Method for detecting monoclonal antibody charge variants

Abstract

The invention discloses a method for detecting a charge variant of a monoclonal antibody. The method comprises the following steps: preparing a monoclonal antibody to be detected into a test solution; the test solution comprises a monoclonal antibody to be detected, a methylcellulose solution, urea, amino acid, an ampholyte carrier and an isoelectric point marker; detecting the test solution by adopting a full-column imaging capillary isoelectric focusing method, and determining the content percentage of the charge variant of the monoclonal antibody to be detected according to an isoelectric focusing map; wherein the isoelectric point of the amino acid is similar to the boundary isoelectric point of a main peak and an alkali peak in an isoelectric focusing map of the monoclonal antibody to be detected. According to the method, the separation degree between the main peak and the alkali peak of the to-be-detected sample can be remarkably improved. The sensitivity is good, the precision is high, and while the separation degree of a main peak and an alkali peak is improved in a targeted manner, the separation degree and proportion of an acid peak and the main peak, the isoelectric point of a sample and the like are basically not influenced.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a method for detecting charge variants of monoclonal antibodies. Background Art

[0002] For monoclonal antibodies (mAbs), charge variants are one of their critical quality attributes (CQAs) and need to be evaluated throughout the development and product life cycle to meet regulatory requirements. Detection methods targeting antibody charge distribution, such as capillary isoelectric focusing (CIEF), ion exchange chromatography (IEX), and capillary zone electrophoresis (CZE), have made great progress. Among them, full-column imaging capillary isoelectric focusing (iCIEF), one of the CIEF methods, has gradually become the "gold standard" for the detection of monoclonal antibody charge variants due to its high sensitivity, fast detection speed, and good resolution. However, in the current development of biosimilars of some monoclonal antibody drugs, it was found that when the original registered iCIEF method was used to analyze the purity of its charge variants, under the conventional uniform pH gradient, the main peak and the first base peak were highly overlapped, and the separation between the two was low, which could not meet the regulatory requirements for the separation of quantitative methods. In addition, the spectrum was inconsistent with the registered spectrum of the original drug, and it was difficult to accurately quantify the proportion of base peaks, which may have affected the accuracy of the iCIEF analysis method. Moreover, the original base peak level is very low, and under lower separation conditions, it cannot be effectively transferred to the preparative iCIEF to complete high-purity base peak collection. Summary of the invention

[0003] Based on this, it is necessary to provide a method for detecting charge variants of monoclonal antibodies.

[0004] The first aspect of the present application provides a method for detecting a monoclonal antibody charge variant, comprising the following steps:

[0005] The monoclonal antibody to be tested is prepared into a test solution; the test solution comprises the monoclonal antibody to be tested and a methylcellulose solution, urea, amino acids, an ampholyte carrier and an isoelectric point marker;

[0006] The test solution is tested by full-column imaging capillary isoelectric focusing, and the content percentage of the charge variant of the monoclonal antibody to be tested is determined according to the isoelectric focusing spectrum;

[0007] The isoelectric point of the amino acid is similar to the boundary isoelectric point of the main peak and the alkaline peak in the isoelectric focusing spectrum of the monoclonal antibody to be tested.

[0008] In some embodiments, the concentration of the amino acid in the test solution is 0.05 mM to 0.3 mM.

[0009] In some embodiments, the amino acids include natural amino acids and unnatural amino acids; optionally, the amino acids include histidine.

[0010] In some embodiments, the concentration of the monoclonal antibody to be tested in the test solution is 0.2 mg / mL to 0.6 mg / mL; optionally, the monoclonal antibody to be tested includes an IgG antibody.

[0011] In some embodiments, the amphoteric electrolyte carrier is an amphoteric electrolyte carrier with a pH of 3 to 10; optionally, the volume fraction of the amphoteric electrolyte carrier in the test solution is 4% to 5%.

