A rapid analysis method for fusion protein samples

Abstract

The present invention discloses a method for analyzing a fusion protein sample, which adopts a pretreatment process before analyzing the sample by reduction capillary electrophoresis technology. Specifically, the pretreatment process includes the following steps: taking the fusion protein sample for denaturation and then changing the liquid, and performing sugar cutting treatment after changing the liquid. This analysis method overcomes the problems of poor peak shape and low response value caused by glycosylation of fusion protein, and provides an efficient and accurate fusion protein purity analysis method.

Description

Technical Field

[0001] The invention relates to the field of biological separation analysis, and in particular to a method for detecting the purity of a fusion protein sample by using reduction capillary electrophoresis technology. Background Art

[0002] Antibodies are protein macromolecules with complex structural characteristics. They are mainly composed of two functional regions, Fab and Fc. Glycosylation modification exists at specific sites of the Fab and Fc segments. The sugar chains formed by glycosylation modification only account for 3% of the entire antibody. Although the proportion is not high, the sugar chain structure plays an extremely important role. Glycosylation modification not only plays an important role in maintaining the structure of antibodies, but also some specific glycosylation modification types and different glycosylation modification degrees will affect the safety characteristics of antibodies. Moreover, glycosylation modification also affects the effectiveness of drugs.

[0003] Multifunctional fusion proteins also contain cytokines, which makes the structure of multifunctional fusion proteins more complex than antibodies. Their glycosylation is highly heterogeneous. In the glycosylation of multifunctional fusion proteins, due to the diversity of glycosylation types and connection methods, the most complex and diverse protein structure in nature is formed. The glycosylation structure of multifunctional fusion protein drugs is of great significance to the efficacy and safety of drugs.

[0004] However, the glycosylation modification of these multifunctional fusion proteins may affect the purity detection of multifunctional fusion protein samples during sample pretreatment, instrument sampling and analysis, and thus it is impossible to obtain true and accurate purity detection results. Therefore, in order to ensure that the key quality attributes of multifunctional fusion protein samples are accurately detected, it is very necessary to develop corresponding efficient and accurate antibody purity analysis methods.

[0005] Capillary electrophoresis is one of the most commonly used methods for purity analysis of antibody drugs or fusion protein drugs. This method adds an excess of sodium dodecyl sulfate to the sample to make all protein molecules have the same charge-to-mass ratio, and introduces the protein molecules with the same charge-to-mass ratio into a capillary filled with a separation gel. By applying voltage at both ends of the capillary, the separation of protein components with different molecular weights in the sample is completed.

[0006] At present, we have found in experiments that the glycosylation in the fusion protein sample formulation has a significant impact on the capillary electrophoresis purity detection. When a capillary electrophoresis instrument is used to detect a glycosylated fusion protein sample, an aggregate peak that cannot be confirmed by other technical means will appear in the test result spectrum, thereby affecting the accurate analysis of the purity of the multifunctional fusion protein sample. At present, there are no reports or solutions for the impact of glycosylated fusion protein samples on capillary electrophoresis detection. The purpose of the present invention is to solve this problem. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a method for detecting the purity of fusion protein samples using reduction capillary electrophoresis technology, which can overcome the interference of glycosylation of fusion protein and accurately detect the purity of fusion protein sample components in a prescription.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0009] A rapid analysis method for fusion protein samples adopts capillary electrophoresis analysis technology to analyze the fusion protein samples and comprises the step of performing sugar cutting treatment on the fusion protein samples, wherein the fusion protein comprises cytokines.

[0010] In an alternative embodiment, the cytokine is IL-15Rα.

[0011] In an optional embodiment, the glycolytic treatment is achieved by removing glycosylation modifications from the fusion protein sample using enzyme cleavage.

[0012] In an optional embodiment, the type of enzyme used in the sugar-cleaving treatment is one or more of PNGaseF, endoglycosidase H, and recombinant endoglycosidase F2.

[0013] In an alternative embodiment, the analytical method requires denaturation treatment prior to sugar cleavage.

[0014] In an alternative embodiment, the denaturation step requires the addition of a denaturing solution.

