Method for detecting interleukin 4 heteroplasmon and application thereof

Abstract

The invention relates to a method for detecting interleukin 4 variants and application of the method. The method comprises the following steps: diluting an interleukin 4 sample with an amphoteric electrolyte solution to obtain a sample solution to be detected; performing capillary electrophoresis-mass spectrometry online combined detection on the to-be-detected sample solution; the volume fraction of the amphoteric electrolyte solution is 0.2-5%; the conditions of capillary electrophoresis separation comprise that a capillary isoelectric focusing electrophoresis mode is adopted; the capillary tube is a neutral coating capillary tube; and the separation voltage is 1-100 kV. The detection method has the advantages of high separation efficiency and accurate analysis result, and can comprehensively obtain the information of the heteroplasmon in the interleukin 4 sample. In addition, the isoelectric point data of each interleukin 4 heteroplasmon can be accurately obtained by the method, so that accurate characterization of the sample is realized. The method disclosed by the invention can be applied to detection of interleukin drugs so as to meet the requirements on quality control of interleukin 4 in the market.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and more specifically, to a method for detecting interleukin-4 isoforms and an application thereof. Background Art

[0002] Interleukin, abbreviated as interleukin (IL), is a class of cytokines that play an important role in the body's immune regulation and homeostasis. Interleukin-4 is one of the multi-effect cytokines, secreted by type II helper T cells, which not only plays an important role in promoting B cell activation and antibody production, but also plays a key role in regulating humoral immunity and adaptive immunity.

[0003] Interleukin-4 is a glycoprotein that contains an N-glycosylation site. Complex and diverse glycosylation modifications significantly increase their structural heterogeneity, leading to functional differences, which have an important impact on its activity and stability as a drug molecule. In addition, during drug production, transportation and storage, proteins may also undergo a variety of changes, including changes in post-translational modifications and degradation. These changes may affect the structure and function of the protein, and thus affect the quality and stability of the drug. Therefore, accurate characterization of interleukin-4 heterogeneities is of great significance for drug quality control.

[0004] Capillary isoelectric focusing is a special micro-separation technology, which injects a sample mixture containing carrier ampholytes, buffers and auxiliary additives into a capillary. When a DC voltage is applied to both ends of the capillary, the carrier ampholytes can form a pH gradient within a certain range in the tube, and the sample components migrate toward the cathode or anode according to their charge. When the sample component migrates to the pH position corresponding to its isoelectric point (pI), its net charge is zero. Macroscopically, the component will focus at this point and stop further migration, thereby achieving effective separation of the components in complex samples.

[0005] Chinese patent CN117677848A discloses a method for characterizing charge variants of proteins of interest. The method first subjects a sample of the protein to capillary isoelectric focusing and collects fractions, then subjects the fractions to desalting size exclusion chromatography, and finally uses mass spectrometry to characterize the charge variants of the protein. The method has complex operating steps, partial sample loss in multiple steps, a long analysis time, increased experimental costs and equipment requirements, and multi-step processing may change the original structure of the protein, thereby affecting the accuracy of the final result. In addition, the patent application does not separate interleukin-4 isoforms, and there are relatively few documents on the separation of interleukin-4 isoforms using capillary isoelectric focusing.

[0006] Therefore, it is necessary to develop a detection method with high separation efficiency and accurate analysis results for interleukin-4 isoforms to meet the needs of interleukin-4 quality control in the market. Summary of the invention

[0007] The primary purpose of the present invention is to overcome the problem that there are few existing methods for detecting interleukin-4 isoforms, and to provide a method for detecting interleukin-4 isoforms. The method dilutes an interleukin-4 sample with an amphoteric electrolyte solution to obtain a sample solution to be tested; the sample solution to be tested is subjected to capillary electrophoresis-mass spectrometry online combined detection; the volume fraction of the amphoteric electrolyte solution is 0.2-5%; the conditions for capillary electrophoresis separation include: using a capillary isoelectric focusing electrophoresis mode; the capillary is a neutral coated capillary; and the separation voltage is 1-100 kV. The detection method has high separation efficiency and accurate analysis results, and can comprehensively obtain the isoform information in the interleukin-4 sample. In addition, the method can also accurately obtain the isoelectric point data of each interleukin-4 isoform, thereby realizing accurate characterization of the sample. The method of the present invention can be applied to the detection of interleukin drugs to meet the needs of quality control of interleukin-4 in the market.

