Method for detecting interleukin 2 heteroplasmon and application thereof

Abstract

The invention relates to a method for detecting interleukin 2 variants and application of the method. The method comprises the following steps: diluting an interleukin 2 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 is high in separation efficiency and accurate in analysis result, and in addition, isoelectric point data of each interleukin 2 variant can be accurately obtained by the method, so that accurate characterization of a 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 2 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-2 heteromorphs and an application thereof. Background Art

[0002] Interleukin, or IL for short, is a type of cytokine that plays an important role in the body's immune regulation and homeostasis. Interleukin-2 is one of the important T cell growth factors, which mainly stimulates the proliferation of natural killer cells and enhances their cytotoxicity, thereby enabling B cells to proliferate more effectively and produce more antibodies. Therefore, interleukin-2 plays an important role as a drug molecule in the treatment of various cancers and immune diseases.

[0003] Interleukin-2, as a glycoprotein, has an O-glycosylation site, and complex and diverse glycosylation modifications can significantly increase its structural heterogeneity. On the one hand, O-glycosylation can affect the folding process of interleukin-2 and change its three-dimensional structure, thereby affecting the stability and function of interleukin-2 as a drug molecule; on the other hand, glycosylation modification can change the charge on the surface of the protein, thereby changing the isoelectric point of interleukin-2, resulting in changes in the migration behavior of the protein in analytical techniques such as electrophoresis. In addition, during drug production, transportation and storage, the degradation and post-translational modification of protein products can increase the structural heterogeneity of proteins. However, changes in the structural heterogeneity of proteins can affect the structure and function of proteins, thereby affecting the quality and stability of drugs. Therefore, the detection and analysis of interleukin-2 heterogeneities are 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 the charge variants of a protein 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 2 and its isoforms, and there are relatively few documents on the separation of interleukin 2 and its 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 2 and its isoforms to meet the needs of quality control of interleukin 2 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-2 isoforms, and to provide a method for detecting interleukin-2 isoforms. The method dilutes an interleukin-2 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. In addition, the method can also accurately obtain the isoelectric point data of each interleukin-2 isoform, thereby achieving 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-2 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-2 and its heteromorphs comprises the following steps:

[0012] S1. diluting the interleukin-2 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 2 heterogeneous forms 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-2 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.

[0019] Preferably, the volume fraction of the amphoteric electrolyte solution is 0.5-2%.

[0020] Preferably, the ampholyte is at least one of ampholytes 6-9, ampholytes 3-10 or ampholytes 5-8.

[0021] More preferably, the ampholyte is ampholyte 3 to 10. The isoelectric point range of the ampholyte can not only focus and separate interleukin-2 and its isomers, but also well cover the isoelectric point range of the internal standard protein.

[0022] Preferably, the concentration of the interleukin-2 sample in the sample solution to be tested is 0.01-0.2 mg / mL.

[0023] Preferably, the capillary is made of elastic quartz.

[0024] Preferably, the inner diameter of the capillary is 40-50 μm.

[0025] Preferably, the capillary tube has a length of 65 to 130 cm.

[0026] Preferably, the capillary electrophoresis conditions also include: the injection method is gas pressure injection.

[0027] More preferably, the gas pressure of the gas pressure injection is 500-2000 mbar.

[0028] More preferably, the time of the gas pressure injection is 10 to 120 seconds.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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 2 heterogeneous forms, and the peak is stable, which is conducive to constructing a fitting curve of migration time and isoelectric point, can ensure the accuracy of pH distribution and can accurately obtain isoelectric point data of interleukin 2 heterogeneous forms. 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.

[0033] More preferably, the concentration of Cytochrome C in the internal standard protein solution is 0.01-0.3 mg / mL.

[0034] More preferably, the concentration of Infliximab in the internal standard protein solution is 0.1-0.3 mg / mL.

