environment, the negative charge density on the surface of telpoeptide molecules is too high. Although large-scale flocculent aggregation can be avoided through electrostatic repulsion, uneven local charge distribution leads to a small number of molecules forming small aggregates due to charge attraction. This indicates that telpoeptide has the strongest aggregation tendency under acidic conditions at pH 5.5, and the weakest aggregation tendency in the neutral to weakly alkaline range of pH 7.0-8.0. pH regulation can effectively intervene in its aggregation behavior by changing the molecular charge state and the degree of hydrophobic exposure.

[0106] Experimental Example 3: Test on the effect of light on peptide aggregation tendency.

[0107] Test samples: Comparative examples 3-6 for detecting aggregation trends.

[0108] Test method: Based on the peptide solutions of comparative examples 3-6, the particle size distribution and polydispersity index were detected. Particles smaller than 10 nm were monomers, particles between 10-1000 nm were polymers, and particles larger than 1000 nm were flocculent aggregates.

[0109] The results of the test on the effect of light on the aggregation trend of peptides are shown in Table 3.

[0110] Table 3. Results of the test on the effect of light on peptide aggregation tendency

[0111] Comparative Example 3, treated with aluminum foil wrapping to shield from light, showed that the telpoeptide molecules were not subjected to photoinduced structural damage, their peptide bond conformation remained stable, the interaction between the hydrophilic groups on the molecular surface and the buffer water molecules was not disrupted, and no additional hydrophobic regions were exposed. The probability of intermolecular contact binding through non-covalent interactions was extremely low, maintaining a stable monomeric state. In Comparative Example 4, 2 hours of light exposure and Comparative Example 5, 4 hours of light exposure, although not directly inducing large-scale aggregation, caused slight changes in the local peptide bond conformation of some telpoeptide molecules due to light energy, exposing a small number of hydrophobic groups and intermolecular non-covalent phases. The interaction strength was slightly improved, as evidenced by the simultaneous increase in particle size and polydispersity index. Although still within the monomer range, the risk of aggregation gradually increased with prolonged illumination. In Comparative Example 6, illumination for 6 hours led to the accumulation of conformational changes in the telpolide molecules, exposing a large number of hydrophobic regions. The hydrophobic interactions and hydrogen bonding between molecules were significantly enhanced, and the aggregation network formed by non-covalent interactions was initially constructed, eventually forming a polymer. This indicates that the aggregation tendency of telpolide significantly increased with prolonged illumination, and that light-induced aggregation of telpolide is one of the key factors in maintaining its solution stability.

[0112] Experimental Example 4: Test on the effect of solubilizers on peptide aggregation tendency.

[0113] Test samples: Examples 9-12 and Comparative Examples 7-9 for the detection of aggregation trends.

[0114] Test method: Based on the peptide solutions of Examples 5, 9-12 and Comparative Examples 7-9, the particle size distribution and polydispersity index were detected. Particles smaller than 10 nm were monomers, particles with a size of 10-1000 nm were polymers, and particles with a size greater than 1000 nm were flocculent aggregates.

[0115] The results of the test on the effect of cosolvents on the aggregation trend of peptides are shown in Table 4.

[0116] Table 4. Test results of the effect of cosolvents on peptide aggregation tendency

[0117] In Example 5, the telpoeptide molecules were dispersed in the buffer solution and easily aggregated spontaneously through hydrophobic interactions and hydrogen bonds, eventually forming flocculent aggregates with the most severe aggregation. In Example 9, diethylene glycol-3-aminopropyl ether and N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine were added as a composite cosolvent to reduce direct intermolecular contact and jointly inhibit aggregation, thus significantly reducing the particle size compared to Example 5. In Example 10, the amount of N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine was increased to further enhance the encapsulation of the hydrophobic region, continuously alleviating the aggregation and resulting in a slightly lower particle size than in Example 9. In Example 11, 2-(2-(2-morpholinoethoxy)ethoxy)ethanol was additionally introduced as a composite cosolvent to enhance the stability of the hydration layer, forming a denser interaction network, significantly weakening the intermolecular aggregation driving force, and further reducing the particle size. Example 12 increased the amount of 2-(2-(2-morpholinoethoxy)ethoxy)ethanol, which further enhanced the combined effect with diethylene glycol-3-aminopropyl ether and N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine, resulting in the smallest particle size among Examples 9-12 and the best aggregation inhibition effect. Comparative Example 7 only added diethylene glycol-3-aminopropyl ether, which could not effectively block hydrogen bond-mediated aggregation, and the particle size was higher than that of Example 9. Comparative Example 8 only added N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine, and the molecules were still prone to local aggregation due to hydrophobic interactions, resulting in a similarly high particle size. Comparative Example 9 used unmodified L-arginine, which lacked functional modification groups such as methoxy and trimethylbenzenesulfonyl groups in its structure. The interaction strength with telpoeptide was weak, and it could not effectively cover the hydrophobic region, resulting in an aggregation inhibition effect far lower than that of Example 9.