[0015] In an optional embodiment, the denaturing solution is a guanidine hydrochloride Tris-HCl buffer solution.

[0016] In an optional embodiment, the composition of the guanidine hydrochloride Tris-HCl buffer solution is 20-40g guanidine hydrochloride, 0.5-5.0g tris is added, water is added to 20-50mL, the pH is adjusted to 7.0-8.0 with 5-7mol / L HCl, and water is added to 30-50mL.

[0017] In an optional embodiment, the composition of the guanidine hydrochloride Tris-HCl buffer solution is 25-35g guanidine hydrochloride, 1.0-2.0g tris is added, water is added to 30-40mL, the pH is adjusted to 7.0-8.0 with 5-7mol / L HCl, and water is added to 35-45mL.

[0018] In an optional embodiment, the analytical method further requires a solution replacement treatment after the denaturation treatment.

[0019] In an optional embodiment, the liquid replacement step requires the addition of an enzyme digestion buffer.

[0020] In an optional embodiment, the enzyme cleavage buffer is 0.1-10 mol / L urea dissolved in 10-30 mmol / LPB buffer, with a pH of 5.5-6.5.

[0021] In an optional embodiment, the enzyme cleavage buffer is 0.5-5 mol / L urea dissolved in 10-30 mmol / L PB buffer, with a pH of 5.5-6.5.

[0022] In an optional embodiment, the enzyme cleavage buffer is 1-3 mol / L urea dissolved in 15-25 mmol / LPB buffer, with a pH of 5.5-6.5.

[0023] In an optional embodiment, the enzyme cleavage buffer is 1-1.5 mol / L urea dissolved in 15-25 mmol / L PB buffer, with a pH of 5.5-6.5.

[0024] In an alternative embodiment, the fusion protein comprises a first heavy chain, a second heavy chain, a first light chain, and a second light chain.

[0025] In an alternative embodiment, the fusion protein further comprises the IL-15 receptor IL-15Rα.

[0026] In alternative embodiments, the first heavy chain comprises IL-15Rα and the second heavy chain comprises IL-15.

[0027] In alternative embodiments, the first heavy chain comprises IL-15 and IL-15Rα, and / or the second heavy chain comprises IL-15 and IL-15Rα.

[0028] In an alternative embodiment, the IL-15Rα and the IL-15 form an IL-15Rα / IL-15 complex.

[0029] In alternative embodiments, the first light chain and the second light chain are the same or different light chains.

[0030] In an optional embodiment, the fusion protein further comprises an Fc region and a mutant thereof, wherein the Fc region is selected from IgG1, IgG2, IgG3 and / or IgG4.

[0031] In an optional embodiment, the first heavy chain amino acid sequence of the fusion protein is selected from SEQ ID NO:1; the second heavy chain amino acid sequence of the fusion protein is selected from SEQ ID NO:2; and / or the amino acid sequences of the first light chain and the second light chain of the fusion protein are selected from SEQ ID NO:3.

[0032] In an optional embodiment, for specific fusion protein and related formulation information, please refer to patent 202110010966.3.

[0033] In an optional embodiment, for specific fusion protein and related formulation information, please refer to patent 202110010974.8.

[0034] Furthermore, a rapid analysis method for a fusion protein sample comprises the following steps:

[0035] Step (1): taking a fusion protein sample, denaturing it, and then changing the solution, and then performing sugar cutting treatment;

[0036] Step (2): Take an appropriate amount of fusion protein solution and dilute it with water to a fusion protein concentration of about 1.0-5.0 mg / mL;

[0037] Step (3): Preparation of test solution

[0038] Add an appropriate amount of loading buffer, an appropriate amount of β-mercaptoethanol, and a buffer solution to 40-200 μL of the diluent prepared in step (2) to prepare a test solution;

[0039] Step (4): Mix the solution, centrifuge it, heat it in a water bath for 5 to 20 minutes, shake it to mix it, and cool it to room temperature; after centrifugation, take the supernatant and load it;

[0040] Step (5): pre-treating the capillary;

[0041] Step (6): reverse polarity electrokinetic injection, injection time 15 to 25 seconds;

[0042] Step (7): the sample chamber temperature is 10-50°C, the capillary temperature is 10-50°C, ultraviolet wavelength detection, and operation under reverse polarity for 10-60 minutes.

