A method for detecting peptides using capillary gel electrophoresis

Abstract

This invention relates to a method for detecting peptides with a molecular weight below 10 kDa using capillary gel electrophoresis. The capillary gel electrophoresis of this invention can accurately characterize peptide impurities, especially aggregates, and serves as an effective supplement to size exclusion chromatography. It overcomes the insufficient resolution of size exclusion chromatography in analyzing peptide aggregates, while simultaneously detecting covalently bound aggregates. As a complementary technique to size exclusion chromatography, it provides multi-dimensional analysis of peptide aggregates.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the analysis of peptides by capillary gel electrophoresis, and more particularly to a method for detecting peptides with a molecular weight of less than 10 kDa using capillary gel electrophoresis. Background Technology

[0002] Aggregation has always been an unavoidable challenge in the research and development of protein or peptide biopharmaceuticals. External or internal factors, such as high temperature and light exposure, can lead to protein or peptide aggregation, thereby reducing the quality and efficacy of such products and, in severe cases, potentially endangering patient safety and health. Therefore, quantitative analysis of aggregation content is of great significance for evaluating the quality, usability, and stability of protein or peptide drug formulations.

[0003] Capillary gel electrophoresis (CGE), an evolution of traditional slab gel electrophoresis, has gradually become a key tool for characterizing the size heterogeneity of molecules in many biopharmaceuticals due to its advantages such as automation, speed, high resolution, and online quantification. However, capillary gel electrophoresis, especially CGE coupled with SDS (CE-SDS), is primarily used for large molecular weight proteins such as antibodies. Its application in small molecular weight peptides (such as insulin, approximately 6 kDa) has been limited. We speculate that this is because peptide sample peaks are easily mixed with system peaks under current methods, leading to analytical difficulties. (2020 Pharmacopoeia of the People's Republic of China) <1393> The description of insulin injection solutions mentions the use of size exclusion chromatography (SUC) to detect high molecular weight species in liquid insulin formulations. However, the low column efficiency caused by adsorption in SUC is unavoidable, resulting in low separation efficiency and the inability to separate different types of aggregates. We believe that high-resolution CE-SDS can be used for the detection of peptide aggregates as a complementary technique to traditional SUC, meeting the current industry needs for peptide biopharmaceutical aggregate analysis. Summary of the Invention

[0004] The technical problem that this invention aims to solve is to provide a method for detecting peptides using capillary gel electrophoresis, since existing capillary gel electrophoresis mainly detects large molecular proteins (greater than 10 kDa). In addition, this technology can overcome the shortcomings of insufficient aggregate resolution in size exclusion chromatography, and can also detect covalently bound aggregates, serving as a complementary technique to size exclusion chromatography.

[0005] The present invention is implemented as follows:

[0006] This invention provides a method for detecting peptides with a molecular weight of less than 10 kDa using capillary gel electrophoresis, comprising the following steps:

[0007] 1) Dilute the polypeptide sample to be tested with sample buffer;

[0008] 2) Incubate the diluted sample from step 1);

[0009] 3) Perform capillary electrophoresis analysis on the samples after incubation in step 2).

[0010] According to a preferred embodiment of the present invention, the sample buffer is selected from Tris-HCl buffer with pH 9.0 and a Tris concentration of 100 mM.

[0011] Furthermore, the sample buffer contains concentrated sodium dodecyl sulfate, with a sodium dodecyl sulfate concentration of 10 mg / mL.

[0012] According to a preferred embodiment of the invention, the incubation temperature is 70°C. The incubation time is 10 minutes.

[0013] According to a preferred embodiment of the present invention, the concentration of the polypeptide is 0.25-1 mg / mL.

[0014] According to a preferred embodiment of the present invention, the incubated sample is injected at a voltage of 6-15 kV for 3-20 s. Preferably, the capillary electrophoresis analysis conditions in step 3) are: the incubated sample is injected at a voltage of 10 kV for 5 s.

[0015] According to a preferred embodiment of the present invention, the molecular weight of the polypeptide sample is in the range of 1-10 kDa.

[0016] The beneficial effects of this invention are as follows:

[0017] This invention optimizes the existing CE-SDS method, enabling the analysis of various peptides and developing a CE-SDS detection method for peptides with molecular weights below 10,000. This invention uses CE-SDS to analyze peptides, increasing the pathways for peptide analysis and detection. It overcomes the insufficient resolution of size exclusion chromatography in analyzing peptide aggregates, and simultaneously detects covalently bound aggregates, serving as a complementary technique to size exclusion chromatography, thus providing multi-dimensional analysis of peptide aggregates. Attached Figure Description

[0018] Figure 1 Results of SE-HPLC analysis of glargine insulin and its degradation products (a. glargine insulin reference standard; b. glargine insulin degraded by light for 1 month);

[0019] Figure 2 CE-SDS spectra of insulin glargine at different injection times (a. injection time 3s; b. injection time 3s; c. injection time 3s; d. injection time 3s);

[0020] Figure 3CE-SDS spectra of insulin glargine at different separation voltages (a separation voltage 6 kV; b separation voltage 8 kV; c separation voltage 10 kV; d separation voltage 12.5 kV; e separation voltage 15 kV);