[0012] In some embodiments, the isoelectric point marker includes one or more of an isoelectric point marker with a pI of 2.0-7.0 and an isoelectric point marker with a pI of 8.0-10.0; optionally, the volume fractions of the isoelectric point marker with a pI of 2.0-7.0 and the isoelectric point marker with a pI of 8.0-10.0 in the test solution are respectively 0.2%-5%.

[0013] In some embodiments, the mass fraction of methylcellulose in the test solution is 0.2%~0.4%.

[0014] In some embodiments, the concentration of urea in the test solution is 1M~3M.

[0015] In some embodiments, the whole-column imaging capillary isoelectric focusing method satisfies one or more of the following conditions:

[0016] The sample tray temperature is 10℃~15℃;

[0017] The pre-focusing voltage is 1000V~1500V, and the pre-focusing time is 1~2min;

[0018] The focusing voltage is 2900V~3100V, and the focusing time is 6~8min; and,

[0019] The loading time is 50s~60s.

[0020] The second aspect of the present application provides a method for quality detection of a monoclonal antibody drug, comprising detecting the charge variants of the monoclonal antibody in the monoclonal antibody drug using the method described in the first aspect of the present application.

[0021] The aforementioned detection method can significantly improve the separation between the main peak and the base peak of the sample to be tested by adding amino acids to produce a space-occupying effect under the action of the electric field. This method has good sensitivity and high precision. While specifically improving the separation between the main peak and the base peak, it has little effect on the separation, ratio and isoelectric point of the acid peak and the main peak. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0023] Figure 1 The iCIEF spectrum of the sample obtained by the detection method of the present application in one embodiment of the present application;

[0024] Figure 2 The iCIEF spectra of samples with different final concentrations of histidine added in one embodiment of the present application, wherein "absorbance" indicates absorbance;

[0025] Figure 3 It is a linear fitting diagram of samples with different concentrations in one embodiment of the present application;

[0026] Figure 4 This is the iCIEF spectrum of the sample obtained in Comparative Example 1;

[0027] Figure 5 This is the iCIEF spectrum of the sample obtained in Comparative Example 2. DETAILED DESCRIPTION

[0028] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0030] The terms "having", "containing" and "including" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0031] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions of the listed contents.

[0032] In this application, if the unit of the data range is followed only by the right endpoint, it means that the units of the left endpoint and the right endpoint are the same. For example, 40~50℃ means that the units of the left endpoint "40" and the right endpoint "50" are both ℃, which have the same meaning as 40℃~50℃. In addition, similar descriptions involving other parameters also apply to the above understanding.

[0033] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0034] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form a new technical solution.

[0035] In the present application, the terms "first", "second", "third", etc. in "the first aspect", "the second aspect", "the third aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0036] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0037] At present, in the process of developing biosimilars of some monoclonal antibody drugs, it was found that when the original registered iCIEF method was used to analyze the purity of its charge variants, the main peak and the first base peak overlapped highly, and the separation between the two was low, which could not meet the regulatory requirements for the separation of quantitative methods. In addition, the spectrum was inconsistent with the registered spectrum of the original drug, and it was difficult to accurately quantify the proportion of base peaks, which may have affected the accuracy of the iCIEF analysis method. In addition, the base peak level of the original drug was very low (<6%), and under the condition of low separation, it could not be effectively transferred to the preparative iCIEF to complete the collection of high-purity base peaks.

[0038] Based on this, the embodiments of the present application at least provide a method for detecting charge variants of monoclonal antibodies.

[0039] In this application, unless otherwise specified, "full-column imaging capillary isoelectric focusing" means that the capillary is filled with amphoteric electrolyte carriers, the anode end is filled with dilute H3PO4, and the cathode end is filled with dilute NaOH. A DC voltage is applied to both ends of the capillary, and a pH gradient that gradually increases from the anode to the cathode is established in the tube. Under the action of the electric field, the antibody electrophoretically migrates in this pH gradient. When it migrates to the pH equal to the pI, the net surface charge is zero, the migration stops, and it is concentrated into a narrow zone, i.e., isoelectric focusing. By real-time full-column imaging of the capillary, ultraviolet absorption signals (at a wavelength of 280 nm) and fluorescence signals (excitation at a wavelength of 280 nm, emission at a wavelength of 320-450 nm) are collected, and spectra are recorded.