[0043] Furthermore, the step (1) is to take an appropriate amount of fusion protein, add denaturing solution, and water bath at 20-50°C for 20-50 minutes. Use an ultrafiltration tube to replace the solution with the enzyme digestion buffer, and replace the solution at least 81 times in total, quantify, and the protein concentration is not less than 7 mg / mL. Take an appropriate amount of the fusion protein after the solution is replaced, add PNGaseF, 10×Glyco Buffer 2, and make up with enzyme digestion buffer. Mix well, centrifuge instantly, and water bath for 5-50 hours.

[0044] Furthermore, the step (1) is to take 1000 μg of fusion protein, add 800 μL of denaturing solution, and incubate at 37°C in a water bath for 30 minutes. Use an ultrafiltration tube to replace the solution with the enzyme digestion buffer, replace the solution at least 81 times in total, and quantify. The final volume is not less than 130 μL, and the protein concentration is not less than 7 mg / mL. Take about 500 μg of fusion protein after the solution is replaced, add 5 μL of PNGaseF, 20 μL of 10×Glyco Buffer 2, and make up to 100 μL of the system with enzyme digestion buffer. Mix well, centrifuge instantly, and incubate at 37°C in a water bath for 16 hours.

[0045] Furthermore, a rapid analysis method for a fusion protein sample comprises the following steps:

[0046] Step (1): Take 1000 μg of fusion protein, add 800 μL of denaturing solution, and incubate at 37°C in a water bath for 30 min. Use an ultrafiltration tube to replace the solution with the enzyme digestion buffer, replace the solution at least 81 times in total, and quantify. The final volume is not less than 130 μL, and the protein concentration is not less than 7 mg / mL.

[0047] Step (2): Take about 500 μg of the fusion protein after the replacement solution, add 5 μL PNGaseF, 20 μL 10× Glyco Buffer 2, and use enzyme digestion buffer to make up to 100 μL system. Mix well, centrifuge briefly, and incubate in a 37°C water bath for 16 hours.

[0048] Step (3): 40uL of fusion protein solution was diluted with water to a fusion protein concentration of about 2.5mg / mL, and the total volume was not less than 40μL;

[0049] Step (4): Preparation of test solution

[0050] Add 40 μL of the diluent prepared in step (3) to 55 μL of loading buffer, 5 μL of β-mercaptoethanol, and add buffer to 100 μL to prepare a test solution;

[0051] Step (5): Mix the solution, centrifuge, heat in a water bath for 5-20 min, oscillate to mix, and cool to room temperature; after centrifugation, take the supernatant and load the sample;

[0052] Step (6): pre-treat the capillary by flushing it with a 0.1 mol / L sodium hydroxide solution at a pressure of 70 psi for 3 minutes, then flushing it with a 0.1 mol / L hydrochloric acid solution at a pressure of 70 psi for 1 minute, and then flushing it with pure water at a pressure of 70 psi for 1 minute;

[0053] Step (7): reverse polarity electrokinetic injection at 5.0 KV for 20 seconds;

[0054] Step (8): Run for 30 minutes at a sample chamber temperature of 25°C, a capillary temperature of 25°C, a detection wavelength of 214 nm, 15.0 KV, and reverse polarity.

[0055] Therefore, the present invention relates to the following technical solutions:

[0056] 1. A rapid analysis method for fusion protein samples, characterized in that the fusion protein samples are analyzed by capillary electrophoresis analysis technology, and the method comprises the step of performing sugar cutting treatment on the fusion protein samples, wherein the fusion protein comprises cytokines.

[0057] 2. The rapid analysis method for fusion protein samples according to Scheme 1 is characterized in that the cytokine is IL-15.

[0058] 3. The rapid analysis method for fusion protein samples according to Scheme 1 or 2 is characterized in that the sugar removal treatment is achieved by enzymatically removing glycosylation modification from the fusion protein sample.

