Method for identifying multiple therapeutic antibodies based on novel specific enzyme digestion method and application

Abstract

The invention relates to a method for identifying multiple therapeutic antibodies based on a novel specific enzyme digestion method and application. The method comprises the following steps: performing enzyme digestion treatment on a sample to be detected, performing denaturation reduction treatment, detecting by adopting liquid chromatography, comparing a detection result with a standard substance, and identifying, a chromatographic column of the liquid chromatography is a polyphenyl chromatographic column, a mobile phase A is a 0.02%-0.5% trifluoroacetic acid aqueous solution, and a mobile phase B is a 0.02%-0.5% trifluoroacetic acid-acetonitrile solution. The invention aims to solve the problems of complex sample preparation, long chromatographic separation time, non-ideal degree of separation and long data analysis time in the existing method, and provides a rapid, stable and controllable identification analysis method for quality control of therapeutic antibodies.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a method and its application for identifying a variety of therapeutic antibodies based on a novel specific enzyme digestion method. Background Technology

[0002] Antibody identification is a regulatory requirement for confirming whether a product's molecular structure conforms to its claimed structure. It is crucial for ensuring antibody consistency and safety, particularly in drug development and quality control of biologics. Currently, peptide mapping is commonly used as an antibody identification method. Although small peptide analysis has high site specificity, complex sample preparation, lengthy chromatographic separations, and time-consuming data processing often hinder its use in routine product quality monitoring. Furthermore, prolonged enzymatic digestion can introduce human error, leading to erroneous conclusions.

[0003] Literature reports that IdeS enzyme digestion followed by high-performance liquid chromatography (HPLC) detection is used as a method for antibody identification and analysis. IdeS enzyme can specifically cleave the hinge region of antibodies, exhibiting high specificity, simple operation, stable and controllable digestion process, and producing only a limited number of antibody fragments, effectively compensating for the shortcomings of peptide mapping methods. However, the currently reported IdeS enzyme digestion-HPLC method still suffers from problems such as long chromatographic separation time, incomplete elution, and unsatisfactory resolution. Therefore, there is an urgent need to establish a rapid, stable, and controllable identification and analysis method for the quality control of therapeutic antibodies. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method and application for identifying various therapeutic antibodies based on a novel specific enzyme digestion method. The purpose of this invention is to solve the problems of complex sample preparation, long chromatographic separation time, unsatisfactory separation degree, and time-consuming data analysis in existing methods, and to provide a rapid, stable, and controllable identification and analysis method for the quality control of therapeutic antibodies.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for identifying a variety of therapeutic antibodies based on a novel specific enzyme digestion, the method comprising digesting the sample to be tested with an enzyme, followed by denaturation and reduction treatment, detecting the sample by liquid chromatography, and identifying the sample by comparing the detection results with a standard.

[0007] The liquid chromatography column is a polystyrene column, mobile phase A is a 0.02%-0.5% trifluoroacetic acid aqueous solution, and mobile phase B is a 0.02%-0.5% trifluoroacetic acid-acetonitrile solution. The 0.02%-0.5% can be, for example, 0.02%, 0.5%, 1%, 1.5%, 2%, 2.5%, or 3%, etc.

[0008] In this invention, the selected chromatographic column was found to have good separation effect on antibody fragments after enzymatic digestion, and a mobile phase with trifluoroacetic acid as the core reagent was constructed. Experiments showed that trifluoroacetic acid can effectively separate antibody fragments. This invention obtains antibody fragments by enzymatic digestion of the hinge region of the antibody, and the type of antibody can be effectively identified by comparing the separated fragments with standards. This method is simple to operate, has high detection accuracy, and is beneficial for the detection of batch samples.

[0009] Preferably, the injection sequence of the liquid chromatography is as follows: first inject 3-5 blank samples, then inject the first standard, then inject the test sample, and finally inject the second standard.

[0010] In this invention, blanks demonstrate the absence of interference, and the injection sequence of standards, samples, and standards can be verified by ensuring that the system suitability meets acceptable standards, thus proving that the instrument, chromatographic column, and other systems are functioning correctly.

[0011] Preferably, the identification standard is to compare the retention times of all main peaks of the test sample with those of all main peaks of the first injection standard. If the ratio is 0.98-1.02, the antibodies are considered to be the same; otherwise, they are considered to be different antibodies. The 0.98-1.02 can be, for example, 0.98, 0.99, 1, 1.01, or 1.02.

