A gel separation buffer for capillary electrophoresis detection

Abstract

The present invention relates to the field of biomedicine, and in particular to a gel separation buffer for capillary electrophoresis detection, characterized in that it comprises: tris(hydroxymethyl)aminomethane (Tris), 3.1-4.6% (w / w) boric acid, disodium ethylenediaminetetraacetate (EDTA), glycerol, a surfactant, 8.0-12.0% (w / w) dextran, wherein the surfactant is sodium dodecyl sulfate (SDS) or sodium hexadecyl sulfate (SHS). The present invention provides a gel separation buffer with good separation, stable baseline and good durability.

Description

Technical Field

[0001] The invention relates to the field of biomedical detection, and in particular to a gel separation buffer for capillary electrophoresis detection. Background Art

[0002] Monoclonal antibody (mAb), also known as mAb, is an antibody produced by only one type of immune cell. At present, monoclonal antibody drugs are widely used in many fields and play an important role in the treatment of autoimmune diseases, tumors, metabolic diseases and other fields. In 2018, the sales of monoclonal antibody drugs accounted for 55.3% of the global biopharmaceutical market. Among the top 20 drugs in global sales in 2020, 9 of the 13 biopharmaceuticals are monoclonal antibodies. With the introduction of new monoclonal antibody drugs and the increase in the penetration rate of antibody drugs, monoclonal antibody drugs have become a research hotspot in recent years.

[0003] Monoclonal antibodies have a large molecular weight of about 150kD, and quality control usually requires multiple means to corroborate each other. Capillary gel electrophoresis is applied in the biotechnology industry and has become a mature technology. It is usually used for the characterization and stability study of therapeutic proteins, especially monoclonal antibodies. CE-SDS can be regarded as SDS-PAGE converted into a capillary form, with better resolution and sensitivity. The components of the gel separation buffer enter the capillary to form a dynamic network structure of the polymer, and the sample is separated under the action of voltage. Under normal circumstances, protein molecules bind to SDS molecules at a ratio of 1:1.4 under the denaturing effect of SDS. The denatured protein complex formed has a uniform charge / size ratio and migrates in the network structure of the gel separation buffer. The migration rate is proportional to the molecular weight, and the proteins are separated in order from small to large molecular weight. When the molecule contains more disulfide bonds or sugars, the arrangement order of the proteins may be opposite to the molecular weight.

[0004] Capillary gel electrophoresis is an important quality control method for biopharmaceuticals, but there are also many problems in its application. The method currently uses a commercial gel separation buffer kit (for example, purchased from Beckman Co.), which has unsatisfactory detection results for most samples and is only applicable to proteins with a molecular weight below 200kD. The separation between protein molecule fragments and intact protein peaks is poor, resulting in inaccurate fragment values, and low resolution for monitoring disulfide bond subtypes of IgG2 antibodies. In addition, commercial kits are expensive, have a long lead time, and have very few options, which limits the development of detection methods.

[0005] US7381317B2-Methods and compositions for capillary electrophoresis (CE) discloses a CE-SDS gel separation buffer, but does not disclose the separation effect that can be obtained by using it to detect IgG2 monoclonal antibodies, and the applicable detection objects have certain limitations.

[0006] At present, there is no good domestic gel separation buffer in the industry, nor is there a gel separation buffer with better separation effect that can be used to replace several commonly used imported commercial gel separation buffers. Therefore, it is urgent to develop a gel separation buffer for capillary electrophoresis detection to provide an important monitoring function for the process development and analysis of macromolecular proteins. Summary of the invention

[0007] In view of the above situation, the present invention provides a gel separation buffer for capillary electrophoresis detection. The gel separation buffer has the advantages of good separation, stable baseline and good durability.

[0008] The purpose of the present invention is achieved through the following technical scheme: a gel separation buffer for capillary electrophoresis detection, characterized in that it comprises: tris(hydroxymethyl)aminomethane (Tris), 3.1-4.6% (w / w) boric acid, disodium ethylenediaminetetraacetate (EDTA), glycerol, a surfactant, 8.0-12.0% (w / w) dextran, wherein the surfactant is sodium dodecyl sulfate (SDS), sodium hexadecyl sulfate (SHS) or sodium hexadecyl sulfate (containing 40% sodium octadecyl sulfate).