[0059] 4. The rapid analysis method for fusion protein samples according to Scheme 3 is characterized in that the type of enzyme used in the sugar cleavage treatment is one or more of PNGaseF, endoglycosidase H, and recombinant endoglycosidase F2.

[0060] 5. The rapid analysis method for fusion protein samples according to any one of Schemes 1-4 is characterized in that the analysis method requires denaturation treatment before sugar cutting treatment.

[0061] 6. The rapid analysis method for fusion protein samples according to Scheme 5 is characterized in that the denaturation step requires the addition of a denaturing solution.

[0062] 7. The rapid analysis method for fusion protein samples according to Scheme 6 is characterized in that the denaturing liquid is a guanidine hydrochloride Tris-HCl buffer solution.

[0063] 8. The rapid analysis method for fusion protein samples according to any one of Schemes 5-7 is characterized in that the analysis method also requires liquid replacement after denaturation treatment.

[0064] 9. The rapid analysis method for fusion protein samples according to Scheme 8 is characterized in that the liquid replacement step requires the addition of an enzyme cleavage buffer.

[0065] 10. A rapid analysis method for a fusion protein sample according to any one of Schemes 1-9, characterized in that the fusion protein comprises a first heavy chain, a second heavy chain, a first light chain and a second light chain.

[0066] 11. A rapid analysis method for fusion protein samples according to any one of Schemes 2-10, characterized in that the fusion protein also contains the IL-15 receptor IL-15Rα.

[0067] 12. The rapid analysis method of the fusion protein sample according to Scheme 10 or 11 is characterized in that the first heavy chain contains IL-15Rα and the second heavy chain contains IL-15.

[0068] 13. The rapid analysis method of the fusion protein sample according to Scheme 10 or 11 is characterized in that the first heavy chain contains IL-15 and IL-15Rα, and / or the second heavy chain contains IL-15 and IL-15Rα.

[0069] 14. A rapid analysis method for fusion protein samples according to any one of Schemes 11-13, characterized in that the IL-15Rα and the IL-15 constitute an IL-15Rα / IL-15 complex.

[0070] 15. The rapid analysis method for fusion protein samples according to any one of Schemes 10-14 is characterized in that the first light chain and the second light chain are the same or different light chains.

[0071] 16. A rapid analysis method for fusion protein samples according to any one of Schemes 1-15, characterized in that the fusion protein further comprises an Fc region and a mutant thereof, and the Fc region is selected from IgG1, IgG2, IgG3 and / or IgG4.

[0072] 17. A rapid analysis method for fusion protein samples according to any one of Schemes 1-16, characterized in that the first heavy chain amino acid sequence of the fusion protein is selected from SEQ ID NO:1; the second heavy chain amino acid sequence of the fusion protein is selected from SEQ ID NO:2; and / or the amino acid sequences of the first light chain and the second light chain of the fusion protein are selected from SEQ ID NO:3.

[0073] Beneficial Effects

[0074] The present invention has the following beneficial effects:

[0075] 1. The present invention provides a method for detecting the purity of a multifunctional fusion protein sample using reduction capillary electrophoresis technology. By performing deglycosylation treatment in the traditional reduction capillary electrophoresis purity detection method, the problems of peak deformation, tailing effect during protein separation, and difficulty in accurate quantification when the purity of such drugs is determined by the traditional reduction capillary electrophoresis are solved.

[0076] 2. The present invention provides a method for detecting the purity of a multifunctional fusion protein sample using reduction capillary electrophoresis technology. In the traditional reduction capillary electrophoresis purity detection method, a liquid replacement process is performed to prevent precipitation caused by mixing guanidine hydrochloride and SDS.

[0077] 3. The present invention helps to detect and control the key quality attributes of multifunctional fusion protein samples during the research and development and production stages.

[0078] the term

[0079] The "fusion protein" of the present invention refers to antibodies, antibody fusion proteins, antibody-cytokine fusion proteins, fusion proteins containing cytokines, and the like.