[0012] Preferably, the method further includes the identification of the detection system, wherein the retention times of all main peaks of the second standard are compared with those of all main peaks of the first standard. If the ratios are all between 0.98 and 1.02, the instrument, chromatographic column, and other systems are deemed to be functioning normally. The range of 0.98-1.02 can be, for example, 0.98, 0.99, 1, 1.01, or 1.02.

[0013] Preferably, the elution program for the liquid chromatography is as follows (mobile phase A and mobile phase B total 100%):

[0014] 0-2 min, mobile phase A is 70%;

[0015] Over 2-18 minutes, the mobile phase A changed from 70% to 52% at a constant rate.

[0016] Over 18-18.1 min, the mobile phase A changed from 52% to 10% at a constant rate.

[0017] 18.1-20 min, mobile phase A is 10%;

[0018] Over 20-20.1 min, the mobile phase A changed from 10% to 70% at a constant rate.

[0019] 20.1-23 min, mobile phase A is 70%.

[0020] In this invention, an elution procedure applicable to the above-mentioned identification method is constructed. Combined with other detection conditions such as chromatographic column and mobile phase, it can effectively detect the retention time of antibody fragments and accurately identify them, significantly improving the accuracy of detection.

[0021] Preferably, the column temperature of the liquid chromatograph is 65-75℃. For example, it can be 65℃, 66℃, 67℃, 68℃, 69℃, 70℃, 71℃, 72℃, 73℃, 74℃, or 75℃, etc.

[0022] Preferably, the flow rate of the liquid chromatography is 0.3-0.8 mL / min. For example, it can be 0.3 mL / min, 0.4 mL / min, 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, or 0.8 mL / min, etc.

[0023] Preferably, the injection volume of the liquid chromatograph is 3-8 μL. For example, it can be 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, or 8 μL.

[0024] Preferably, the detection wavelength of the liquid chromatography is 210-230 nm. For example, it can be 210 nm, 215 nm, 220 nm, 225 nm, or 230 nm.

[0025] Preferably, the enzymatic digestion includes adjusting the pH of the sample to be tested, adding immunoglobulin G degrading enzyme, and incubating.

[0026] Preferably, the concentration of the sample to be tested is 1-10 μg / μL. For example, it can be 1 μg / μL, 2 μg / μL, 3 μg / μL, 4 μg / μL, 5 μg / μL, 6 μg / μL, 7 μg / μL, 8 μg / μL, 9 μg / μL, or 10 μg / μL, etc.

[0027] Preferably, the pH value is 6.5-7. For example, it can be 6.5, 6.6, 6.7, 6.8, 6.9, or 7, etc.

[0028] Preferably, the concentration of the immunoglobulin G degrading enzyme is 10-15 units / μL. For example, it can be 10 units / μL, 11 units / μL, 12 units / μL, 13 units / μL, 14 units / μL, or 15 units / μL, etc.

[0029] Preferably, the ratio of the sample to be tested to immunoglobulin G degrading enzyme is 1 μg:(1-1.4) Unit. The (1-1.4) can be, for example, 1, 1.1, 1.2, 1.3, or 1.4. Unit refers to the enzyme activity unit, measured in μmol / min. A unit refers to the amount of 1 μg of human IgG that can be digested by one unit after incubation at 37°C for 30 minutes in 10 mM sodium phosphate, 137 mM NaCl, and 2.7 mM KCl at pH 7.4.

[0030] Preferably, the incubation temperature is 35-40℃, and the incubation time is 0.5-1.5h. The 35-40℃ can be, for example, 35℃, 36℃, 37℃, 38℃, 39℃, or 40℃. The 0.5-1.5h can be, for example, 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1.0h, 1.1h, 1.2h, 1.3h, 1.4h, or 1.5h.

[0031] Preferably, the denaturation and reduction process involves adding a denaturant and a reducing agent to the incubated sample before reacting.

[0032] Preferably, the denaturant comprises guanidine hydrochloride.

[0033] Preferably, the reducing agent includes dithiothreitol.

[0034] Preferably, the reaction temperature is 35-40°C and the time is 10-30 min. The 35-40°C can be, for example, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. The 10-30 min can be, for example, 10 min, 15 min, 20 min, 25 min, or 30 min.

[0035] Preferably, the sample to be tested includes any one or a combination of at least two of the following: monoclonal antibody, bispecific antibody sample, antibody-drug conjugate, or fusion protein.