[0035] More preferably, the concentration of Ubiquitin in the internal standard protein solution is 0.01-0.3 mg / mL.

[0036] A method for detecting interleukin-2 heterogeneity comprises the following steps:

[0037] S1. diluting the interleukin-2 sample with an amphoteric electrolyte solution to obtain a sample solution to be tested;

[0038] S2. The sample solution to be tested in step S1 is subjected to capillary electrophoresis-mass spectrometry online detection;

[0039] The volume fraction of the amphoteric electrolyte solution is 0.2-5%;

[0040] 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.

[0041] Preferably, the separation voltage is 10-50 kV.

[0042] Preferably, the volume fraction of the amphoteric electrolyte solution is 0.5-2%.

[0043] Preferably, the ampholyte is at least one of ampholytes 6-9, ampholytes 3-10 or ampholytes 5-8.

[0044] More preferably, the ampholyte is ampholyte 3-10.

[0045] Preferably, the concentration of the interleukin-2 sample in the sample solution to be tested is 0.01-0.2 mg / mL.

[0046] Preferably, the capillary is made of elastic quartz.

[0047] Preferably, the inner diameter of the capillary is 40-50 μm.

[0048] Preferably, the capillary tube has a length of 65 to 130 cm.

[0049] Preferably, the capillary electrophoresis conditions also include: the injection method is gas pressure injection.

[0050] More preferably, the gas pressure of the gas pressure injection is 500-2000 mbar.

[0051] More preferably, the time of the gas pressure injection is 10 to 120 seconds.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] More preferably, the concentration of Cytochrome C in the internal standard protein solution is 0.01-0.3 mg / mL.

[0056] More preferably, the concentration of Infliximab in the internal standard protein solution is 0.1-0.3 mg / mL.

[0057] More preferably, the concentration of Ubiquitin in the internal standard protein solution is 0.01-0.3 mg / mL.

[0058] Preferably, the detection conditions of the mass spectrometry include: a resolution of 17500 to 70000.

[0059] The inventors of the present invention have found that when using mass spectrometry for analysis, low-abundance components in the sample may not be captured if the resolution is too low, which limits the dynamic range of mass spectrometry detection and fails to provide clear peak shapes and accurate peak intensity measurements. The specific resolution of the present invention improves the quality and peak shape characteristics of mass spectrometry data, thereby improving the accuracy of the analysis results of interleukin-2 isoforms.

[0060] Preferably, the online coupling is achieved by a coaxial sheath flow electrospray ion source driven by electroosmotic flow.

[0061] More preferably, the voltage of electrospray in the coaxial sheath liquid electrospray ion source is 1.2 to 3 kV.

[0062] More preferably, the sheath fluid in the coaxial sheath fluid electrospray ion source is the BufferSL solution in the CR3520 kit.

[0063] More preferably, the flow rate of the sheath fluid is <50 nL / min.

[0064] 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.

[0065] 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.

[0066] The present invention also protects the application of the above method in interleukin drug detection.

[0067] Preferably, the interleukin drug detection includes at least one of immune cytokine detection, antibody drug detection, fusion protein detection, recombinant protease detection, polypeptide hormone detection or therapeutic vaccine detection.

[0068] The present invention also protects the use of the above method in detecting and / or distinguishing products obtained by interleukin-2 undergoing structural heterogeneity changes.

[0069] Preferably, the structural heterogeneity change is post-translational modification or degradation.

[0070] 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.

[0071] 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.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] The present invention provides a method for detecting interleukin-2 isomers. In the method, an interleukin-2 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. In addition, the method can also accurately obtain the isoelectric point data of each interleukin-2 isomer, 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-2 in the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 Schematic diagram of the positions of the capillary, spray needle and mass spectrometer ion source.

[0075] Figure 2 The total ion current (TIC) (upper figure) and Base Peak graph (lower figure) of the interleukin 2 sample in Example 1.