[0080] The "fusion protein sample" of the present invention may refer to the fusion protein, or may refer to a combination of the fusion protein and other excipients.

[0081] The "glycoprotein" of the present invention refers to an antibody, a fragment thereof or a fusion protein having multiple glycan sites.

[0082] "Deglycosylation" of the present invention refers to the process of removing sugar entities (glycans) from glycoproteins.

[0083] "SDS" in the present invention refers to sodium dodecyl sulfate.

[0084] The "Tris" or "tris" of the present invention is tris(hydroxymethyl)aminomethane, which is a biological buffer. The "Tris" or "tris" in the present invention is not contradictory.

[0085] The "PNGaseF" of the present invention refers to N-glycosamidase F or peptide N-glycosidase F.

[0086] The "loading buffer" of the present invention is a mixture of Tris and 1% SDS, and the specific preparation method is to weigh 1.211g of Tris and 1.0g of SDS, add 80ml of water to dissolve, mix well, cool, adjust the pH to 9.0±0.2 with concentrated HCl, add water to make up to 100ml, and mix well.

[0087] The "10×Glyco buffer 2" of the present invention is 0.5 mol / L sodium phosphate (pH 7.5, temperature 25°C)

[0088] The "rCE-SDS" of the present invention refers to the reduction capillary electrophoresis technology. In the present invention, rCE-SDS and reduction CE-SDS and reduction CE-SDS are not contradictory.

[0089] The “HC1 chain” of the present invention is the first heavy chain of the fusion protein, ie, the heavy chain whose amino acid sequence is selected from SEQ ID NO: 1.

[0090] The “HC2 chain” of the present invention is the second heavy chain of the fusion protein, ie, the heavy chain whose amino acid sequence is selected from SEQ ID NO: 2.

[0091] The "PB" of the present invention refers to a mixed solution of Na2HPO4 and NaH2PO4.

[0092] "DTT" of the present invention refers to dithiothreitol. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 This is the rCE-SDS spectrum of the test solution without pretreatment.

[0094] Figure 2 These are the rCE-SDS spectra of the test solution using different denaturing solutions and under different denaturing time conditions during the pretreatment process.

[0095] Figure 3 The rCE-SDS spectra of the test solution using different enzyme cleavage buffers during the pretreatment process. DETAILED DESCRIPTION

[0096] The present invention is further described below in conjunction with the accompanying drawings and specific examples, and the protection content of the present invention is not limited to the following examples. It should also be understood that the terms used in the examples of the present invention are to describe specific specific embodiments, rather than to limit the scope of protection of the present invention. Without departing from the spirit and scope of the inventive concept, the changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims and any equivalents thereof are the protection scope of the present invention. In the specification and claims of the present invention, unless otherwise clearly stated in the text, the singular forms "one", "one" and "this" include plural forms. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are the common knowledge and common common sense of those skilled in the art, and the present invention does not specifically limit the content.

[0097] Example 1 Comparative Experiment

[0098] This example is a comparative experiment to investigate the separation effect of the fusion protein sample without any treatment before loading.

[0099] (1) Dilution:

[0100] Take a fusion protein sample and dilute it with water to a protein concentration of about 2.5 mg / mL, with a total volume of no less than 40 μL.

[0101] (2) Preparation of injection solution:

[0102] The injection solution was prepared according to the components and contents described in Table 1.

[0103] Table 1 Injection solution components and contents

[0104]

[0105] Mix the injection solution, centrifuge for 5 seconds, heat the centrifuge tube in a 70°C water bath for 10 minutes, oscillate to mix, and cool to room temperature; centrifuge at 3000rpm for 60 seconds, take 90μL of the supernatant into a micro-sample tube, then put the micro-sample tube into the injection bottle, cover it with a lid, and put it into the Inlet sample tray of the capillary electrophoresis instrument.

[0106] (3) Capillary pretreatment:

[0107] Rinse with 0.1 mol / L sodium hydroxide solution at 70 psi pressure for 3 minutes, then rinse with 0.1 mol / L hydrochloric acid solution at 70 psi pressure for 1 minute, and finally rinse with pure water at 70 psi pressure for 1 minute.