[0036] Secondly, the present invention provides an application of the method described in the first aspect for identifying a variety of therapeutic antibodies based on a novel specific enzyme digestion method in antibody identification.

[0037] Compared with the prior art, the present invention has at least the following beneficial effects:

[0038] This invention optimizes chromatographic conditions for high-performance liquid chromatography (HPLC), establishing a rapid, stable, and controllable identification and analysis method for the quality control of various therapeutic antibodies. This invention addresses key technical challenges of existing methods, such as long chromatographic separation times, incomplete elution, and unsatisfactory resolution, by specifically optimizing gradient elution methods suitable for different molecular types. Furthermore, this invention can be coupled with mass spectrometry (MS) for further characterization and analysis of the molecular weight of enzyme fragments and post-translational modifications. Attached Figure Description

[0039] Figure 1 This is the high-performance liquid chromatography (HPLC) detection chromatogram of Example 1.

[0040] Figure 2 This is the high-performance liquid chromatography (HPLC) detection chromatogram for Example 2.

[0041] Figure 3 This is the high-performance liquid chromatography (HPLC) detection chromatogram for test example 2. Detailed Implementation

[0042] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0043] Example 1

[0044] This embodiment performs antibody identification.

[0045] Take 10 μg of trastuzumab, add ultrapure water to a final volume of 10 μL, and mix well. Adjust the pH to 6.7 with 200 mM Tris-HCl, add 1.0 μL of 12 units / μL IdeS enzyme, and incubate at 37 °C for 1 h. Add 37.5 μL of 8 M guanidine hydrochloride solution and 2 μL of 0.5 M dithiothreitol to the incubated monoclonal antibody, mix well, and incubate at 37 °C for 15 min. Simultaneously prepare a blank sample without adding monoclonal antibody, using 10 μL of ultrapure water, and add the same amounts of 200 mM Tris-HCl, IdeS enzyme, 8 M guanidine hydrochloride solution, and 0.5 M dithiothreitol.

[0046] After incubation, the samples were transferred to HPLC vials for analysis. The system was flushed with 30% acetonitrile solution until a stable baseline was obtained. Mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was 0.1% trifluoroacetic acid-acetonitrile solution. A Waters BioResolve RP column (2.1 x 150 mm, 2.7 μm) was installed, and the column was equilibrated for 90 min using the initial mobile phase ratio. The injection sequence was as follows: first, three injections of blank sample; then, the first injection of standard; then, one injection of the analyte sample; and finally, the second injection of standard.

[0047] The liquid chromatography conditions were: detection wavelength 214 nm, column temperature 70 ℃, flow rate 0.5 mL / min, injection volume 5 μL, and run time 23 min. The elution gradient was:

[0048] 0-2 min, mobile phase A is 70%;

[0049] Over 2-18 minutes, the mobile phase A changed from 70% to 52% at a constant rate.

[0050] Over 18-18.1 min, the mobile phase A changed from 52% to 10% at a constant rate.

[0051] 18.1-20 min, mobile phase A is 10%;

[0052] Over 20-20.1 min, the mobile phase A changed from 10% to 70% at a constant rate.

[0053] 20.1-23 min, mobile phase A is 70%.

[0054] Example 2

[0055] This embodiment performs antibody identification, and its only difference from Example 1 is that the sample being tested is a bispecific antibody sample; otherwise, it is the same as Example 1.

[0056] Test Example 1

[0057] This test case analyzes the above test results.

[0058] The retention times of each main peak in the above embodiments and comparative examples were compared with the retention times of the standard, and the specific results are shown in Table 1.

[0059] Table 1

[0060]

[0061]

[0062] The results show that:

[0063] (1) As can be seen from Example 1, using the detection method of the present invention, the results of Example 1 are obtained through... Figure 1 As can be seen, the standard sample injected in the first injection was used for system detection. The results of the first injection show that the monitoring system is stable. After the IdeS enzyme cleaves the monoclonal antibody, it forms three antibody fragments. The retention times of each peak in the sample were compared with the retention times of each peak in the standard sample. The ratios were found to be between 0.98 and 1.02, which proves that the detection results of this application are highly accurate.

[0064] (2) By comparing Example 1 and Example 2, it can be seen that the detection results of Example 2 are shown in Table 2, and the detection result graph is shown in Figure 2. Figure 2 As shown, the method of the present invention also has a good detection effect on bispecific antibodies.

[0065] Table 2

[0066]

[0067] Test Example 2

[0068] This test case performs multiple antibody detections.

