Method for analyzing antibody drug molecule size variants

Abstract

The invention discloses a method for analyzing an antibody drug molecule size variant. According to the present invention, the antibody drug is analyzed by using the strip sedimentation analysis ultracentrifugation (BS-AUC) for the first time, and the use amount of the sample is less compared with the use amount of the sample used by using the sedimentation rate method; the invention provides a rapid, accurate and high-resolution antibody drug analysis method for the field, and the method has important application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection and analysis. Specifically, the present invention relates to a method for analyzing size variants of antibody drugs. Background Art

[0002] Antibody drug molecular size variants mainly refer to the variation of molecular size caused by various factors during the production and development of antibody drugs. This variation may affect the efficacy and safety of the drug, so it needs to be strictly measured and controlled.

[0003] At present, the methods for determining the size variants of antibody drugs include CE-SDS and size exclusion chromatography (SEC). CE-SDS is a commonly used determination method that can be used for the analysis of the size heterogeneity of monoclonal antibodies. Size exclusion chromatography (SEC) is often preferred for the release, stability and process analysis of monoclonal antibodies due to its advantages such as fast speed, good reproducibility, and quantitative requirements for methodological validation.

[0004] However, given that the determination of size variants of antibody drugs is an important part of antibody drug quality control, it requires the use of high-resolution, high-precision and highly automated analytical methods. Therefore, it is necessary to develop more methods with higher resolution for the determination of size variants of antibody drugs in order to control the quality of antibody drugs. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a novel method for analyzing size variants of antibody drug molecules.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides a method for analyzing size variants of antibody drug molecules.

[0007] Furthermore, the method comprises the following steps: determining the sedimentation coefficient of the antibody drug sample by strip sedimentation analysis ultracentrifugation; drawing a sedimentation coefficient distribution curve; integrating the sedimentation coefficient distribution curve to obtain the proportion of monomers, polymers and fragments in the antibody drug sample.

[0008] Further, the step of drawing a sedimentation coefficient distribution curve includes obtaining the sedimentation coefficient using an analytical zone centrifugation c(s) analysis model of SEDFIT software.

[0009] Furthermore, the strip sedimentation analytical ultracentrifugation method uses an analytical ultracentrifuge, and its relevant parameters are set as follows: temperature, 20° C.; rotation speed, 40000 rpm to 50000 rpm.

[0010] Preferably, the rotation speed is set to 40000 rpm.

[0011] Preferably, the rotor used in the detection process is AN-50Ti.

[0012] Furthermore, the reference substance of the antibody drug sample is a buffer used in diluting the antibody drug sample.

[0013] Furthermore, the buffer solution includes water and PBS.

[0014] Preferably, the buffer is PBS.

[0015] In the present invention, the antibody drug refers to an antibody with specific therapeutic function produced by artificial synthesis or biotechnology. These antibodies can recognize and bind to specific molecules in the body (such as bacteria, viruses or abnormal cells), and treat diseases by targeting specific molecules to block their functions or promote their clearance. Antibody drugs simulate or enhance the functions of the human immune system to fight pathogens or abnormal cells, and are widely used to treat cancer, autoimmune diseases and other diseases.

[0016] In the present invention, molecular size variants refer to the differences in molecular size between different individuals in the same population. Molecular size variants are an important part of antibody drug quality control. In the present invention, the applicant first used the band sedimentation analysis ultracentrifugation method (BS-AUC) to analyze the molecular size variants of antibody drugs, which uses less sample than the sedimentation rate method.

[0017] In the present invention, the sedimentation coefficient value depends on the size, shape, molecular weight and other properties of the particles. Larger particles settle faster and have larger sedimentation coefficient values. The analytical ultracentrifugation experiment monitors the radial concentration distribution of the substance at appropriate time intervals, records the movement of the sample sedimentation boundary during the centrifugation process, and uses fluid mechanics principles and related mathematical models to analyze and obtain the sedimentation coefficient value of the substance.

[0018] In the present invention, the antibody drug sample is added to an analytical ultracentrifuge for detection. The analytical ultracentrifuge is composed of a power system and an optical detection system.