[0008] A further object of the present invention is to provide application of the above method in interleukin drug detection.

[0009] Another object of the present invention is to provide application of the above method in detecting and / or distinguishing products obtained by structural heterogeneity changes of proteins.

[0010] The above-mentioned object of the present invention is achieved by the following technical solutions:

[0011] A method for separating interleukin-4 heterogeneous forms comprises the following steps:

[0012] S1. diluting the interleukin-4 sample with an amphoteric electrolyte solution to obtain a sample solution to be tested;

[0013] S2. Separating the sample solution in step S1 by capillary electrophoresis;

[0014] The volume fraction of the amphoteric electrolyte solution is 0.2-5%;

[0015] The conditions of the capillary electrophoresis include: using the capillary isoelectric focusing electrophoresis mode; the capillary is a neutral coating capillary; and the separation voltage is 1-100 kV.

[0016] The inventors of the present invention have found that the separation voltage of capillary electrophoresis can only provide a suitable electric field strength to achieve efficient separation of interleukin 4 isoforms if it is regulated within a certain range. If the separation voltage is too high, the protein migration speed may be too fast, resulting in broadening of the protein focusing zone and reduced resolution; conversely, if the separation voltage is too low, the protein migration speed will slow down, which may lead to prolonged separation time and even incomplete focusing, reducing separation efficiency.

[0017] The inventors of the present invention have also found that the use of neutral coated capillaries in the present invention can effectively reduce the nonspecific adsorption between interleukin-4 and its heteromeric molecules and the capillary wall, and can eliminate the effect of electroosmotic flow on migration, making protein migration dependent on the electrophoretic speed, thereby improving separation efficiency and reproducibility.

[0018] Preferably, the separation voltage is 10-50 kV, and may be 10 kV, 20 kV, 30 kV, 40 kV or 50 kV.

[0019] Preferably, the volume fraction of the amphoteric electrolyte solution is 0.5-2%, and can be specifically 0.5%, 1%, 1.5% or 2%.

[0020] Preferably, the ampholyte is at least one of ampholytes 8-10.5, ampholytes 3-10 or ampholytes 9-11.

[0021] More preferably, the ampholytes are ampholytes 3-10 and ampholytes 8-10.5. When the ampholytes are ampholytes 3-10 and ampholytes 8-10.5, the separation between different peak regions is clearer, which helps to more effectively separate and analyze interleukin-4 isoforms.

[0022] More preferably, the volume ratio of the ampholytes 3-10 to the ampholytes 8-10.5 is 1:(0.5-20), and can be specifically 1:0.5, 1:2, 1:4, 1:7, 1:10, 1:15 or 1:20.

[0023] Further preferably, the volume ratio of the ampholytes 3-10 to the ampholytes 8-10.5 is 1:(4-7). When the volume ratio of the ampholytes 3-10 to the ampholytes 8-10.5 is controlled within this range, the separation between different peak regions of interleukin-4 isoforms is clearer.

[0024] Preferably, the concentration of the interleukin-4 sample in the sample solution to be tested is 0.05-0.5 mg / mL.

[0025] More preferably, the concentration of the interleukin-4 sample in the test sample solution is 0.1-0.5 mg / mL.

[0026] Further preferably, the concentration of the interleukin 4 sample in the sample solution to be tested is 0.15-0.5 mg / mL. When the concentration of the interleukin 4 sample is controlled within this range, the separation effect of interleukin 4 isoforms is better, which is more conducive to the response of the mass spectrometry signal, and thus the interleukin 4 isoform information can be more comprehensively obtained.

[0027] Preferably, the capillary is made of elastic quartz.

[0028] Preferably, the inner diameter of the capillary is 40-50 μm.

[0029] Preferably, the capillary tube has a length of 65 to 130 cm.

[0030] Preferably, the capillary electrophoresis conditions also include: the injection method is gas pressure injection.