[0108] (4) Pre-filling of capillaries:

[0109] The SDS gel separation buffer was flushed at 70 psi for 10 min.

[0110] (5) Sample injection:

[0111] Reverse polarity electrokinetic injection at 5.0 KV, injection time 20 seconds.

[0112] (6) Separation:

[0113] Run at 15.0KV for 30 minutes, reverse polarity.

[0114] (7) Sample room conditions:

[0115] Sample chamber temperature: 25°C;

[0116] Capillary temperature: 25°C;

[0117] Detection wavelength: 214nm.

[0118] Depend on Figure 1 It can be seen from the rCE-SDS spectrum that the HC2 peak has a poor shape. The main reason is that there are multiple sugar modifications on the HC2 chain. Direct reduction capillary electrophoresis results in poor peak shape and low response value during protein separation.

[0119] Example 2 Investigation of denaturing fluid

[0120] In order to solve the problem of poor peak shape of HC2, this example examined the peak shape of the fusion protein sample after denaturation, liquid replacement, and sugar removal.

[0121] Prepare denaturation buffer A, denaturation buffer B, and enzyme digestion buffer A according to the following ratio:

[0122] Denaturing solution A: Add 1.21 g tris to 30.57 g guanidine hydrochloride, add water to 35 mL, adjust the pH to 7.6 with 6 mol / L HCl, and add water to 40 mL.

[0123] Denaturing solution B (i.e. SDS DTT denaturing solution): 5% (m / v) SDS, 0.4 mol / L DTT.

[0124] Enzyme digestion buffer A: 1 mol / L urea dissolved in 20 mmol / L PB buffer, pH 6.0.

[0125] Take 500μg of fusion protein, add 400μL of denaturing solution, and place in a 37℃ water bath; use an ultrafiltration tube to replace the solution with enzyme digestion buffer A, and replace the solution at least 81 times in total, and quantify; the final volume of the solution after the solution replacement is not less than 130μL, and the protein concentration is not less than 7mg / mL. Take about 500μg of fusion protein after the solution replacement, add 5μL PNGaseF, 20μL 10×Glyco buffer 2, and make up to 100μL system with enzyme digestion buffer; mix well, centrifuge for 5s, and place in a 37℃ water bath for 16h. Load the sample in the manner of Example 1.

[0126] The different denaturing solutions and denaturing times used in this example are shown in Table 2.

[0127] Table 2 Different denaturation solutions and denaturation times

[0128] Serial number Denaturing fluid Denaturation time (hours) 1 Denaturing Fluid A 0.5 2 Denaturing Fluid A 1 3 Denaturing Fluid B 0.5

[0129] Figure 2 The rCE-SDS spectra of the test solution under different denaturation conditions. It can be seen from the rCE-SDS spectrum that when using denaturation solution B, the HC1 chain peak and the HC2 chain peak cannot be separated. When using denaturation solution A, whether the denaturation time is 0.5 hour or 1 hour, the HC1 chain peak and HC2 chain peak of the test solution are completely separated, and the peak shape of the HC2 chain peak is very good and the response value is high.

[0130] Example 3 Investigation of enzyme digestion buffer

[0131] This example investigates the effect of the concentration of the enzyme digestion buffer on the rCE-SDS spectrum of the test solution during the solution change.

[0132] Take 500μg of fusion protein, add 400μL of denaturing solution A, and place in a water bath at 37℃ for 0.5 hours; use an ultrafiltration tube to replace the solution with the enzyme digestion buffer; the final volume of the solution after the solution replacement is not less than 130μL, and the protein concentration is not less than 7mg / mL. Take about 500μg of fusion protein after the solution replacement, add 5μL PNGaseF, 20μL 10×Glyco buffer 2, and make up to 100μL system with enzyme digestion buffer; mix well, centrifuge for 5s, and place in a water bath at 37℃ for 16h. Load the sample in the same manner as in Example 1.

[0133] Prepare Enzyme Digestion Buffer A and Enzyme Digestion Buffer B according to the following ratio:

[0134] Enzyme digestion buffer A: 1 mol / L urea dissolved in 20 mmol / L PB buffer, pH 6.0.

