A method for analyzing bispecific antibody assembly by combining high-resolution mass spectrometry and capillary electrophoresis

Abstract

The present invention discloses a method for analyzing the assembly of bispecific antibodies by combining high-resolution mass spectrometry and capillary electrophoresis technology, comprising performing intact molecular weight and reduced molecular weight detection and analysis on bispecific antibodies by mass spectrometry technology, performing non-reduced CE-SDS and reduced CE-SDS sample detection and analysis on bispecific antibodies by capillary electrophoresis technology, and confirming the molecular assembly form of the bispecific antibodies and the composition ratio analysis of each molecular form by comprehensive analysis of the results of the two methods.

Description

Technical Field

[0001] The present invention relates to a method for analyzing double antibody assembly, and in particular to a method for analyzing double antibody assembly by combining high-resolution mass spectrometry and capillary electrophoresis technology. Background Art

[0002] Bispecific antibodies (BsAb) are artificial antibodies that can specifically bind to two antigens or antigenic epitopes at the same time. Bispecific antibodies do not exist under natural conditions, but are prepared through cell fusion or recombinant DNA technology. Due to their specificity and dual functionality, they have become a research hotspot in the field of antibody engineering and have broad application prospects in the fields of tumor treatment and autoimmune diseases.

[0003] During the development of bispecific antibodies of different configurations, how to obtain molecules with correct structures at high yields is a key and difficult point in drug development. For example, asymmetric bispecific antibody structures are usually composed of two different heavy chains and light chains, or two different heavy chains and the same light chain. Therefore, impurities caused by multiple mismatches are easily generated during the assembly process. These impurities are often similar to the physical and chemical properties of the target structure and are difficult to remove. Therefore, it is very important to identify whether the bispecific antibody is assembled correctly and to identify the form of incorrect assembly in the early stage of drug research.

[0004] At present, the main methods for bispecific antibody assembly analysis are size exclusion chromatography (SEC), ion exchange chromatography (CEX), gel electrophoresis (SDS-PAGE), capillary electrophoresis (CE), liquid chromatography-mass spectrometry (LC-MS), etc. However, for bispecific antibodies with different structures, there will be mismatched forms with small differences in molecular weight, and the physical and chemical properties are also similar. It is difficult for the above methods to achieve the ideal separation effect, and the adjacent spectral peaks cannot confirm the specific assembly form. Even if the assembly form is determined by mass spectrometry molecular weight identification, the response of the mass spectrometry signal cannot fully reflect the sample content and cannot provide effective and comprehensive support for molecular design optimization. Therefore, it is necessary to develop efficient and accurate analytical detection methods to provide characterization and analysis support for molecular design and functional verification. Summary of the invention

[0005] The present invention provides a method for analyzing the assembly of bispecific antibodies, which combines high-resolution mass spectrometry and capillary electrophoresis technology to analyze the assembly of bispecific antibodies. On the one hand, the method confirms the existence of various assembly forms through high-resolution mass spectrometry, and on the other hand, the capillary electrophoresis technology assists in analyzing the content ratio of each assembly form, thereby achieving comprehensive qualitative and quantitative analysis.

[0006] The present invention provides a method for analyzing bispecific antibody assembly by combining high-resolution mass spectrometry and capillary electrophoresis technology, using high-resolution mass spectrometry technology to perform intact molecular weight and reduced molecular weight detection and analysis on bispecific antibody samples, and using capillary electrophoresis technology to perform non-reduced CE-SDS and reduced CE-SDS sample detection and analysis on bispecific antibody samples;

[0007] Wherein, in the high-resolution mass spectrometry detection, the detection conditions of the chromatographic part are as follows:

[0008] Mobile phase A was an aqueous solution containing 0.1% FA;

[0009] Mobile phase B was acetonitrile (ACN) solution containing 0.1% FA;

[0010] The chromatographic column was a reverse phase column with a diameter of 21 mm, a length of 50 mm, and a particle size of 4 μm;

[0011] The gradient elution conditions for high-resolution mass spectrometry detection of the complete molecular weight of the dual antibody are as follows:

[0012] 0min: starting from 10% B;

[0013] 3min-7min: from 10% B to 90% B;

[0014] 7min-8min: maintain 90% B;

[0015] 8min-8.1min: from 90%B to 10%B;

[0016] 8.1min-10min: maintain 10% B;

[0017] The flow rate was maintained at 0.3 mL / min.