[0031] More preferably, the gas pressure of the gas pressure injection is 500-2000 mbar.

[0032] More preferably, the time of the gas pressure injection is 10 to 120 seconds.

[0033] More preferably, the injection step is: first inject a section of alkaline cathode liquid into the capillary injection port, then inject the sample solution to be tested, and finally move the capillary injection port to the acidic anode liquid.

[0034] Further preferably, the acidic anolyte is the Buffer A solution in the CR3520 kit, and the alkaline cathode solution is the Buffer B solution in the CR3520 kit.

[0035] Preferably, the sample solution to be tested also includes an internal standard protein solution, and the internal standard protein is selected from at least one of Cytochrome C, Infliximab or Ubiquitin.

[0036] The inventors of the present invention have found that the three internal standard proteins, Cytochrome C, Infliximab and Ubiquitin, do not interfere with the peak of interleukin-4 isoforms, and the peak is stable, which is conducive to constructing a fitting curve of migration time and isoelectric point, ensuring the accuracy of pH distribution and accurately obtaining isoelectric point data of interleukin-4 isoforms. In addition, the internal standard protein of the present invention can be used to evaluate the operating conditions of the entire electrophoresis system, including the integrity of the capillary, the stability of the electrophoresis conditions, and the consistency of sample preparation, ensuring the accuracy of the experimental results.

[0037] More preferably, the concentration of Cytochrome C in the internal standard protein solution is 0.01-0.3 mg / mL.

[0038] More preferably, the concentration of Infliximab in the internal standard protein solution is 0.1-0.3 mg / mL.

[0039] More preferably, the concentration of Ubiquitin in the internal standard protein solution is 0.01-0.3 mg / mL.

[0040] A method for detecting interleukin-4 isoforms, comprising the following steps:

[0041] S1. diluting the interleukin-4 sample with an amphoteric electrolyte solution to obtain a sample solution to be tested;

[0042] S2. The sample solution to be tested in step S1 is subjected to capillary electrophoresis-mass spectrometry online detection;

[0043] The volume fraction of the amphoteric electrolyte solution is 0.2-5%;

[0044] The conditions for capillary electrophoresis separation include: using capillary isoelectric focusing electrophoresis mode; the capillary is a neutral coating capillary; and the separation voltage is 1-100 kV.

[0045] Preferably, the separation voltage is 10-50 kV, and may be 10 kV, 20 kV, 30 kV, 40 kV or 50 kV.

[0046] Preferably, the volume fraction of the amphoteric electrolyte solution is 0.5-2%, and can be specifically 0.5%, 1%, 1.5% or 2%.

[0047] Preferably, the ampholyte is at least one of ampholytes 8-10.5, ampholytes 3-10 or ampholytes 9-11.

[0048] More preferably, the ampholytes are ampholytes 3-10 and ampholytes 8-10.5.

[0049] More preferably, the volume ratio of the ampholytes is 1:(0.5-20) of ampholytes 3-10 and ampholytes 8-10.5, and can be 1:0.5, 1:2, 1:4, 1:7, 1:10, 1:15 or 1:20.

[0050] Further preferably, the volume ratio of the ampholytes is 1:(4-7) for ampholytes 3-10 and ampholytes 8-10.5. When the volume ratio of ampholytes 3-10 and ampholytes 8-10.5 is controlled within this range, the separation between different peak regions of interleukin-4 isomers is clearer.

[0051] Preferably, the concentration of the interleukin-4 sample in the sample solution to be tested is 0.05-0.5 mg / mL.

[0052] More preferably, the concentration of the interleukin-4 sample in the test sample solution is 0.1-0.5 mg / mL.

[0053] Further preferably, the concentration of the interleukin 4 sample in the sample solution to be tested is 0.15-0.5 mg / mL. When the concentration of the interleukin 4 sample is controlled within this range, the separation effect of interleukin 4 isoforms is better, which is more conducive to the response of the mass spectrometry signal, and thus the interleukin 4 isoform information can be more comprehensively obtained.

[0054] Preferably, the capillary is made of elastic quartz.