[0040] In this application, unless otherwise specified, "charge variant" also refers to charge isomers, which are different molecular forms formed due to changes in the charge carried by biological macromolecules (such as proteins, antibodies, etc.).

[0041] In some embodiments of the present application, the separation between the main peak of the sample and the charge variant base peak can be improved by adding a certain concentration of a zwitterionic substance to the premix, wherein the isoelectric point of the added zwitterionic substance is similar to the boundary isoelectric point between the main peak of the sample and the first base peak, and the zwitterionic substance can be an amino acid.

[0042] In some embodiments of the present application, according to the IgG4 monoclonal antibody icIEF detection method, the boundary pI between the main peak and the first alkaline peak is 7.60, so in the sample solution, the amphoteric substance histidine with a pI of exactly 7.60 is added, so that under the action of the electric field, in the amphoteric electrolyte of Pharmalyte 3-10, a space-occupying effect is produced, and the main peak and the first alkaline peak are squeezed to both ends, thereby improving the separation between the two peaks. Unlike the commonly used method for improving the separation, this method has little effect on the separation, ratio and isoelectric point of the acid peak and the main peak while improving the separation between the main peak and the first alkaline peak in a targeted manner. The method was verified by indicators such as precision (repeatability, intermediate precision) and linear range, and the results showed that the requirements of relevant guidelines and verification regulations were met.

[0043] In a first aspect of the present application, a method for detecting charge variants of a monoclonal antibody is provided, comprising the following steps:

[0044] S100: preparing a test monoclonal antibody to be tested into a test solution; the test solution comprises the test monoclonal antibody and a methylcellulose solution, urea, amino acids, an ampholyte carrier and an isoelectric point marker;

[0045] S200: detecting the test solution by full-column imaging capillary isoelectric focusing, and determining the content percentage of the charge variant of the monoclonal antibody to be tested according to the isoelectric focusing spectrum;

[0046] The isoelectric point of the amino acid is similar to the boundary isoelectric point of the main peak and the alkaline peak in the isoelectric focusing spectrum of the monoclonal antibody to be tested.

[0047] In the present application, unless otherwise specified, "urea" is used as a co-solvent to increase the solubility of the monoclonal antibody to be tested.

[0048] In the present application, unless otherwise specified, "amphoteric electrolyte carrier" refers to a substance that can exhibit both acidity and alkalinity within a certain pH range and can form a stable pH gradient under the action of an electric field.

[0049] In this application, unless otherwise specified, "isoelectric point marker" refers to a compound with a known isoelectric point, which is used to determine the isoelectric point position of proteins or other amphiphilic molecules in isoelectric focusing.

[0050] In some embodiments, the concentration of the amino acid in the test solution is 0.05 mM to 0.3 mM. In a non-limiting manner, the concentration of the amino acid in the test solution can be, but is not limited to, 0.05 mM, 0.10 mM, 0.15 mM, 0.20 mM, 0.25 mM, 0.30 mM or a value or range between any two of the above values.

[0051] In some embodiments, the amino acid is selected according to the separation requirements of the monoclonal antibody to be tested, wherein the isoelectric point of the selected amino acid is 3 to 9. Further, the amino acid includes natural amino acids and non-natural amino acids, for example, the amino acid may be but is not limited to histidine.

[0052] In some embodiments, the concentration of the monoclonal antibody to be tested in the test solution is 0.2 mg / mL to 0.6 mg / mL. In a non-limiting manner, the concentration of the monoclonal antibody to be tested in the test solution can be, but is not limited to, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, or a value or range between any two of the above values.