[0009] As one of the embodiments, the pH of the gel separation buffer is 8.1±0.1, and as an exemplary description, includes but is not limited to 8.0, 8.1, and 8.2.

[0010] As one of the embodiments, the gel separation buffer is calculated by weight percentage (w / w):

[0011] The Tris content is 5.5-6.7%, preferably 5.72-6.67%;

[0012] The boric acid content is 3.15-4.56%;

[0013] The EDTA content is 0.1-0.2%, preferably 0.13-0.15%;

[0014] The glycerol content is 7.9-8.5%, preferably 8.05-8.39%;

[0015] The surfactant content is 0.05-0.2%, preferably 0.08-0.18%;

[0016] The dextran content is 8.21-11.80%.

[0017] The present invention also provides a method for preparing the above-mentioned gel separation buffer, comprising the following steps:

[0018] (a) preparing Tris-borate buffer;

[0019] (b) adding a surfactant to the buffer obtained in step (a);

[0020] (c) adding dextran to the solution obtained in step (b), stirring thoroughly to dissolve.

[0021] As one of the implementation schemes, the above steps are calculated by weight percentage (w / w):

[0022] (a) Weigh 5.72-6.67% Tris and 3.15-4.56% boric acid, add 90 ml of deionized water, adjust pH to 8.1±0.10 (25°C±1.5°C), stir to dissolve, add 0.13-0.15% EDTA, stir to dissolve, and then add 8.05-8.39% glycerol;

[0023] (b) adding 0.08-0.18% of a surfactant;

[0024] (c) After the surfactant is completely dissolved, 8.21-11.80% dextran is slowly added to the buffer solution while stirring. Stirring is continued at room temperature for 18-24 hours, and the buffer solution is filtered using a 0.22 μm filter system.

[0025] The present invention also provides a method for detecting an analyte in a sample, comprising contacting the analyte with the gel separation buffer and performing detection and separation.

[0026] As one of the embodiments, the method comprises the following steps:

[0027] Prepare a sample solution containing the analyte: prepare a non-reducing sample solution and a reducing sample solution respectively; the non-reducing sample solution is: dilute the analyte with a sample buffer to a solution containing about 1 mg / ml protein, add iodoacetamide solution, mix well, and then heat; the reducing sample solution is: dilute the analyte with a sample buffer to a solution containing about 1.25 mg / ml protein, add 2-mercaptoethanol, mix well, and then heat;

[0028] Load the sample and perform capillary electrophoresis detection.

[0029] As one of the implementation schemes, the sample buffer is a 40mmol / L phosphate buffer containing 1% SDS, pH 6.5; the concentration of the iodoacetamide solution is 800mmol / L; the electrophoresis detection conditions are: detection wavelength: 214nm; column temperature: 25°C; injection voltage: 5kV; injection time: 20s (reducing conditions) / 40s (non-reducing conditions); separation voltage: 15kV; separation time: 38min.

[0030] As one of the embodiments, the method comprises the following steps:

[0031] 1) Solution preparation: prepare iodoacetamide solution, phosphate sample buffer and gel separation buffer respectively; the iodoacetamide solution is: weigh 74 mg of iodoacetamide powder, add 500 μl of water to dissolve, and obtain 800 mmol / L iodoacetamide solution; the phosphate sample buffer is: weigh 1.69 g of anhydrous disodium hydrogen phosphate and 0.95 g of anhydrous sodium dihydrogen phosphate, dissolve in 500 ml of deionized water, mix well, and adjust the pH to 6.5 with phosphoric acid. Take 25 ml of the solution, add 0.25 g of SDS, stir and mix well to obtain 40 mmol / L phosphate sample buffer;