[0055] Preferably, the inner diameter of the capillary is 40-50 μm.

[0056] Preferably, the capillary tube has a length of 65 to 130 cm.

[0057] Preferably, the capillary electrophoresis conditions also include: the injection method is gas pressure injection.

[0058] More preferably, the gas pressure of the gas pressure injection is 500-2000 mbar.

[0059] More preferably, the time of the gas pressure injection is 10 to 120 seconds.

[0060] More preferably, the injection step is: first inject a section of alkaline cathode liquid into the capillary injection port, then inject the sample solution to be tested, and finally move the capillary injection port to the acidic anode liquid.

[0061] Further preferably, the acidic anolyte is the Buffer A solution in the CR3520 kit, and the alkaline cathode solution is the Buffer B solution in the CR3520 kit.

[0062] Preferably, the sample solution to be tested also includes an internal standard protein solution, and the internal standard protein is selected from at least one of Cytochrome C, Infliximab or Ubiquitin.

[0063] More preferably, the concentration of Cytochrome C in the internal standard protein solution is 0.01-0.3 mg / mL.

[0064] More preferably, the concentration of Infliximab in the internal standard protein solution is 0.1-0.3 mg / mL.

[0065] More preferably, the concentration of Ubiquitin in the internal standard protein solution is 0.01-0.3 mg / mL.

[0066] Preferably, the detection conditions of the mass spectrometry include: a resolution of 17500 to 70000.

[0067] Preferably, the online coupling is achieved by a coaxial sheath flow electrospray ion source driven by electroosmotic flow.

[0068] More preferably, the voltage of electrospray in the coaxial sheath liquid electrospray ion source is 1.2 to 3 kV.

[0069] More preferably, the sheath fluid in the coaxial sheath fluid electrospray ion source is the BufferSL solution in the CR3520 kit.

[0070] More preferably, the flow rate of the sheath fluid is <50 nL / min.

[0071] More preferably, the distance between the tip of the electrospray needle in the coaxial sheath liquid electrospray ion source and the mass spectrometer is 1 to 10 mm.

[0072] More preferably, the distance between the tip of the electrospray needle and the tip of the capillary in the coaxial sheath liquid electrospray ion source is 0.1 to 3 mm.

[0073] The present invention also protects the application of the above method in interleukin drug detection.

[0074] The present invention also protects the use of the above method in detecting and / or distinguishing products obtained by interleukin-4 undergoing structural heterogeneity changes.

[0075] Preferably, the structural heterogeneity change is post-translational modification or degradation.

[0076] More preferably, the post-translational modification product is one or more of a glycosylation modification product, a phosphorylation modification product, a ubiquitination modification product, a methylation modification product or an acetylation modification product.

[0077] More preferably, the degradation product is one or more of a protein truncated form product, an amino acid degradation product, a polypeptide degradation product or an oxidation product.

[0078] Compared with the prior art, the present invention has the following beneficial effects:

[0079] The present invention provides a method for detecting interleukin-4 isoforms. In the method, an interleukin-4 sample is diluted with an amphoteric electrolyte solution to obtain a sample solution to be tested; the sample solution to be tested is subjected to capillary electrophoresis-mass spectrometry online combined detection; the volume fraction of the amphoteric electrolyte solution is 0.2-5%; the conditions for capillary electrophoresis separation include: using a capillary isoelectric focusing electrophoresis mode; the capillary is a neutral coating capillary; and the separation voltage is 1-100 kV. The detection method has high separation efficiency and accurate analysis results, and can comprehensively obtain the isoform information in the interleukin-4 sample. In addition, the method can also accurately obtain the isoelectric point data of each interleukin-4 isoform, thereby realizing accurate characterization of the sample. The method of the present invention can be applied to the detection of interleukin drugs to meet the needs of quality control of interleukin-4 in the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 Schematic diagram of the positions of the capillary, spray needle and mass spectrometer ion source.

[0081] Figure 2 This is the total ion current (TIC) of the interleukin 4 sample in Example 1.

[0082] Figure 3 This is the average mass spectrum of the interleukin-4 isoforms and the internal standard protein in Example 1.