[0019] The power system described above may include a driving system, a vacuum system, a control system, a protection system, a rotor and a sample cell.

[0020] In some embodiments of the present invention, the rotor has multiple models, which can be divided into AN-60Ti with 4 holes and AN-50Ti with 8 holes. During the detection process, the rotor with the corresponding number of holes can be selected according to the number of samples. In the present invention, AN-50Ti with 8 holes is preferred. The material of the rotor is generally titanium alloy.

[0021] In addition, the sample pool consists of a center piece, a window, a gasket, a threaded ring and an outer shell; the material of the center piece is generally aluminum alloy or resin, and more preferably, the material of the center piece is resin; the material of the window is selected from sapphire or quartz, and more preferably, the material of the window is sapphire.

[0022] Furthermore, the optical detection system includes one of an ultraviolet / visible light absorption detector and an interference light detector.

[0023] The UV / visible light absorption detector can record the absorbance of the sample at different positions in the rotor radial direction during the centrifugation process to complete the detection when the sample absorbs within a certain wavelength. The interference light detector can detect the change in optical path length due to the different refractive indices of the reference solution and the sample when light passes through the reference solution and the sample, and the point of equal optical path length on the collector is displaced. The displacement distance of the interference fringes is proportional to the solute concentration. By monitoring the displacement of the interference fringes, the change of the solute concentration in the rotor radial direction over time can be obtained.

[0024] In some preferred embodiments of the present invention, a UV / visible light absorption detector is selected to detect the sample. Specifically, after the test sample is loaded, ultraviolet light is used for detection and data is recorded.

[0025] Further, the sample loading volume is 10 μL. After loading, the detection is performed at 20°C, 40000rpm~50000rpm with a wavelength of 250~300nm, and a data is collected every 60~240s; preferably, after loading, the detection is performed at 20°C, 40000rpm with a wavelength of 280nm, and a data is collected every 180s.

[0026] In addition, since the test sample may contain buffer components, in order to ensure the accuracy of the results, a control group with the same buffer component (such as PBS) solution as the sample was set up to eliminate the interference of PBS solution as a solvent on the results. The pH of the PBS solution is 7.4±0.5, and the sample volume is 10μL.

[0027] The data obtained through the above test were processed by analytical zone centrifugation c(s) model using SEDFIT software to obtain the sedimentation coefficient distribution diagram of the test sample.

[0028] The analytical zone centrifugation c(s) model in SEDFIT software is a model used to analyze the sedimentation coefficient distribution of samples in strip sedimentation experiments. It is the core of experimental data analysis. In SEDFIT software, the analytical zone centrifugation c(s) model obtains the sedimentation coefficient distribution by fitting experimental data. This distribution can reflect the sedimentation characteristics of different components in the sample.

[0029] Through the sedimentation coefficient distribution, we can obtain the sedimentation coefficient of each component in the sample, and then use the known relationship between molecular weight and sedimentation coefficient (such as the Svedberg equation) to determine the molecular weight of each component.

[0030] The sedimentation coefficient distribution can reflect the relative content of each component in the sample. If there are multiple components in the sample, the c(s) distribution will show multiple peaks. By observing the peak shape and peak area of ​​the c(s) distribution, we can determine the purity of the sample.

[0031] When fitting the analytical zone centrifugation c(s) model, some parameters (such as fitting range, resolution, etc.) need to be set. The setting of these parameters will affect the quality and accuracy of the fitting results. Therefore, these parameters need to be carefully set and adjusted before fitting.

[0032] In a specific implementation scheme of the present invention, the relevant parameters of the model are: Resolution: 150; s min: 0.1; s max: 20; frictional ratio: 1.2 (checked); Baseline: 0 (checked); Fit RI Noise: checked; Fit Time Independent Noise: checked; Meniscus: checked; Bottom: 7.2 (unchecked); Confidence level: 0.68; Partial spec. Volume: 0.73; Buffer density: 1.536; Buffer Viscosity: 0.01251.

[0033] The second aspect of the present invention provides application of the method described in the first aspect of the present invention in quality control of antibody drugs.