[0032] 2) Preparation of stock solution: prepare non-reducing stock solution and reducing stock solution respectively; the non-reducing stock solution is: the non-reducing sample is diluted with water to a solution containing about 2 mg / ml protein, and diluted with sample buffer to a solution containing about 1 mg / ml protein; the reducing stock solution is: the reducing sample is diluted with water to a solution containing about 2.5 mg / ml protein, and diluted with sample buffer to a solution containing about 1.25 mg / ml protein;

[0033] 3) Preparation of sample solution: prepare a non-reduced sample solution and a reduced sample solution respectively; the non-reduced sample solution is prepared by adding 5 μl of 800 mmol / L iodoacetamide solution to the non-reduced stock solution obtained in step 2), vortexing for 30 seconds, centrifuging for 20 seconds, sealing with a sealing film, water bathing at 65°C for 10 minutes, and cooling at room temperature for 5 minutes; the reduced sample solution is prepared by adding 5 μl of 2-mercaptoethanol to the reduced stock solution obtained in step 2), vortexing for 30 seconds, centrifuging for 20 seconds, sealing with a sealing film, water bathing at 65°C for 10 minutes, and cooling at room temperature for 5 minutes;

[0034] 4) Loading:

[0035] a. Pretreatment of the capillary: flush with 0.1mol / L sodium hydroxide solution at 70psi pressure for 10min, then flush with 0.1mol / L hydrochloric acid solution at 50psi pressure for 5min, and finally flush with pure water at 50psi pressure for 2min. This should be done before each run;

[0036] b. Pre-filling of capillaries: The gel separation buffer is flushed at 70 psi pressure for 10 min before each run; the capillary column is a non-coated fused silica capillary column, 50 μm*30 cm

[0037] c. The detection conditions are as follows: detection wavelength: 214 nm; column temperature: 25°C; injection voltage: 5 kV; injection time: 20 s (reducing conditions) / 40 s (non-reducing conditions); separation voltage: 15 kV; separation time: 38 min.

[0038] As one of the embodiments, the analyte is a macromolecular protein, preferably a monoclonal antibody, more preferably IgG2 or IgG4.

[0039] As one of the embodiments, the IgG2 includes Romosozumab and its analogs.

[0040] As one of the embodiments, the IgG4 is Dupilumab and its analogs.

[0041] The present invention also provides an application of the method in protein drug monitoring.

[0042] As one of the implementation schemes, the protein drug monitoring includes culture medium optimization, clone screening, formulation stability study, purification process, protein characterization, related impurity detection, protein structure identification and quality control of protein drug products.

[0043] The technical effects achieved by the present invention are as follows:

[0044] 1. The gel separation buffer for capillary detection provided by the present invention is different from the prior art and has the advantages of good separation, stable baseline and good durability. The fragment values ​​and disulfide bond isomer ratio values ​​obtained by detection are reduced, eliminating a large number of false values, thereby ensuring the accuracy of the data and the feasibility of the process, and also enhancing the monitoring ability of disulfide bond isomers. Under reducing conditions, the peak time difference between the heavy chain and the light chain is longer, the baseline is more stable, and the heavy chain tailing is effectively suppressed.

[0045] 2. The gel separation buffer of the present invention has good durability and still has a good separation effect after being placed for 3 months.

[0046] 3. The gel separation buffer of the present invention can be used to detect monoclonal antibodies, such as Romosozumab and its analogs, Dupilumab and its analogs. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 :Gel separation buffer control 1 is used to detect the non-reducing CE spectrum of Romo monoclonal antibody

[0048] Figure 2 :Gel separation buffer control 1 is used to detect the reductive CE spectrum of Romo monoclonal antibody

[0049] Figure 3 :Gel separation buffer A and B, control 2 used to detect non-reducing CE spectra of Romo monoclonal antibody

[0050] Figure 4 :Gel separation buffer A and B, control 2 used to detect the reductive CE spectrum of Romo monoclonal antibody

[0051] Figure 5 :Gel separation buffer C was used to detect the non-reducing CE spectrum of Romo monoclonal antibody

[0052] Figure 6 :Gel separation buffer C is used to detect the reduced CE spectrum of Romo monoclonal antibody