[0083] Figure 4 This is the TIC diagram of the interleukin-4 sample in Example 2.

[0084] Figure 5 This is the average mass spectrum of the interleukin-4 isoforms and the internal standard protein in Example 2.

[0085] Figure 6 This is the TIC diagram of the interleukin-4 sample in Example 3.

[0086] Figure 7 This is the average mass spectrum of the interleukin-4 isoforms and the internal standard protein in Example 3.

[0087] Figure 8 This is the fitting curve of the migration time and isoelectric point of the internal standard protein in Example 3.

[0088] Fig. 9 This is the TIC diagram of the interleukin-4 sample in Example 4.

[0089] Fig.10 This is the average mass spectrum of the interleukin-4 isoforms and the internal standard protein in Example 4. DETAILED DESCRIPTION

[0090] In order to more clearly and completely describe the technical solution of the present invention, the present invention is further described in detail through specific embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention. Various changes can be made within the scope of the rights of the present invention.

[0091] Example 1

[0092] This embodiment provides a method for detecting interleukin-4 isoforms, comprising the following steps:

[0093] S1. Dilute the interleukin-4 sample (purchased from Beijing Biopsies Biotechnology Co., Ltd.) with an amphoteric electrolyte solution, add the internal standard protein solution, and obtain a sample solution to be tested;

[0094] S2. The sample solution to be tested in step S1 is subjected to capillary electrophoresis-mass spectrometry online detection;

[0095] The concentration of the interleukin 4 sample in the sample solution to be tested is 0.15 mg / mL; the ampholyte is ampholyte 3-10 (provided by Cytiva, model: 17-0456-01), the volume fraction of the ampholyte solution is 1%, and it is prepared by the following method: 10 μL of ampholyte 3-10 and 990 μL of diluent are mixed, and the diluent is the Buffer S35 solution in the CR3520 kit; the internal standard protein is Ubiquitin, and the concentration of Ubiquitin is 0.03 mg / mL.

[0096] The conditions for the capillary electrophoresis-mass spectrometry online detection are as follows:

[0097] A neutral-coated capillary (provided by CMP Scientific, P / N: E-SC-PS1-360 / 150-50-100-B1, with a capillary inner diameter of 50 μm and a capillary length of 100 cm) was connected to the capillary electrophoresis system, and the capillary electrophoresis instrument was connected online to the mass spectrometer using a Yongdao Zhiyuan EMASS-II CE-MS online coupled ion source;

[0098] Capillary electrophoresis instrument model: ECE-001;

[0099] Mass spectrometer model: Q Exactive Plus;

[0100] Distance between the spray needle tip and the mass spectrometer: 2.511 mm;

[0101] Distance from capillary tip to needle tip: 1.016mm

[0102] Ion source electrospray voltage: +2.4 kV;

[0103] Sheath fluid: Buffer SL in the CR3520 kit (provided by CMP Scientific, the same below);

[0104] Sheath fluid flow rate: <50nL / min;

[0105] Separation voltage: +27kV;

[0106] The injection steps are as follows: first, a section of alkaline cathode solution (Buffer B of the CR3520 kit) is injected into the capillary injection port, and the injection pressure and time are 950 mbar and 30 s respectively, and then the sample solution to be tested is injected, and the injection pressure and time are 950 mbar and 90 s respectively, and finally, the capillary injection port is moved to the acidic anode solution (Buffer A of the CR3520 kit);

[0107] Ion mode: positive ion mode;

[0108] Mass spectrometry data type: Profile;

[0109] Data-dependent scanning, level one is full scan;

[0110] The first-level parameters are set as:

[0111] Resolution: 70000;

[0112] Automatic gain control: 3E6;

[0113] Maximum injection time: 150ms;

[0114] Microscans: 10;

[0115] Scanning range: 1000~3000m / z;

[0116] The secondary parameters are set as:

[0117] Resolution: 35000;

[0118] Automatic gain control: 5E5;

[0119] Maximum injection time: 600ms.