[0034] A third aspect of the present invention provides a system for analyzing size variants of antibody drug molecules.

[0035] Further, the system comprises: Determination module: Determination of the sedimentation process of antibody drug samples by strip sedimentation analysis ultracentrifugation; Data processing module: Draw a sedimentation coefficient distribution curve for the data obtained by the determination module; integrate the sedimentation coefficient distribution curve to obtain the proportion of monomers, polymers and fragments in the antibody drug sample.

[0036] Furthermore, the system also includes a parameter setting module for setting parameters of the analytical ultracentrifuge.

[0037] A fourth aspect of the present invention provides a computer device and a computer-readable storage medium.

[0038] Furthermore, the device includes a memory and a processor, the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the method described in the first aspect of the present invention is implemented.

[0039] Furthermore, the computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the method described in the first aspect of the present invention is implemented.

[0040] In the present invention, the processor and the memory can be set separately or integrated together. For example, the memory can include random access memory, flash memory, read-only memory, programmable read-only memory, non-volatile memory or register, etc. The processor can be a central processing unit (CPU), etc. Or a graphic processing unit (GPU) The memory can store executable instructions. The processor can execute the executable instructions stored in the memory to implement the various processes described herein.

[0041] It can be understood that the memory in this embodiment can be a volatile memory or a non-volatile memory, or can include both volatile and non-volatile memories. Among them, the non-volatile memory can be a ROM (Read-Only Memory), a PROM (Programmable ROM), an EPROM (Erasable PROM), an EEPROM (Electrically EPROM) or a flash memory. The volatile memory can be a RAM (Random Access Memory), which is used as an external cache. By way of example and not limitation, many forms of RAM are available, such as SRAM (Static RAM), DRAM (Dynamic RAM), SDRAM (Synchronous DRAM), DDRSDRAM (Double Data Rate SDRAM), ESDRAM (Enhanced SDRAM), SLDRAM (Synchlink DRAM), and DRRAM (Direct Rambus RAM). The memories described herein are intended to include, but are not limited to, these and any other suitable types of memories.

[0042] In some embodiments, the memory stores the following elements, upgrade packages, executable units or data structures, or a subset thereof, or an extended set thereof: an operating system and an application program.

[0043] The operating system includes various system programs, such as a framework layer, a core library layer, a driver layer, etc., which are used to implement various basic services and process hardware-based tasks. The application program includes various application programs, which are used to implement various application services. The program for implementing the method of the embodiment of the present invention can be included in the application program.

[0044] In an embodiment of the present invention, the processor calls a program or instruction stored in a memory, specifically, a program or instruction stored in an application program, and the processor is used to execute the method steps provided in the first aspect.

[0045] In the present invention, the computer-readable storage medium may include but is not limited to various known and unknown types of non-volatile memories.

[0046] Advantages and beneficial effects of the present invention: The present invention uses BS-AUC to analyze antibody drugs for the first time, and uses less sample than the sedimentation rate method. The present invention provides a fast, accurate, high-resolution antibody drug analysis method in the art, which has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is the sedimentation curve of monoclonal antibody sample; Figure 2 This is the sedimentation coefficient distribution curve of monoclonal antibody samples. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0049] The instruments and reagents used in the embodiments of the present invention are as follows: Instrument: Analytical ultracentrifuge Optima AUC (Beckman Coulter) Reagents: 10× phosphate buffered saline (PBS, Cytiva, catalog number BR100673); 10× monoclonal antibody buffer deuterated PBS; heavy water (D2O, Solebol, catalog number SAH9000, specification 25ml / bottle) Example

[0050] 1. Experimental Methods 1. Buffer preparation: dilute 10×PBS 10 times with D2O to obtain heavy water PBS buffer, and dilute 10×PBS 10 times with ddH2O double pure water to obtain PBS buffer for standby use.

[0051] 2. Sample dilution: Dilute the antibody sample to OD with PBS buffer. 280 Approximately 1.