[0053] Figure 7 :Gel separation buffer D was used to detect the non-reducing CE spectrum of Romo monoclonal antibody

[0054] Figure 8 : Non-reducing CE spectrum of gel separation buffer C durability test

[0055] Fig. 9 : Reductive CE spectrum of gel separation buffer C durability test

[0056] Fig.10 :Comparison of non-reducing CE spectra of gel separation buffer C and gel separation buffer control 1 for detecting Dupi monoclonal antibody

[0057] Fig.11 :Comparison of the reductive CE spectra of gel separation buffer C and gel separation buffer control 1 for detecting Dupi monoclonal antibody DETAILED DESCRIPTION

[0058] The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0059] Those skilled in the art should understand that the reagents used in the following examples, unless otherwise specified, were purchased from the market.

[0060] Romosozumab fermentation broth was obtained with reference to CN103435698B, and Romosozumab injection (batch numbers 202311001, 202311002, and 202311003) was obtained according to the instructions formula disclosed by the FDA (https: / / www.accessdata.fda.gov / drugsatfda_docs / label / 2019 / 761062s000lbl.pdf).

[0061] Example 1 Gel separation buffer (control 1) prepared with reference to US20090314638A1 was used to detect Romosozumab monoclonal antibody

[0062] 1. Solution preparation

[0063] (1) 800mmol / L iodoacetamide solution: weigh 74mg of iodoacetamide powder, place in a 1.5ml centrifuge tube, add 500μl of water to dissolve, prepare freshly before use, and avoid exposure to light;

[0064] (2) 40mmol / L phosphate sample buffer: weigh 1.69g of anhydrous disodium hydrogen phosphate and 0.95g of anhydrous sodium dihydrogen phosphate, dissolve in 500ml of deionized water, mix well, and adjust the pH to 6.5 with phosphoric acid. Take 25ml of the solution, add 0.25g of SDS (sodium dodecyl sulfate), stir and mix well, and filter with a 0.22μm filter membrane before use;

[0065] (3) 2-mercaptoethanol solution;

[0066] (4) 0.1 mol / L hydrochloric acid solution;

[0067] (5) 0.1 mol / L sodium hydroxide solution;

[0068] (6) Gel separation buffer:

[0069] a. Prepare triborate glycerol buffer: weigh 7.23g Tris (trishydroxymethylaminomethane), 3.75g boric acid, add 90ml deionized water, adjust pH 8.1±0.10 (25℃±1.5℃), stir to dissolve, add 0.16g EDTA (disodium ethylenediaminetetraacetic acid), stir to dissolve, and then add 9.86g glycerol. b. Prepare gel separation buffer: add 0.11g SDS to triborate glycerol buffer solution, wait for SDS powder to completely dissolve, and slowly add 9.01g dextran to the buffer while stirring, weigh, the total weight of the solution is 117g. Continue stirring at room temperature for 18 to 24 hours. Filter the buffer with a 0.22μm filter system. Store the gel separation buffer in a clean container at room temperature.

[0070] 2. Preparation of stock solution

[0071] (1) System suitability stock solution: Take an appropriate amount of Romosozumab (abbreviated as Romo) original injection solution as the system suitability sample. The non-reduced sample is diluted with sample buffer to a solution containing approximately 1 mg / ml protein; the reduced sample is diluted with sample buffer to a solution containing approximately 1.25 mg / ml protein.

[0072] (2) Test sample stock solution: Take an appropriate amount of Romo original injection solution and dilute the non-reduced sample with sample buffer to a test sample solution containing approximately 1 mg / ml protein; dilute the reduced sample with sample buffer to a test sample solution containing approximately 1.25 mg / ml protein.

[0073] (3) Blank stock solution: Take an appropriate amount of solvent equal to the amount of Romo original injection solution in the test sample stock solution and prepare it using the same preparation method.

[0074] 3. Sample solution preparation

[0075] (1) Non-reduced sample solution: Take 95 μl of system suitability stock solution, test sample stock solution and blank stock solution, add 5 μl of 800 mmol / L iodoacetamide solution to each, vortex for 30 seconds, centrifuge for 20 seconds, seal with sealing film, and place in a water bath at 65°C for 10 minutes. Cool at room temperature for 5 minutes, transfer 70 μl of sample solution to a microinjection tube, and then place it in a universal bottle and cover the bottle cap.