[0135] Enzyme digestion buffer B: 1.5 mol / L urea dissolved in 20 mmol / L PB buffer, pH 6.0.

[0136] The enzyme digestion buffers used in this example are respectively enzyme digestion buffer A and enzyme digestion buffer B. The rCE-SDS spectrum of the fusion protein sample is as follows: Figure 3 As shown in the results, no matter whether the enzyme digestion buffer A or the enzyme digestion buffer B is used, the peak shape of the HC2 chain of the fusion protein sample is very good.

[0137] Example 4 Improved Experiment on Precipitation

[0138] Take 500 μg of fusion protein, add 400 μL of denaturing solution, and place in a 37°C water bath; use an ultrafiltration tube to replace the solution with enzyme digestion buffer A, with a final concentration of 2.5 mg / mL. Take about 500 μg of fusion protein after the solution replacement, add 5 μL of PNGaseF, 20 μL of 10×Glyco buffer 2, and use enzyme digestion buffer to make up to 100 μL system; mix well, centrifuge for 5 seconds, and place in a 37°C water bath for 16 hours. Load the sample in the same manner as in Example 1.

[0139] Take 500 μg of fusion protein and add 400 μL of denaturing solution A, incubate at 37°C in a water bath for 30 min, and replace the solution with enzyme digestion buffer A, with a final concentration of 2.5 mg / mL. Add 2.5 μL of PNGaseF and 20 μL of 10× Glyco buffer 2, and incubate at 37°C in a water bath for 16 h.

[0140] The test solution was prepared according to the method of Example 1. After adding 55 μL of loading buffer, the solution became turbid and a white precipitate was produced after centrifugation. The following improved experiments were performed: Method 1: Lower the temperature and place the fusion protein after sugar cleavage at -20°C. It was still turbid. Method 2: Increase the number of liquid changes (at least 81 times) to make the liquid change thorough and no precipitation occurs.

[0141] In summary, before using the reduction capillary electrophoresis method to detect the purity of the fusion protein sample, the fusion protein sample is denatured, the liquid is replaced and the sugar is cut, which not only solves the problem of precipitation of the fusion protein sample, but also solves the problem of poor peak shape of the HC2 chain peak and low response value.

[0142] It should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. 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 present invention.

Claims

1. A rapid analysis method for fusion protein samples, characterized in that: The fusion protein sample is analyzed by capillary electrophoresis analysis technology, and the method includes a step of performing sugar cleavage treatment on the fusion protein sample, wherein the sugar cleavage treatment is achieved by removing glycosylation modification by enzymatic cleavage of the fusion protein sample, wherein the enzyme used in the sugar cleavage treatment is PNGaseF; the analysis method needs to undergo denaturation treatment before the sugar cleavage treatment; the denaturation step needs to add a denaturing solution, wherein the denaturing solution is a guanidine hydrochloride Tris-HCl buffer solution; the analysis method also needs to undergo a liquid replacement treatment after the denaturation treatment; The liquid replacement step requires the addition of an enzyme cleavage buffer, which is 1 mol / L urea dissolved in 20 mmol / L PB buffer, with a pH of 6.0; the fusion protein comprises a first heavy chain, a second heavy chain, a first light chain, and a second light chain, wherein the first light chain and the second light chain are the same light chain; the fusion protein comprises a cytokine, wherein the cytokine is IL-15; the fusion protein also comprises the IL-15 receptor IL-15Rα; the IL-15Rα and the IL-15 constitute an IL-15Rα / IL-15 complex; the fusion protein also comprises an Fc region and a mutant thereof, wherein the Fc region is selected from IgG1, IgG2, IgG3 and / or IgG4; the first heavy chain amino acid sequence of the fusion protein is as shown in SEQ ID NO: 1; the second heavy chain amino acid sequence of the fusion protein is as shown in SEQ ID NO: 2; and the amino acid sequences of the first light chain and the second light chain of the fusion protein are as shown in SEQ ID NO: 3.

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