[0076] Figure 3 This is the average mass spectrum of the peaks of interleukin-2, its isoforms and the internal standard protein in Example 1.

[0077] Figure 4 This is a graph showing the heterogeneous distribution and abundance characteristics of the interleukin-2 sample in Example 1.

[0078] Figure 5 The TIC graph (upper graph) and Base Peak graph (lower graph) of the interleukin-2 sample in Example 2.

[0079] Figure 6 This is the average mass spectrum of the peaks of interleukin-2, its isoforms and the internal standard protein in Example 2.

[0080] Figure 7 This is a diagram showing the heterogeneous distribution and abundance characteristics of the interleukin-2 sample in Example 2.

[0081] Figure 8It is the fitting curve of the migration time and isoelectric point of the internal standard protein in Example 2.

[0082] Fig. 9 The TIC graph (upper graph) and Base Peak (lower graph) of the interleukin-2 sample in Example 3.

[0083] Fig.10 This is the average mass spectrum of the peaks of interleukin-2, its isoforms and the internal standard protein in Example 3.

[0084] Fig.11 This is a diagram showing the heterogeneous distribution and abundance characteristics of the interleukin-2 sample in Example 3. DETAILED DESCRIPTION

[0085] 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.

[0086] Example 1

[0087] This embodiment provides a method for detecting interleukin-2 isoforms, comprising the following steps:

[0088] S1. Dilute the interleukin-2 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;

[0089] S2. The sample solution to be tested in step S1 is subjected to capillary electrophoresis-mass spectrometry online detection;

[0090] The concentration of the interleukin 2 sample in the sample solution to be tested is 0.025 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 Cytochrome C, and the concentration of Cytochrome C is 0.01 mg / mL.

[0091] The conditions for capillary electrophoresis-mass spectrometry online detection are as follows:

[0092] A neutral-coated capillary (provided by CMP Scientific, P / N: E-SC-PS1-360 / 150-50-100-B1, capillary inner diameter of 50 μm, 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 the Yongdao Zhiyuan EMASS-II CE-MS online coupled ion source;

[0093] Capillary electrophoresis instrument model: ECE-001;

[0094] Mass spectrometer model: Q Exactive Plus;

[0095] Distance between the spray needle tip and the mass spectrometer: 2.934 mm;

[0096] Distance from capillary tip to needle tip: 0.993mm

[0097] Ion source electrospray voltage: +2.4 kV;

[0098] Sheath fluid: Buffer SL in the CR3520 kit (provided by CMP Scientific, the same below);

[0099] Sheath fluid flow rate: <50nL / min;

[0100] Separation voltage: +27kV;

[0101] 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);

[0102] Ion mode: positive ion mode;

[0103] Mass spectrometry data type: Profile;

[0104] Data-dependent scanning, level one is full scan;

[0105] The first-level parameters are set as:

[0106] Resolution: 70000;

[0107] Automatic gain control: 3E6;

[0108] Maximum injection time: 150ms;

[0109] Microscans: 10;

[0110] Scanning range: 1000~3000m / z;

[0111] The secondary parameters are set as:

[0112] Resolution: 35000;

[0113] Automatic gain control: 5E5;

[0114] Maximum injection time: 600ms.

[0115] Figure 2 The TIC graph (upper graph) and Base Peak graph (lower graph) of the interleukin-2 sample in Example 1; Figure 3 The average mass spectra of the interleukin-2 isoforms and the internal standard protein in Example 1. Figures 2-3 It can be seen that interleukin 2 (peak 1) and its two isoforms (peak 2 and peak 3) can be clearly separated in the spectrum, and the internal standard protein Cytochrome C has a stable peak and does not interfere with the peak of interleukin 2 isoforms. Figure 4 The isoform distribution and abundance characteristic diagram of the interleukin-2 sample in Example 1, including the peak area of ​​each peak and the corresponding percentage. Figure 4 It can be seen that the interleukin 2 isoform corresponding to peak 2 has the lowest abundance, and the relative abundance of the interleukin 2 isoform corresponding to peak 3 is about twice that of interleukin 2 (peak 1). This shows that the method of the present invention has high separation efficiency and accurate analysis results, and can more comprehensively understand the distribution and abundance characteristics of the isoforms in the interleukin 2 sample.