[0076] (2) Reduction of sample solution: Take 95 μl of system suitability stock solution, test sample stock solution and blank stock solution, add 5 μl of 2-mercaptoethanol to each, vortex for 30 seconds, centrifuge for 20 seconds, seal with sealing film, and place in a water bath at 65°C for 10 minutes. Cool at room temperature for 5 minutes, transfer 70 μl of sample to a microinjection tube, and then place it in a universal bottle and cover the bottle cap.

[0077] 4. Determination method

[0078] Capillary column: non-coated fused silica capillary column, 50μm*30cm

[0079] Pretreatment of the capillary: flush with 0.1mol / L sodium hydroxide solution at 70psi pressure for 10min, then flush with 0.1mol / L hydrochloric acid solution at 50psi pressure for 5min, and finally flush with pure water at 50psi pressure for 2min. This should be performed before each run.

[0080] Pre-filling of the capillary: Flush with gel separation buffer at 70 psi for 10 min before each run.

[0081] Detection wavelength: 214 nm; column temperature: 25°C; injection voltage: 5 kV; injection time: 20 s (reducing conditions) / 40 s (non-reducing conditions); separation voltage: 15 kV; separation time: 38 min.

[0082] 5. Results Analysis

[0083] Non-reductive results such as Figure 1 As shown, according to the area normalization method, the data detection results are shown in Table 1, and the reduction results are shown in Figure 2 shown.

[0084] Table 1 Gel separation buffer control 1 test sample results

[0085] name Romo original injection Fragments 1.95 IgG2-1 17.95 IgG2-2 80.10

[0086] Example 2 Gel Separation Buffer A and B and Gel Separation Buffer Control 2 for Detection of Romosozumab Monoclonal Antibody

[0087] Gel Separation Buffer Control 2 was prepared as follows:

[0088] Weigh 7.27g Tris, 2.01g boric acid, add 90ml deionized water, adjust pH 8.1±0.10 (25℃±1.5℃), stir to dissolve, add 0.17g EDTA, stir to dissolve, then add 9.96g glycerol. Add 0.10g SDS to the above buffer solution, wait until the SDS powder is completely dissolved, slowly add 10.03g dextran to the buffer solution while stirring, weigh, the total weight of the solution is 118g. Continue stirring at room temperature for 18 to 24 hours. Filter the buffer using a 0.22μm filter system. Store the gel separation buffer in a clean container at room temperature.

[0089] Gel separation buffer A was prepared as follows:

[0090] Weigh 7.27g Tris, 4.00g boric acid, add 90ml deionized water, adjust pH 8.1±0.10 (25℃±1.5℃), stir to dissolve, add 0.16g EDTA, stir to dissolve, then add 10.13g glycerol. Add 0.10g SDS to the above buffer solution, wait until the SDS powder is completely dissolved, slowly add 10.07g dextran to the buffer solution while stirring, weigh, the total weight of the solution is 121g. Continue stirring at room temperature for 18 to 24 hours. Filter the buffer using a 0.22μm filter system. Store the gel separation buffer in a clean container at room temperature.

[0091] Gel separation buffer B was prepared as follows:

[0092] Weigh 7.27g Tris, 5.57g boric acid, add 90ml deionized water, adjust pH 8.1±0.10 (25℃±1.5℃), stir to dissolve, add 0.17g EDTA, stir to dissolve, then add 10.24g glycerol. Add 0.11g SDS to the above buffer solution, wait until the SDS powder is completely dissolved, slowly add 10.02g dextran to the buffer solution while stirring, weigh, the total weight of the solution is 122g. Continue stirring at room temperature for 18 to 24 hours. Filter the buffer using a 0.22μm filter system. Store the gel separation buffer in a clean container at room temperature.