[0021] Figure 4 Results of CE-SDS analysis of insulin glargine and its degradation products (a. insulin glargine reference standard; b. insulin glargine degraded by light for 1 month);

[0022] Figure 5 CE-SDS diagram of glargine insulin degradation products and protein markers (a) glargine insulin degraded by light after 1 month; b) protein marker with a molecular weight of 21 kDa;

[0023] Figure 6 Mobility-molecular weight curves of insulin glargine monomers, aggregates, and protein markers in CE-SDS;

[0024] Figure 7 MALDI-TOF-MS spectrum of glargine insulin degraded by light for 1 month;

[0025] Figure 8 Results of CE-SDS analysis of human insulin and its degradation products (a) human insulin control; b) human insulin degraded by light for 1 month;

[0026] Figure 9 Results of CE-SDS analysis of glucagon;

[0027] Figure 10 CE-SDS analysis results of oxytocin;

[0028] Figure 11 Results of CE-SDS analysis of thymopentin;

[0029] Figure 12 Results of CE-SDS analysis of a mixture of oxytocin, glucagon, and insulin glargine samples. Detailed Implementation

[0030] The present invention will be further illustrated below through specific embodiments. It must be noted that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0031] Example 1: Size Exclusion-High Performance Liquid Chromatography Analysis of Glargine Insulin (6063 Da) and its Aggregates

[0032] Undegraded and light-damaged insulin glargine samples were diluted to 1 mg / mL with ultrapure water. SE-HPLC analysis was performed using a G2000S column at 25℃ and a UV detector at 276 nm. The mobile phase contained 20 mM sodium phosphate and 150 mM arginine (pH 7.4). 20 μL of sample was then injected at a flow rate maintained at 0.8 mL / min. Results are as follows: Figure 1 The spectrum of the undamaged sample showed only one monomer peak, while the light-damaged sample showed an aggregate peak in addition to the insulin glargine monomer.

[0033] Example 2: Comparison of CE-SDS analysis of glargine insulin at different injection times

[0034] Glargine insulin samples were diluted to 1 mg / mL with electrophoresis sample buffer (10 mg / mL SDS, 100 mM Tris, pH 9.0), incubated in a water bath at 70 °C for 10 minutes, and analyzed by capillary gel electrophoresis. Samples were injected at 10.0 kV for 3 s, 5 s, 10 s, and 20 s, and separated at 10.0 kV. The monitoring wavelength was 220 nm.

[0035] The results show that, Figure 2 Under different injection times, insulin glargine can achieve complete separation from the system peak. As the injection time decreases, the number of plates and the resolution increase (Table 1), while the signal-to-noise ratio decreases. Therefore, the optimal injection time is 5 s.

[0036] Table 1. Comparison of plate number, resolution, and signal-to-noise ratio of glargine insulin main peak and system peak at different injection times.

[0037] Injection time (s) Number of plates Resolution Signal-to-noise ratio 20 5574±106 2.71±0.02 1289±226 10 9314±56 3.63±0.07 592±160 5 15951±201 4.18±0.10 302±78 3 29680±521 4.98±0.13 202±9

[0038] Example 3: Comparison of CE-SDS analysis of glargine insulin under different separation voltages

[0039] Glargine insulin samples were diluted to 1 mg / mL with electrophoresis sample buffer (10 mg / mL SDS, 100 mM Tris, pH 9.0), incubated in a water bath at 70 °C for 10 min, and analyzed by capillary gel electrophoresis. Samples were injected at 10.0 kV for 5 s and separated at 6, 8, 10, 12.5, and 15 kV, respectively, with a monitoring wavelength of 220 nm.

[0040] The results show that, Figure 3 Under different injection voltages, insulin glargine can be completely separated from the system peak (Table 2), with the preferred separation voltage being 10kV.

[0041] Table 2 Comparison of plate number, resolution, and signal-to-noise ratio of glargine insulin main peak and system peak under different separation voltages.

[0042] Separation voltage (kV) Number of plates Resolution Signal-to-noise ratio 6 15885±22 4.22±0.07 182±22 8 16101±585 4.19±0.05 241±69 10 15951±201 4.18±0.10 302±78 12.5 15491±130 4.03±0.09 183±31 15 15459±464 3.92±0.15 96±21

[0043] The optimized analytical conditions for peptides obtained through the above methods are as follows: 10kV injection for 5s, and separation at 10kV voltage.

[0044] Example 4: CE-SDS analysis of insulin glargine and its aggregates

[0045] Undamaged insulin glargine samples and light-damaged samples were diluted to 1 mg / mL with electrophoresis sample buffer (10 mg / mL SDS, 100 mM Tris, pH 9.0), incubated in a water bath at 70 °C for 10 minutes, and analyzed by capillary gel electrophoresis. The samples were injected at 10.0 kV for 5 seconds and separated at 10.0 kV. The monitoring wavelength was 220 nm.