[0120] Figure 2 is the TIC diagram of the interleukin-4 sample in Example 1; Figure 3 The average mass spectra of the interleukin-4 isoforms and the internal standard protein in Example 1. Figures 2-3It can be seen that the different peak regions of interleukin-4 isoforms are clearly separated, which is helpful for efficient separation and detection of these isoforms. In addition, the internal standard protein Ubiquitin has a stable peak and will not interfere with the separation and detection of interleukin-4 isoforms. Table 1 shows the separation and detection information of interleukin-4 isoforms obtained by the method of Example 1. As can be seen from Table 1, the method of Example 1 can detect 20 interleukin-4 isoforms, which shows that the method of the present invention has high separation efficiency and accurate analysis results, and can comprehensively obtain the isoform information in the interleukin-4 sample.

[0121] Table 1 Separation and detection information of interleukin-4 heterogeneous forms in Example 1

[0122]

[0123]

[0124] Example 2

[0125] The present embodiment provides a method for detecting interleukin-4 isoforms. The difference from Example 1 is that the volume fraction of the ampholyte solution is 1%, which is prepared by the following method: 2 μL of ampholyte 3-10, 8 μL of ampholyte 8-10.5 and 990 μL of diluent are mixed, and the diluent is the Buffer S35 solution in the CR3520 kit (the volume ratio of ampholyte 3-10 to ampholyte 8-10.5 is 1:4); the internal standard proteins are Cytochrome C, Ubiquitin and Infliximab, and the concentration of Cytochrome C is 0.05 mg / mL, the concentration of Ubiquitin is 0.01 mg / mL, and the concentration of Infliximab is 0.1 mg / mL.

[0126] Figure 4 is the TIC diagram of the interleukin-4 sample in Example 2; Figure 5 is the average mass spectrum of the interleukin-4 isoforms and the internal standard protein in Example 2. Figures 4-5 It can be seen that under the conditions where the ampholytes are ampholytes 3-10 and ampholytes 8-10.5, the separation between different peak regions of interleukin 4 isoforms is clearer than that in Example 1. In addition, the three internal standard proteins (Cytochrome C, Ubiquitin and Infliximab) have stable peaks and do not interfere with the separation and detection of interleukin 4 isoforms. Table 2 is the separation and detection information of interleukin 4 isoforms obtained by the method of Example 2. It can be seen from Table 2 that the method of Example 2 can detect 21 interleukin 4 isoforms, which shows that the method of the present invention has high separation efficiency and accurate analysis results, and can comprehensively obtain the isoform information in the interleukin 4 sample.

[0127] Table 2 Separation and detection information of interleukin-4 heterogeneous forms in Example 2

[0128]

[0129]

[0130] Example 3

[0131] The present embodiment provides a method for detecting interleukin-4 isoforms. The difference from Example 1 is that the volume fraction of the ampholyte solution is 1%, which is prepared by the following method: 1.25 μL of ampholyte 3-10, 8.75 μL of ampholyte 8-10.5 and 990 μL of diluent are mixed, and the diluent is the Buffer S35 solution in the CR3520 kit (the volume ratio of ampholyte 3-10 to ampholyte 8-10.5 is 1:7); the internal standard proteins are Cytochrome C, Ubiquitin and Infliximab, and the concentration of Cytochrome C is 0.05 mg / mL, the concentration of Ubiquitin is 0.01 mg / mL, and the concentration of Infliximab is 0.1 mg / mL.

[0132] Figure 6 is the TIC diagram of the interleukin-4 sample in Example 3; Figure 7 The average mass spectra of the interleukin-4 isoforms and the internal standard protein in Example 3. Figures 6-7 It can be seen that increasing the ratio of amphoteric electrolytes 8-10.5 in the amphoteric electrolyte solution provides a more refined focusing environment for interleukin 4 heterogeneities, and the separation between the peaks in the peak regions ④ and ⑤ is clearer than that in Example 2, and the overall detection signal is improved, which is beneficial to improving the separation and detection efficiency of interleukin 4 heterogeneities. In addition, the three internal standard proteins (Cytochrome C, Ubiquitin and Infliximab) have stable peaks and will not interfere with the separation and detection of interleukin 4 heterogeneities. Table 3 shows the separation and detection information of interleukin 4 heterogeneities obtained by the method of Example 3. As can be seen from Table 3, the method of Example 3 can detect 21 interleukin 4 heterogeneities. Figure 8 is the fitting curve of the migration time and isoelectric point of the internal standard protein in Example 3. Figure 8 It can be seen that the linear fitting degree of the curve is high, R 2 =0.9533, and the isoelectric point data of each interleukin 4 isoform can be accurately obtained. This shows that the method of the present invention has high separation efficiency and accurate analysis results, can comprehensively obtain the isoelectric point information of the interleukin 4 sample, and can also effectively provide the isoelectric point information of the protein isoform, thereby realizing the accurate characterization of the sample.