[0052] 3. Analytical ultracentrifugation experiment 3.1 Sample cell assembly and sample addition: Place the window assembly and BS-AUC centerpiece vertically into the sample cell shell along the positioning strips on the sample cell shell, add 10 μL of PBS buffer and diluted sample respectively, then put in another window assembly, threaded ring gasket and threaded ring, tighten them fully with a torque wrench, and add 350 μL / 340 μL of PBS heavy water buffer to the reference or sample area respectively.

[0053] Balance the sample cell axially and symmetrically, and control the weight difference within 0.5g after balancing. Place the sample cell and the special balance piece into the rotor, and make sure the scale lines are aligned.

[0054] 3.2 Program setting and operation: Create a new experimental method on the equipment operating software, and select the corresponding rotor according to the speed and flux requirements to perform the analytical ultracentrifugation band sedimentation experiment. The experimental program settings are shown in Table 1.

[0055] Table 1 Experimental procedure settings

[0056] 3.3 Data analysis and statistics: Open SEDFIT software, import the experimental data of the target sample, select the analytical zone centrifugation c(s) model, and enter relevant parameters to calculate and fit the data.

[0057] Table 2 Analysis parameter settings

[0058] 2. Experimental Results Figure 1 is the sedimentation curve of the monoclonal antibody sample during the strip sedimentation process. The sedimentation data were analyzed to obtain Figure 2 The sedimentation coefficient distribution curve of the monoclonal antibody sample. By integrating the curve, it can be obtained that the monomer proportion of the monoclonal antibody sample is 94.24%, the polymer proportion is 5.0%, and the fragment proportion is 0.76%.

[0059] It is obvious to those skilled in the art that the embodiments of the present invention are not limited to the details of the exemplary embodiments described above, and that the embodiments of the present invention can be implemented in other specific forms without departing from the spirit or essential features of the embodiments of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive, and the scope of the embodiments of the present invention is defined by the appended claims rather than the above description, and therefore it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the embodiments of the present invention.

[0060] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for analyzing size variants of antibody drugs, characterized in that: The method comprises the following steps: determining the sedimentation coefficient of the antibody drug sample by using a strip sedimentation analysis ultracentrifugation method; drawing a sedimentation coefficient distribution curve; and integrating the sedimentation coefficient distribution curve to obtain the proportions of monomers, polymers and fragments in the antibody drug sample.

2. The method according to claim 1, characterized in that The step of drawing the sedimentation coefficient distribution curve includes obtaining the sedimentation coefficient distribution curve using the analytical zone centrifugation c(s) analysis model of the SEDFIT software.

3. The method according to claim 1, characterized in that The strip sedimentation analytical ultracentrifugation method uses an analytical ultracentrifuge, and its relevant parameters are set as follows: temperature, 20°C; speed, 40000rpm~50000rpm; Preferably, the rotation speed is set to 40000 rpm; Preferably, the rotor used in the detection process is AN-50Ti.

4. The method according to claim 1, characterized in that: The absorbance of the antibody drug sample is 0.6~2; Preferably, the absorbance of the antibody drug sample is 1.

5. The method according to claim 1, characterized in that The reference substance of the antibody drug sample is the buffer used in diluting the antibody drug sample.

6. The method according to claim 5, characterized in that The buffer comprises water and PBS; Preferably, the buffer is PBS.

7. Use of the method according to any one of claims 1 to 6 in the quality control of antibody drugs.

8. A system for analyzing size variants of antibody drugs, characterized in that: The system comprises: Determination module: Determination of the sedimentation process of antibody drug samples by strip sedimentation analysis ultracentrifugation; Data processing module: Draw a sedimentation coefficient distribution curve for the data obtained by the determination module; integrate the sedimentation coefficient distribution curve to obtain the proportion of monomers, polymers and fragments in the antibody drug sample.

9. The system according to claim 8, characterized in that The system also includes a parameter setting module for setting parameters of the analytical ultracentrifuge.

10. A computer device and a computer readable storage medium, characterized in that: The device comprises a memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the method according to any one of claims 1 to 6 is implemented; The computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the method according to any one of claims 1 to 6 is implemented.

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