[0053] In some embodiments, the monoclonal antibodies to be tested include but are not limited to IgG1, IgG2, IgG3, and IgG4.

[0054] In some embodiments, the ampholyte carrier is an ampholyte carrier with a pH of 3 to 10.

[0055] In some embodiments, the volume fraction of the amphoteric electrolyte carrier in the test solution is 4% to 5%. In a non-limiting manner, the volume fraction of the amphoteric electrolyte carrier in the test solution can be, but is not limited to, 4%, 4.5%, 5%, or a value or range between any two of the above values.

[0056] In some embodiments, the isoelectric point marker includes one or more of an isoelectric point marker with a pI of 2.0 to 7.0 and an isoelectric point marker with a pI of 8.0 to 10.0. Further, the isoelectric point marker includes one or more of an isoelectric point marker with a pI of 4.10 and an isoelectric point marker with a pI of 9.50.

[0057] In some embodiments, the volume fraction of the isoelectric point marker in the test solution is 0.2% to 5%. Without limitation, the volume fraction of the isoelectric point marker in the test solution can be 0.2%, 1%, 2%, 3%, 4%, 5%, or a value or range between any two of the above values.

[0058] In some embodiments, the mass fraction of methylcellulose in the test solution is 0.2% to 0.4%. In a non-limiting manner, the mass fraction of methylcellulose in the test solution can be, but is not limited to, 0.2%, 0.3%, 0.4%, or a value or range between any two of the above values.

[0059] In some embodiments, the concentration of urea in the test solution is 1 M to 3 M. Without limitation, the concentration of urea in the test solution can be, but is not limited to, 1 M, 2 M, 3 M, or a value or range between any two of the above values.

[0060] In some embodiments, in step S200, the temperature of the sample tray in the whole-column imaging capillary isoelectric focusing method is 10°C to 15°C; further, the temperature of the sample tray is 10°C.

[0061] In some embodiments, the pre-focusing voltage in the whole-column imaging capillary isoelectric focusing method is 1000V~1500V, and the pre-focusing time is 1~2min; further, the pre-focusing voltage is 1500V, and the pre-focusing time is 1min.

[0062] In some embodiments, the focusing voltage in the whole-column imaging capillary isoelectric focusing method is 2900V~3100V, and the focusing time is 6~8min; further, the focusing voltage is 3000V, and the focusing time is 7min.

[0063] In some embodiments, the loading time of the sample to be tested in the whole-column imaging capillary isoelectric focusing method is 50s to 60s. Further, the loading time of the sample to be tested is 55s.

[0064] In a second aspect of the present application, a method for detecting the quality of a monoclonal antibody drug is provided, comprising detecting the charge variants of the monoclonal antibody in the monoclonal antibody drug using the method of the first aspect of the present application.

[0065] Some examples are provided below.

[0066] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0067] In the following examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operating accuracy are allowed.

[0068] Example 1

[0069] 1. Key instruments and equipment

[0070] Table 1

[0071]

[0072] 2. Key Reagents

[0073] Table 2

[0074]

[0075] 3. Key consumables

[0076] Table 3

[0077]

[0078] 4. Sample information

[0079] Table 4

[0080]

[0081] 5. Inspection steps

[0082] (1) Solution preparation:

[0083] 10 M urea solution: Weigh 0.6 g of urea, add 580 μL of ultrapure water to dissolve, vortex to aid dissolution, and wait until it is fully dissolved to obtain a 10 M urea solution. Filter through a 0.22 μm filter membrane, and place at room temperature. Prepare before use.

[0084] 10 mM histidine solution: weigh 15.52 mg L-histidine, dissolve in 10 mL ultrapure water, and vortex to aid dissolution.

[0085] (2) Preparation of premix:

[0086] Prepare the premix according to the formula in Table 5 based on the number of samples. The volume should be increased proportionally according to the number of samples and actual needs, and mixed thoroughly (the volume unit of each reagent is μL).