[0118] Experimental Example 5: Long-term stability test of cosolvents for peptide solutions.

[0119] Test samples: peptide solutions prepared in Examples 5, 9-12, and Comparative Examples 7-9.

[0120] Test method: The polypeptide solutions prepared in Examples 9-12 and Comparative Examples 7-9 were stored in a sealed container at 4°C in the dark. Samples were taken at 0, 30, 60 and 90 days of storage. Dynamic light scattering technology was used with a scattering angle of 90°C and a detection temperature of 25°C to determine the particle size distribution and polydispersity index of the samples at each time point. Particles smaller than 10 nm were identified as monomers, 10-1000 nm as polymers, and larger than 1000 nm as flocculent aggregates. The aggregation state of each sample at different storage times was recorded.

[0121] The results of the long-term stability test of the cosolvent for the peptide solution are shown in Table 5.

[0122] Table 5. Results of long-term stability tests of co-solvents for peptide solutions

[0123] Example 5: No co-solvent was added. The telpoeptide molecules were dispersed solely by the buffer solution. During long-term storage, the molecules easily aggregated spontaneously through hydrophobic interactions and hydrogen bonds, resulting in the worst solution stability. The polydispersity index was highest on day 30. Example 9: Diethylene glycol-3-aminopropyl ether and N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine were added as a composite co-solvent. Both inhibited aggregation, improving long-term stability, and the polydispersity index decreased compared to Example 5. Example 10: Based on Example 9, the amount of N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine was increased. More functional groups enhanced the encapsulation ability of the hydrophobic regions of the telpoeptide, further reducing the polydispersity index. Example 11: 2-(2-(2-morpholinoethoxy)ethoxy)ethanol was additionally introduced to enhance stability and, combined with… N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine forms a denser interaction network, resulting in a lower polydispersity index than in Example 10. Example 12, by increasing the amount of 2-(2-(2-morpholinoethoxy)ethoxy)ethanol, further enhanced the effect, achieving the best inhibition of aggregation, with the lowest polydispersity index on day 30. Comparative Example 7 only added diethylene glycol-3-aminopropyl ether as a single cosolvent, and Comparative Example 8 only added N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine as a single cosolvent, resulting in weak interaction with telpoeptide, with a polydispersity index of 0.54 on day 30, also higher than in Examples 9-12. Comparative Example 9 used unmodified L-arginine, lacking functional modification groups and hydration layer protection, resulting in a polydispersity index far higher than in Examples 9-12 on day 30, making it difficult to effectively inhibit long-term peptide aggregation.

[0124] Experimental Example 6: Testing of peptide aggregation trend using an insoluble particulate detector.

[0125] Test sample: Peptide solution prepared in Comparative Example 6.

[0126] Test method: The polypeptide solution prepared in Comparative Example 6 was tested for insoluble particles using an insoluble particle detector.

[0127] The test results of the insoluble particulate detector on the aggregation trend of peptides are shown in Table 6.

[0128] Table 6. Test results of peptide aggregation trend using an insoluble particulate detector.

[0129] From the perspective of the number of insoluble particles, the sample is qualified, indicating that from the dimension of the number of macroscopic micron-sized particles, there is no serious macroscopic foreign matter pollution or large-scale coarse particle aggregation in the sample; however, since the aggregation of tirzepatide gradually forms multimers from monomers and then to flocculent aggregates, the core particle sizes in the initial and middle stages of its aggregation are 10-1000 nm, which is much smaller than the minimum effective detection scale of 10 μm of the insoluble particle detector. As a result, this technology cannot identify the aggregation behavior at this stage, let alone distinguish the different aggregation states of monomers, multimers, and flocculent aggregates. Compared with the dynamic light scattering technology adopted in the present invention, the insoluble particle detector can only reflect the macroscopic particle pollution situation and cannot distinguish the aggregation trend, further confirming the limitations of traditional detection technologies in the research of polypeptide aggregation trend and highlighting the necessity and advantages of the detection method based on dynamic light scattering in the present invention.

[0130] Test Example 7: Testing the aggregation trend of polypeptides by scanning electron microscope.

[0131] Test sample: The polypeptide solution prepared in Comparative Example 6.

[0132] Test method: The polypeptide solution prepared in Comparative Example 6 was detected by scanning electron microscope.