[0069] To test the specificity of the method of this invention for different samples, this test example uses the method of Example 1 to detect three fusion proteins, one antibody-drug conjugate, and three bispecific antibodies: utsenoside, goussetzumab, daratumumab, secukinumab, pembrolizumab, cetuximab, and trastuzumab. Specific test results are as follows: Figure 3 As shown, the method of this invention can effectively detect the enzyme-digested fragments. Different antibody molecules have different characteristic spectra, and the identification of antibody molecules can be achieved based on these characteristics.

[0070] In summary, this invention obtains antibody fragments by enzymatic digestion of the hinge region of the antibody, and effectively identifies the type of antibody by separating and detecting the fragments and comparing them with standards. This method is simple to operate, has high detection accuracy, and is beneficial for the detection of batch samples.

[0071] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for identifying multiple therapeutic antibodies based on a novel specific enzyme digestion method, characterized in that, The method includes enzymatic digestion of the sample to be tested, followed by denaturation and reduction treatment, detection by liquid chromatography, and identification by comparing the detection results with a standard. The liquid chromatography column is a polystyrene column, mobile phase A is a 0.02%-0.5% trifluoroacetic acid aqueous solution, and mobile phase B is a 0.02%-0.5% trifluoroacetic acid-acetonitrile solution.

2. The method for identifying multiple therapeutic antibodies based on a novel specific enzyme digestion method according to claim 1, characterized in that, The injection sequence for the liquid chromatography is as follows: first inject 3-5 blank samples, then inject the first standard, then inject the test sample, and finally inject the second standard. Preferably, the identification standard is to compare the retention times of all main peaks of the sample to be tested with those of all main peaks of the first injection standard. If the ratio is 0.98-1.02, they are judged to be the same antibody; otherwise, they are different antibodies.

3. The method for identifying multiple therapeutic antibodies based on a novel specific enzyme digestion method according to claim 1 or 2, characterized in that, The elution procedure for the liquid chromatography is as follows: 0-2 min, mobile phase A is 70%; Over 2-18 minutes, the mobile phase A changed from 70% to 52% at a constant rate. Over 18-18.1 min, the mobile phase A changed from 52% to 10% at a constant rate. 18.1-20 min, mobile phase A is 10%; Over 20-20.1 min, the mobile phase A changed from 10% to 70% at a constant rate. 20.1-23 min, mobile phase A is 70%.

4. The method for identifying multiple therapeutic antibodies based on a novel specific enzyme digestion method according to any one of claims 1-3, characterized in that, The column temperature for the liquid chromatography is 65-75℃; Preferably, the flow rate of the liquid chromatography is 0.3-0.8 mL / min; Preferably, the injection volume of the liquid chromatograph is 3-8 μL; Preferably, the detection wavelength of the liquid chromatography is 210-230 nm.

5. The method for identifying multiple therapeutic antibodies based on a novel specific enzyme digestion method according to any one of claims 1-4, characterized in that, The enzymatic digestion involves adjusting the pH of the sample to be tested, adding immunoglobulin G degrading enzyme, and incubating.

6. The method for identifying multiple therapeutic antibodies based on a novel specific enzyme digestion method according to claim 5, characterized in that, The concentration of the sample to be tested is 1-10 μg / μL; Preferably, the pH value is 6.5-7; Preferably, the concentration of the immunoglobulin G degrading enzyme is 10-15 units / μL.

7. The method for identifying multiple therapeutic antibodies based on a novel specific enzyme digestion method according to claim 5 or 6, characterized in that, The ratio of the sample to be tested to immunoglobulin G degrading enzyme was 1 μg:(1-1.4) Unit; Preferably, the incubation temperature is 35-40℃ and the incubation time is 0.5-1.5h.

8. The method for identifying multiple therapeutic antibodies based on a novel specific enzyme digestion method according to any one of claims 1-7, characterized in that, The denaturation and reduction process involves adding a denaturant and a reducing agent to the incubated sample and then reacting the sample.

9. The method for identifying multiple therapeutic antibodies based on a novel specific enzyme digestion method according to claim 8, characterized in that, The denaturing agent includes guanidine hydrochloride; Preferably, the reducing agent comprises dithiothreitol; Preferably, the reaction temperature is 35-40℃ and the time is 10-30 min.

10. The application of the method for identifying multiple therapeutic antibodies based on a novel specific enzyme digestion method according to any one of claims 1-9 in antibody identification.

Images