[0087] Table 5: Premix (taking one sample as an example)

[0088]

[0089] (3) Adjust the sample concentration to 4.0 mg / mL using ultrapure water.

[0090] (4) Combine 10 μL of sample (4.0 mg / mL, final concentration 0.4 mg / mL) with 90 μL of premixed solution, vortex three times for 5 seconds each time, centrifuge at 12,000 rpm for 5 min to remove any insoluble particles, carefully pipette 70 μL of supernatant and add to a 96-well plate, centrifuge at 2,500 rpm for 5 min, and perform assay according to the parameters in Table 6.

[0091] (5) Open the Compass for iCE software of the Maurice instrument and perform data analysis.

[0092] Table 6

[0093]

[0094] The experimental results are as follows Figure 1 As shown, the results show that the separation degree between the main peak and the first base peak in the spectrum measured by the method of the present application is good, which is consistent with the registered spectrum of the original manufacturer.

[0095] Example 2

[0096] 1. Investigate the effect of 10 mM histidine addition

[0097] According to the formula in Table 7, prepare the premix according to the number of samples. The volume should be increased proportionally according to the number of samples and actual needs, and the mixture should be fully mixed (the volume unit of each reagent is μL). The final concentrations of His were investigated as 0.00, 0.05, 0.10, 0.15, 0.20, 0.25, and 0.30 mM. The experimental results are shown in Figure 7. Figure 2 As shown, the results showed that 0.20 mM was the optimal concentration, and the pretreatment method corresponding to the premix solution was confirmed.

[0098] Table 7

[0099]

[0100] 2. Precision-Repeatability

[0101] Under the same operating conditions and in the same sequence, three different protein concentrations were designed (the samples were diluted to 2 mg / ml, 4 mg / ml and 6 mg / ml, respectively, and after mixing with the premix at a ratio of 1:9, the corresponding final protein concentrations were 0.2 mg / ml, 0.4 mg / ml and 0.6 mg / ml, respectively). Three samples were prepared in parallel by the same person at the same concentration, and the repeatability of the results obtained by at least 9 samples was determined. The experimental results are shown in Table 8 below. When the final concentrations of the protein samples were 0.2 mg / mL, 0.4 mg / mL and 0.6 mg / mL, respectively, the RSDs of the peak area percentages of the acid peak, main peak and alkali peak among the three groups of parallel samples at the same concentration were all less than 2%, and the total RSDs at different concentrations were all less than 5%, and the ranges were all within 1%. The peak area RSDs of the acid peak, main peak and alkali peak were also within 5%.

[0102] Table 8

[0103]

[0104] 3. Precision-Intermediate Precision

[0105] The intermediate precision of the results between different dates, different instruments, different icIEF cartridges, and different operators was investigated. Two operators each tested at least three groups of samples in parallel. The experimental results are shown in Table 9 below. Among the nine groups of parallel samples with different dates, different instruments, different icIEF cartridges, and different operators, the RSD of the acid peak area ratio was only 0.4%, the main peak was 0.2%, and the alkali peak was 1.5%, all less than 2%; the range of the acid peak, main peak, and alkali peak area ratios were all less than 0.5%, and the range of pI was 0.0. In addition, within the same sequence, the peak area deviations of the acid peak, main peak, and alkali peak were small, and their RSDs were all within 5%; the peak area deviations of each peak between different sequences increased, but the total RSD was still within 5%. Using different instruments and different cartridges, the main peak height exceeded 500 mAU.