[0018] Furthermore, before the high-resolution mass spectrometry was used to detect the reduced molecular weight of the bispecific antibody, the bispecific antibody sample was pretreated as follows: 50-100 μg of the sample was taken, 4-6 M guanidine hydrochloride, 10-50 mM dithiothreitol (DTT) were added, and the mixture was incubated at 60° C. for 30-60 min. The sample was replaced into a 50 mM NH 4 HCO 3 solution using a desalting column, and the supernatant was taken after high-speed centrifugation and added to a sample tube for detection;

[0019] The sample is preferably 100 μg, the guanidine hydrochloride is preferably 6 M, the DTT is preferably 30 Mm, and the incubation is preferably 40 min.

[0020] Furthermore, when the high-resolution mass spectrometry is used to detect the reduced molecular weight of the dual antibody, the gradient elution conditions of the chromatographic part are as follows:

[0021] 0min: Start from 25% B;

[0022] 1min-13min: from 25% B to 32% B;

[0023] 13min-14min: from 32% B to 80% B;

[0024] 14min-16min: maintain 80% B;

[0025] 16min-16.1min: from 80%B to 25%B;

[0026] 16.1min-20min: maintain 25% B;

[0027] The flow rate was maintained at 0.25 mL / min.

[0028] Furthermore, the mass spectrometry detection conditions are as follows:

[0029] Use positive ion Full MS-SIM scan mode, Microscans set to 10, Resolution set to 17500, AGC target set to 3e6, Maximum IT set to 200ms, Scan range set to 500-4000, Sheath gas flow (arb) set to 30, Aux gas flow rate (arb) set to 10, Sweep gas flow rate (arb) set to 0, Spray voltage (kV) set to 3.8, Capillary temperature (℃) set to 300, S-lens RF level set to 60, Aux gas heater temperature (℃) set to 300.

[0030] Furthermore, the capillary electrophoresis technique is non-reducing CE-SDS capillary electrophoresis detection;

[0031] Before the non-reduced CE-SDS capillary electrophoresis analysis, the sample was pretreated as follows:

[0032] Dilute the sample with water to 2 mg / mL, add 35.5 μL 1X Sample Buffer and 2 μL 25X molecular weight standard into the tube; add 2.5 μL 250 mM iodoacetamide solution; add 10 μL diluted sample, vortex to mix, the total volume is 50 μL; after centrifugation, heat in a 70°C water bath for 10 min; place at 5±3°C for 5 min, vortex to mix, and centrifuge at 12000 rpm for 5 min; take out 45 μL sample and add to a 96-well plate; centrifuge at 2000 rpm for 5 min.

[0033] Further, the capillary electrophoresis technique is reduced CE-SDS capillary electrophoresis detection;

[0034] Before the reduced CE-SDS capillary electrophoresis analysis, the sample was pretreated as follows:

[0035] Dilute the sample with water to 2 mg / mL. Add 35.5 μL 1X Sample Buffer and 2 μL 25X molecular weight standard to the tube; add 2.5 μL mercaptoethanol solution; add 10 μL diluted sample, vortex to mix, total volume 50 μL; after centrifugation, heat in 70°C water bath for 10 min; place at 5±3°C for 5 min, vortex to mix, centrifuge at 12000 rpm for 5 min; take out 45 μL sample and add to 96-well plate; centrifuge at 2000 rpm for 5 min.

[0036] Furthermore, the CE-SDS capillary electrophoresis detection injection voltage is set to 4600V, the injection time is 20s, and the separation voltage is set to 5750V, and the separation time is 30min-40min.