[0093] Referring to the method described in detail in Example 1, the Romo original injection was tested using the gel separation buffer control 2, gel separation buffer A and B of this example. The non-reduced results are as follows: Figure 3 As shown, according to the area normalization method, the data detection results are shown in Table 2, and the reduction results are shown in Figure 4 shown.

[0094] Table 2 Gel separation buffer control 2, A and B test sample results

[0095]

[0096] The above spectra and data detection results show that the gel separation buffer control 2 failed to achieve the purpose of detection, while the peaks of gel separation buffers A and B were relatively consistent. Compared with the gel separation buffer control 1 of Example 1, the separation degree of the fragments before the main peak was better, which reduced the false value and reduced the percentage of fragments. The increase in the amount of boric acid also has a direct impact on the stability of the baseline, making the value more accurate.

[0097] Example 3 Gel separation buffer C is used to detect Romosozumab monoclonal antibody

[0098] Gel separation buffer C was prepared as follows:

[0099] Weigh 7.27g Tris, 4.00g boric acid, add 90ml deionized water, adjust pH 8.1±0.10 (25℃±1.5℃), stir to dissolve, add 0.17g EDTA, stir to dissolve, then add 10.22g glycerol. Add 0.10g SDS to the above buffer solution, wait until the SDS powder is completely dissolved, slowly add 15.02g dextran to the buffer solution while stirring, weigh, the total weight of the solution is 127g. Continue stirring at room temperature for 18 to 24 hours. Filter the buffer using a 0.22μm filter system. Store the gel separation buffer in a clean container at room temperature.

[0100] Romo original injection and Romo injection (batch numbers 202311001, 202311002, and 202311003) were tested with reference to the method described in detail in Example 1. The non-reduction results are as follows: Figure 5 As shown, according to the area normalization method, the data detection results are shown in Table 3, and the reduction results are shown in Figure 6 shown.

[0101] Table 3 Results of gel separation buffer C test samples

[0102]

[0103] The above spectra and data detection results show that compared with Example 1, under non-reducing conditions, the fragment values ​​and disulfide bond subtype isomer ratio values ​​obtained by using gel separation buffer C for detection have decreased, eliminating a large number of false values, thereby ensuring the accuracy of the data and the feasibility of the process. In addition, the separation degree of IgG2 disulfide bond subtype isomers has also been significantly improved, enhancing the monitoring ability of disulfide bond subtype isomers. At the same time, it can be seen that the spectrum baseline is more stable, ensuring the accuracy of the integration effect. Under reducing conditions, the peak time difference between the heavy chain and the light chain is longer, the baseline is more stable, and the heavy chain tailing is effectively suppressed.

[0104] Example 4 Gel separation buffer D is used to detect Romosozumab monoclonal antibody

[0105] Gel separation buffer D was prepared as follows:

[0106] Weigh 7.27g Tris, 4.01g boric acid, add 90ml deionized water, adjust pH 8.1±0.10 (25℃±1.5℃), stir to dissolve, add 0.17g EDTA, stir to dissolve, then add 9.13g glycerol. Add 0.20g SHS (sodium hexadecyl sulfate (containing 40% sodium octadecyl sulfate)) to the above buffer solution, wait until the SHS powder is completely dissolved, slowly add 9.01g dextran to the buffer solution while stirring, weigh, the total weight of the solution is 109g. Continue stirring at room temperature for 18 to 24 hours. Filter the buffer using a 0.22μm filter system. Store the gel separation buffer in a clean container at room temperature.

[0107] Referring to the method described in detail in Example 1, the original Romo injection solution was tested using the gel separation buffer control 1 of Example 1 and the gel separation buffer D of this example. The non-reducing results are as follows: Figure 7 shown.

[0108] The above spectral results show that compared with the gel separation buffer control 1, the gel separation buffer D (containing 0.2% SHS) has a significant effect on the separation of the disulfide bond subtypes of the main peak.

[0109] Example 5 Durability test of gel separation buffer C

[0110] Referring to the method described in detail in Example 1, the Romo original injection was tested using gel separation buffer C with different storage times. The storage times of the three gel separation buffers C were 3 months, 1 month, and 0 days, respectively. The non-reduction results are as follows: Figure 8 As shown, the reduction results are as follows Fig. 9 As shown, according to the area normalization method, the data detection results are shown in Table 4.