[0106] Table 9

[0107]

[0108] 4. Linear

[0109] The final concentrations of the investigated protein samples were 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL and 0.6 mg / mL respectively: the samples were diluted with ultrapure water to 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL and 6.0 mg / mL respectively, and then the above experimental method was used for the experiment. The experimental results are shown in Tables 10 and Figure 3The linear results showed that in the final concentration range of 0.2-0.6 mg / mL, the linear equations were: (1) Acid peak: y=(8298.3±130.1)x+(73.8±55.1), linear correlation coefficient R 2 =0.999, the residual sum of squares is 5074.4; (2) Main peak: y=(15557.2±266.6)x+(149.3±113.1), linear correlation coefficient R 2 =0.999, the residual sum of squares is 21315.6; (3) Alkali peak: y=(1108.3±10.7)x-(14.3±4.5), linear correlation coefficient R 2 =0.999, and the residual sum of squares was 34.1. The RSDs of the peak area percentages of the acid peak, main peak, and base peak were all less than 5%.

[0110] Table 10

[0111]

[0112] Comparative Example 1

[0113] The sample was tested using the method of Example 1 of the present application, except that the concentration of histidine was 0 mM. The whole column imaging capillary isoelectric focusing spectrum was measured as follows: Figure 4 The results show that the resolution of the main peak and the first base peak is low.

[0114] Comparative Example 2

[0115] A third party organization (Fucheng Biotechnology) was commissioned to test the sample using the method of Example 1 of the present application, except that the concentration of histidine was 0 mM. The measured full-column imaging capillary isoelectric focusing map is as follows: Figure 5 The results show that the resolution of the main peak and the first base peak is low.

[0116] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0117] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the scope of the claims.

Claims

1. A method for detecting charge variants of a monoclonal antibody, characterized in that: It includes the following steps: The monoclonal antibody to be tested is prepared into a test solution; the test solution comprises the monoclonal antibody to be tested and a methylcellulose solution, urea, amino acids, an ampholyte carrier and an isoelectric point marker; The test solution is tested by full-column imaging capillary isoelectric focusing, and the content percentage of the charge variant of the monoclonal antibody to be tested is determined according to the isoelectric focusing spectrum; The isoelectric point of the amino acid is similar to the boundary isoelectric point of the main peak and the alkaline peak in the isoelectric focusing spectrum of the monoclonal antibody to be tested.

2. The method according to claim 1, characterized in that The concentration of the amino acid in the test solution is 0.05 mM to 0.3 mM.

3. The method according to claim 2, characterized in that The amino acids include natural amino acids and unnatural amino acids; optionally, the amino acids include histidine.

4. The method according to claim 1, characterized in that The concentration of the monoclonal antibody to be tested in the test solution is 0.2 mg / mL to 0.6 mg / mL; optionally, the monoclonal antibody to be tested includes IgG antibody.

5. The method according to any one of claims 1 to 4, characterized in that: The amphoteric electrolyte carrier is an amphoteric electrolyte carrier with a pH of 3 to 10; Optionally, the volume fraction of the amphoteric electrolyte carrier in the test solution is 4% to 5%.

6. The method according to any one of claims 1 to 4, characterized in that: The isoelectric point marker includes one or more of an isoelectric point marker with a pI of 2.0 to 7.0 and an isoelectric point marker with a pI of 8.0 to 10.0; Optionally, the volume fractions of the isoelectric point marker with a pI of 2.0 to 7.0 and the isoelectric point marker with a pI of 8.0 to 10.0 in the test solution are respectively 0.2% to 5%.

7. The method according to any one of claims 1 to 4, characterized in that The mass fraction of methyl cellulose in the test solution is 0.2%~0.4%.

8. The method according to any one of claims 1 to 4, characterized in that The concentration of urea in the test solution is 1M~3M.

9. The method according to claim 1, characterized in that The whole-column imaging capillary isoelectric focusing method satisfies one or more of the following conditions: The sample tray temperature is 10℃~15℃; The pre-focusing voltage is 1000V~1500V, and the pre-focusing time is 1~2min; The focusing voltage is 2900V~3100V, and the focusing time is 6~8min; and, The loading time is 50s~60s.

10. A method for quality detection of monoclonal antibody drugs, characterized in that: The method comprises detecting the charge variants of the monoclonal antibody in the monoclonal antibody drug by using the method according to any one of claims 1 to 9.

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