[0133] Table 3 Separation and detection information of interleukin-4 heterogeneous forms in Example 3

[0134]

[0135]

[0136] Example 4

[0137] This embodiment provides a method for detecting interleukin-4 isoforms. The difference from Embodiment 1 is that the concentration of the interleukin-4 sample in the sample solution to be tested is 0.1 mg / mL.

[0138] Fig. 9 is the TIC diagram of the interleukin-4 sample in Example 4; Fig.10 The average mass spectra of the interleukin-4 isoforms and the internal standard protein in Example 4. Figures 9-10 It can be seen that the different peak regions of interleukin 4 isoforms are clearly separated, and the peak of the internal standard protein Ubiquitin is stable, which will not interfere with the peak of interleukin 4 isoforms. Table 4 is the separation and detection information of interleukin 4 isoforms detected by the method of Example 4. It can be seen from Table 4 that although some low-abundance interleukin 4 isoforms (such as interleukin 4 isoforms with molecular weights of 18286Da, 19078Da and 19825Da) were not detected, 18 interleukin 4 isoforms can still be detected, which shows that the method of the present invention has high separation efficiency and accurate analysis results, and can comprehensively obtain the isoform information in the interleukin 4 sample.

[0139] Table 4 Separation and detection information of interleukin-4 heterogeneous forms in Example 4

[0140]

[0141]

[0142] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for separating interleukin-4 heterogeneous forms, comprising the following steps: S1. diluting the interleukin-4 sample with an amphoteric electrolyte solution to obtain a sample solution to be tested; S2. Separating the sample solution in step S1 by capillary electrophoresis; The volume fraction of the amphoteric electrolyte solution is 0.2-5%; The conditions of the capillary electrophoresis include: using the capillary isoelectric focusing electrophoresis mode; the capillary is a neutral coating capillary; and the separation voltage is 1-100 kV.

2. The method according to claim 1, characterized in that: The separation voltage is 10-50 kV.

3. The method according to claim 1, characterized in that: The ampholyte is at least one of ampholytes 8-10.5, ampholytes 3-10 or ampholytes 9-11.

4. The method according to claim 1, characterized in that: The concentration of the interleukin-4 sample in the sample solution to be tested is 0.05-0.5 mg / mL.

5. The method according to claim 1, characterized in that: The sample solution to be tested also includes an internal standard protein solution, and the internal standard protein is selected from at least one of Cytochrome C, Infliximab or Ubiquitin.

6. A method for detecting interleukin-4 isoforms, comprising the following steps: S1. diluting the interleukin-4 sample with an amphoteric electrolyte solution to obtain a sample solution to be tested; S2. The sample solution to be tested in step S1 is subjected to capillary electrophoresis-mass spectrometry online detection; The volume fraction of the amphoteric electrolyte solution is 0.2-5%; The conditions for capillary electrophoresis separation include: using capillary isoelectric focusing electrophoresis mode; the capillary is a neutral coating capillary; and the separation voltage is 1-100 kV.

7. The method according to claim 6, characterized in that: The detection conditions of the mass spectrometer include: a resolution of 17500 to 70000.

8. The method according to claim 6, characterized in that: The online coupling is achieved through a coaxial sheath flow electrospray ion source driven by electroosmotic flow.

9. Use of the method according to any one of claims 1 to 8 in the detection of interleukin drugs.

10. Use of the method according to any one of claims 1 to 8 in detecting and / or distinguishing products obtained through structural heterogeneity changes of interleukin-4.

Images