[0111] Table 4 Durability test results of gel separation buffer C

[0112]

[0113] The above spectra and data detection results show that the use of gel separation buffer C to detect Romo original injection has good durability, a stable baseline, and basically consistent peak shapes.

[0114] Example 6 Gel separation buffer C is used to detect Dupilumab monoclonal antibody

[0115] The original Dupi injection was tested using gel separation buffer C according to the method described in detail in Example 1. The non-reduced results are as follows: Fig.10 As shown, the reduction results are as follows Fig.11 shown.

[0116] The spectral results show that the use of gel separation buffer C has the advantages of good separation, good baseline, long peak time difference between heavy chain and light chain under reducing conditions, and no tailing.

[0117] It is to be understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present invention, but the present invention is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the principles and essence of the present invention, and these modifications and improvements are also considered as protections of the present invention.

Claims

1. A gel separation buffer for capillary electrophoresis detection, characterized in that: The invention comprises, by mass percentage, 5.5-6.7% tris(hydroxymethyl)aminomethane, 3.1-4.6% boric acid, 0.1-0.2% EDTA, 7.9-8.5% glycerol, 0.05-0.2% surfactant, 8.0-12.0% dextran, and a pH value of 8.1±0.1; the surfactant is sodium dodecyl sulfate, sodium hexadecyl sulfate, or a mixture of sodium hexadecyl sulfate containing 40% sodium octadecyl sulfate.

2. The method for preparing the gel separation buffer according to claim 1, characterized in that: The following steps are involved: (a) Prepare Tris-borate buffer; (b) adding a surfactant to the buffer obtained in step (a); (c) Add dextran to the solution obtained in step (b) and stir thoroughly to dissolve.

3. The method according to claim 2, characterized in that The method comprises the following steps, calculated by weight percentage: (a) Weigh 5.72-6.67% Tris and 3.15-4.56% boric acid, add deionized water, adjust the pH to 8.1±0.10, stir to dissolve, add 0.13-0.15% EDTA, stir to dissolve, and then add 8.05-8.39% glycerol; (b) adding 0.08-0.18% of a surfactant; (c) After the surfactant is completely dissolved, slowly add 8.21-11.80% dextran to the buffer solution while stirring. Continue stirring at room temperature for 18-24 hours and filter the buffer solution using a 0.22 μm filter system.

4. A method for detecting an analyte in a sample, characterized in that: The method comprises contacting an analyte with the gel separation buffer according to claim 1 and performing detection and separation, wherein the analyte is a macromolecular protein.

5. The method according to claim 4, characterized in that The following steps are involved: Prepare a sample solution containing the analyte: prepare a non-reducing sample solution and a reducing sample solution respectively; the non-reducing sample solution is: dilute the analyte with a sample buffer to a solution containing 1 mg / ml protein, add iodoacetamide solution, mix well, and then heat; the reducing sample solution is: dilute the analyte with a sample buffer to a solution containing 1.25 mg / ml protein, add 2-mercaptoethanol, mix well, and then heat; Load the sample and perform capillary electrophoresis detection; The sample buffer is 40 mmol / L phosphate buffer containing 1% SDS, pH 6.5; the concentration of the iodoacetamide solution is 800 mmol / L; the electrophoresis detection conditions are: detection wavelength: 214 nm, column temperature: 25°C, injection voltage: 5 kV, injection time: 20 s under reducing conditions / 40 s under non-reducing conditions, separation voltage: 15 kV, separation time: 38 min. The method according to claim 4 , wherein the macromolecular protein is a monoclonal antibody. The method according to claim 6 , wherein the monoclonal antibody is IgG2 or IgG4.

8. Use of the method according to claim 4 in protein drug monitoring.

9. The use according to claim 8, characterized in that The protein drug monitoring includes culture medium optimization, clone screening, formulation stability study, purification process, protein characterization, related impurity detection, protein structure identification and quality control of protein drug products.

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