[0046] The results show that, Figure 4 CE-SDS analysis of insulin glargine can separate two types of aggregates, after the introduction of another 21kDa protein marker ( Figure 5 The migration-molecular weight curves confirmed that the two aggregates were insulin glargine dimer and trimer, respectively. Figure 6 Furthermore, MADLI-TOF-MS was used to demonstrate that the molecular weight of the polymer conforms to both the insulin glargine dimer and trimer. Figure 7 Furthermore, CE-SDS exhibits superior separation and column efficiency compared to SE-HPLC, demonstrating that capillary gel electrophoresis can be used to detect peptide aggregates.

[0047] Example 5: CE-SDS analysis of human insulin (5808 Da) in its aggregates

[0048] Undamaged human insulin samples and light-damaged samples were diluted to 1 mg / mL with electrophoresis sample buffer (10 mg / mL SDS, 100 mM Tris, pH 9.0), incubated in a water bath at 70 °C for 10 minutes, and analyzed by capillary gel electrophoresis. The samples were injected at 10.0 kV for 5 s and separated at 10.0 kV. The monitoring wavelength was 220 nm.

[0049] The results show that, Figure 8 Human insulin has a good peak shape in CE-SDS. CE-SDS can effectively separate dimers and trimers when analyzing human insulin. Capillary gel electrophoresis can be used to detect peptide aggregates.

[0050] Example 6: CE-SDS analysis of glucagon (3485 Da)

[0051] Glucagon samples were diluted to 0.25 mg / mL with electrophoresis sample buffer (10 mg / mL SDS, 100 mM Tris, pH 9.0), incubated in a water bath at 70 °C for 10 minutes, and analyzed by capillary gel electrophoresis. Samples were injected at 10.0 kV for 5 seconds and separated at 10.0 kV. The monitoring wavelength was 220 nm.

[0052] The results show that, Figure 9 Glucagon shows good peak shape in CE-SDS, and capillary gel electrophoresis can be used to detect small molecule peptide samples.

[0053] Example 7: CE-SDS analysis of oxytocin (1007 Da)

[0054] Oxytocin samples were diluted to 0.25 mg / mL with electrophoresis sample buffer (10 mg / mL SDS, 100 mM Tris, pH 9.0), incubated in a water bath at 70 °C for 10 minutes, and analyzed by capillary gel electrophoresis. Samples were injected at 10.0 kV for 5 seconds and separated at 10.0 kV. The monitoring wavelength was 220 nm.

[0055] The results show that, Figure 10 Oxytocin exhibits a good peak shape in CE-SDS, and capillary gel electrophoresis can be used to detect small molecule peptide samples.

[0056] Example 8: CE-SDS analysis of thymopentin (680 Da)

[0057] Thymopentin samples were diluted to 0.25 mg / mL with electrophoresis sample buffer (10 mg / mL SDS, 100 mM Tris, pH 9.0), incubated in a water bath at 70 °C for 10 minutes, and analyzed by capillary gel electrophoresis. Samples were injected at 10.0 kV for 5 seconds and separated at 10.0 kV. The monitoring wavelength was 220 nm.

[0058] The results show that, Figure 11 In CE-SDS, the main peak and the system peak of thymopentin overlapped, making further separation impossible. Capillary gel electrophoresis can detect small molecule peptides or peptide samples with a molecular weight range of 1-10 kDa.

[0059] Example 9: CE-SDS Analysis of Peptide Mixtures

[0060] Oxytocin, glucagon, and insulin glargine samples were diluted to 0.25 mg / mL with electrophoresis sample buffer (10 mg / mL SDS, 100 mM Tris, pH 9.0), incubated in a 70°C water bath for 10 minutes, and then the three samples were mixed for capillary gel electrophoresis analysis. The samples were injected at 10.0 kV for 5 seconds and separated at 10.0 kV. The monitoring wavelength was 220 nm.

[0061] The results show that, Figure 12 Oxytocin, glucagon, and insulin glargine samples were separated from each other in CE-SDS, and capillary gel electrophoresis can be used to detect mixtures of peptides.

[0062] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for detecting peptides using capillary gel electrophoresis, characterized in that, The polypeptide has a molecular weight range of 1-10 kDa, and is one or more of glargine insulin and its aggregates, human insulin and its aggregates, glucagon, and oxytocin; the method includes the following steps: 1) Dilute the peptide sample to be tested with sample buffer; the sample buffer is selected from Tris-HCl buffer with pH 9.0±0.1, the Tris concentration is 100 mM, the sample buffer contains sodium dodecyl sulfate, the sodium dodecyl sulfate concentration is 10 mg / mL; the concentration of peptide in the diluted sample is 0.25-1 mg / mL. 2) Incubate the diluted sample from step 1) at 70ºC for 10 minutes. 3) Perform capillary electrophoresis analysis on the samples after incubation in step 2). The conditions for capillary electrophoresis analysis are: the incubated samples are injected at a voltage of 6-15kV for 3-20 s.

2. The method according to claim 1, characterized in that, The conditions for capillary electrophoresis analysis in step 3) are: the incubated sample is injected at 10kV for 5 s.

3. The method according to claim 1, characterized in that, Glargine insulin aggregates are diploid and triploid, while human insulin aggregates are diploid and triploid.

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