[0116] Example 2

[0117] This embodiment provides a method for detecting interleukin-2 isoforms. The difference from Embodiment 1 is that the internal standard proteins are Cytochrome C, Ubiquitin and Infliximab, the concentration of Cytochrome C is 0.01 mg / mL, the concentration of Ubiquitin is 0.01 mg / mL, and the concentration of Infliximab is 0.1 mg / mL.

[0118] Figure 5 The TIC graph (upper graph) and Base Peak graph (lower graph) of the interleukin-2 sample in Example 2; Figure 6 is the average mass spectrum of the interleukin-2 isoforms and the internal standard protein in Example 2. Figures 5-6 It can be seen that in the spectrum, interleukin 2 (peak 1) and its two isoforms (peak 2 and peak 3) can be clearly separated, and under this condition, the internal standard proteins Cytochrome C, Ubiquitin and Infliximab have stable peaks and do not interfere with the peaks of interleukin 2 isoforms. Figure 7The isoform distribution and abundance characteristic diagram of the interleukin-2 sample in Example 2, including the peak area and corresponding percentage of each peak. Figure 7 It can be seen that the interleukin-2 isoform corresponding to peak 2 has the lowest abundance, and the relative abundance of the interleukin-2 isoform corresponding to peak 3 is about twice that of interleukin-2 (peak 1). Figure 8 is the fitting curve of the migration time and isoelectric point of the internal standard protein in Example 2. Figure 8 It can be seen that the linear fitting degree of the curve is high, R 2 = 1, and the isoelectric point data of each interleukin 2 isoform can be accurately obtained. This shows that this method not only has high separation efficiency and accurate analysis results, but also can provide a more comprehensive understanding of the distribution and abundance characteristics of isoforms in interleukin 2 samples, and can also effectively provide isoelectric point information of protein isoforms, thereby achieving accurate characterization of samples.

[0119] Example 3

[0120] This embodiment provides a method for detecting interleukin-2 isoforms. The difference from Embodiment 1 is that in the mass spectrometry detection condition, the resolution of the primary scan is 17500.

[0121] Fig. 9 The total ion current (upper figure) and Base Peak (lower figure) of the interleukin 2 sample in Comparative Example 1. Fig.10 The average mass spectra of the interleukin-2 isoforms and the internal standard protein in Comparative Example 1. Figures 9-10 It can be seen that interleukin 2 (peak 1) and its two isoforms (peak 2 and peak 3) can be clearly separated in the spectrum, and the internal standard protein Cytochrome C has a stable peak and does not interfere with the peak of interleukin 2 isoforms. Fig.11 The isoform distribution and abundance characteristic diagram of the interleukin-2 sample in Example 3, including the peak area and corresponding percentage of each peak. Fig.11 It can be seen that the interleukin 2 isoform corresponding to peak 2 has the lowest abundance, and the relative abundance of the interleukin 2 isoform corresponding to peak 3 is about twice that of interleukin 2 (peak 1). This shows that the method of the present invention has high separation efficiency and accurate analysis results, and can more comprehensively understand the distribution and abundance characteristics of the isoforms in the interleukin 2 sample.

[0122] 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-2 and its heterogeneous forms, comprising the following steps: S1. diluting the interleukin-2 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 6-9, ampholytes 3-10 or ampholytes 5-8.

4. The method according to claim 1, characterized in that: The capillary electrophoresis conditions also include: the injection method is gas pressure injection.

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-2 heterogeneity, comprising the following steps: S1. diluting the interleukin-2 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 by structural heterogeneity of interleukin-2.

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