[0133] It was found that tirzepatide presented uniformly dispersed rod-shaped particles with a diameter of about 40 nm, and only the static size and appearance characteristics of the particles could be obtained from the dimension of microscopic morphology. The scanning electron microscope detection requires complex pretreatment of the sample, and the pretreatment process may destroy the original dispersion state and aggregation equilibrium of tirzepatide in the solution, resulting in the observed rod-shaped particles not being the true existence form of the polypeptide in the solution, and there is a risk of sample state distortion; at the same time, the scanning electron microscope belongs to a static imaging technology, which can only capture the morphology and size of polypeptide particles at the moment of detection, and cannot track the dynamic aggregation process of tirzepatide evolving from monomers to multimers and flocculent aggregates in real time, making it difficult to reflect the time-dependent changes of the aggregation trend; moreover, the scanning electron microscope can only provide the morphology and single size data of the particles, and cannot output key parameters such as particle size distribution and polydispersity index like the dynamic light scattering technology, cannot distinguish single-dispersed multimers from mixed systems with different aggregation degrees, let alone accurately quantify the aggregation degree, and cannot judge whether the 40-nm rod-shaped particles are in a stable multimer form or an intermediate transition state during the aggregation process. This result further confirms the limitations of traditional microscopic imaging technologies in the research of polypeptide aggregation trend and highlights the significant advantages of the detection method based on dynamic light scattering in the present invention in ensuring the authenticity, dynamics, and quantification of detection.

[0134] Test Example 8: Testing the effect of shaker oscillation on the aggregation trend of polypeptides.

[0135] Test sample: Detection of the aggregation trend in Comparative Examples 10-14.

[0136] Test method: Based on the peptide solution of Comparative Example 10-14, the particle size distribution and polydispersity index were detected. Particles smaller than 10 nm were monomers, particles between 10-1000 nm were polymers, and particles larger than 1000 nm were flocculent aggregates.

[0137] The results of the test on the effect of shaker oscillation on the aggregation trend of peptides are shown in Table 7.

[0138] Table 7. Test results of the effect of shaking on peptide aggregation tendency

[0139] In Comparative Examples 10-14, the telpoeptide solutions prepared by simply standing for 4 hours without shaking exhibited particle sizes smaller than 10 nm, maintaining a stable monomeric state without aggregation. Under standing conditions, telpoeptide molecules diffused solely through their slow Brownian motion, resulting in low intermolecular collision frequency and weak interaction strength. Comparative Examples 1-5 show that shaking accelerates the movement rate of telpoeptide molecules through mechanical action, significantly increasing the effective intermolecular collision frequency, rapidly triggering and capturing peptide aggregation behavior, evaluating the aggregation trend of peptides at different concentrations, and ensuring detection efficiency and accuracy.

[0140] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0141] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A method for predicting polypeptide aggregation trends, characterized in that: include, The peptides were dispersed in a buffer solution and shaken on a shaker for 2-6 hours to prepare a peptide solution. The peptide solution is detected by dynamic light scattering, and aggregation is determined when the detected particle size is larger than the theoretical size of the peptide monomer. The buffer solution comprises one of the following: a disodium hydrogen phosphate solution with pH 6.5-8.5, a 0.9 wt% sodium chloride solution with pH 6.5-8.5, and a tri(2-chloroethyl) phosphate solution with pH 7.0-8.0; The polypeptide includes telpoeptide.

2. The method for predicting polypeptide aggregation trends according to claim 1, characterized in that: The mass-to-volume ratio of the polypeptide to the buffer solution is 0.1-50 mg: 1 mL.

3. The method for predicting polypeptide aggregation trends according to claim 1, characterized in that: The theoretical monomer size of the telpoeptide is 0.1-10 nm.

4. The method for predicting polypeptide aggregation trends according to claim 1, characterized in that: When the particle size of the telpolide is 10-1000 nm, it is determined that a polymer has formed; when the particle size of the telpolide is greater than 1000 nm, it is determined that a flocculent aggregate has formed.

5. The method for predicting polypeptide aggregation trends according to claim 1, characterized in that: The shaking temperature is 2-8℃, and the shaking speed is 10-300rpm.

6. The method for predicting polypeptide aggregation trends according to claim 1, characterized in that: The scattering angle of the dynamic light scattering is 20-130°, and the detection temperature of the dynamic light scattering is 23-27℃.

7. The method for predicting polypeptide aggregation trends according to claim 1, characterized in that: The polypeptide solution includes a cosolvent comprising diethylene glycol-3-aminopropyl ether and N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine.

8. The method for predicting polypeptide aggregation trends according to claim 7, characterized in that: The mass-to-volume ratio of diethylene glycol-3-aminopropyl ether to the buffer solution is 1-5 mg:1 mL.

9. The method for predicting polypeptide aggregation trends according to claim 7, characterized in that: The mass-to-volume ratio of N'-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine to the buffer solution is 1-10 mg:1 mL.

10. The use of the polypeptide aggregation trend prediction method according to any one of claims 1-9 in the stability evaluation of polypeptide raw materials, characterized in that: The peptide active pharmaceutical ingredient includes telpolide, and the stability evaluation includes assessing the storage conditions or process parameters of the peptide active pharmaceutical ingredient.

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