[0037] Furthermore, the bispecific antibody sample is a bispecific antibody with a non-IgG structure or an IgG-like structure; for example, the light chain sequence of the bispecific antibody sample is shown in SEQ ID NO: 1, one heavy chain sequence is shown in SEQ ID NO: 2, and the other heavy chain sequence is shown in SEQ ID NO: 3.

[0038] The bispecific antibody assembly analysis method provided by the present invention combines high-resolution mass spectrometry and capillary electrophoresis technology, which can not only accurately detect various assembly forms contained in the sample, but also quantitatively analyze the proportion of each assembly form, thereby providing strong support for bispecific antibody molecular screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0040] Figure 1 is a mass spectrum of the intact molecular weight of the bispecific antibody described in Example 1 of the present invention;

[0041] Figure 2 is a mass spectrum of the reduced molecular weight of the bispecific antibody described in Example 1 of the present invention;

[0042] Figure 3 This is the non-reduced CE-SDS capillary electrophoresis diagram of the dual antibody described in Example 1 of the present invention;

[0043] Figure 4is the reduced CE-SDS capillary electrophoresis diagram of the dual antibody described in Example 1 of the present invention;

[0044] Figure 5A This is the mass spectrum of the complete molecular weight result of the dual antibody described in Example 2 of the present invention;

[0045] Figure 5B It is the enlarged spectrum of the main peak;

[0046] Fig. 6A This is the mass spectrum of the reduced molecular weight result of the dual antibody described in Example 2 of the present invention;

[0047] Figure 6B This is the enlarged spectrum of the light chain part;

[0048] Figure 7 This is the non-reduced CE-SDS capillary electrophoresis diagram of the dual antibody described in Example 2 of the present invention;

[0049] Figure 8 This is the reduced CE-SDS capillary electrophoresis diagram of the dual antibody described in Example 2 of the present invention. DETAILED DESCRIPTION

[0050] In the following description, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0051] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0052] Although the above disclosed content shows exemplary embodiments of the present invention, it should be noted that, without departing from the scope defined in the claims, various changes and modifications may be made. In addition, although elements of the present invention may be described or required in individual form, it is also conceivable to have multiple elements, unless explicitly limited to a single element.

[0053] Additionally, references to dimensions, sizes, formulations, parameters, shapes, and other quantities and features are not and need not be exact, but may be approximate and / or larger or smaller as necessary, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those skilled in the art. In general, dimensions, sizes, formulations, parameters, shapes, or other quantities or features are "about" or "approximately" whether or not so expressly stated.

[0054] definition

[0055] In order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0056] The term "bispecific antibody" refers to a bispecific antibody (BsAb), which is an artificial antibody that can specifically bind to two antigens or antigenic epitopes at the same time.

[0057] The term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy chains (HC) and two light chains (LC) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chains of antibodies can be divided into κ or λ light chains. The heavy chains can be divided into μ, δ, γ, α or ε, which define the isotype of the antibody as IgM, IgD, IgG, IgA or IgE, respectively. In the light and heavy chains, the variable region is connected to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids.

[0058] The term "double antibody assembly" refers to an antibody assembled from two light chains and two heavy chains. In this context, double antibody assembly can be assembled from, for example, two different heavy chains and light chains, or two different heavy chains and the same light chain.

[0059] The present invention provides a method for analyzing the assembly of double antibodies by combining high-resolution mass spectrometry and capillary electrophoresis technology, using high-resolution mass spectrometry technology to perform intact molecular weight and reduced molecular weight detection and analysis on double antibody samples, and using capillary electrophoresis technology to perform non-reduced CE-SDS (sodium dodecyl sulfate capillary electrophoresis) and reduced CE-SDS sample detection and analysis on double antibody samples;

[0060] Wherein, in the high-resolution mass spectrometry detection, the detection conditions of the chromatographic part are as follows:

[0061] Mobile phase A was an aqueous solution containing 0.1% FA;

[0062] Mobile phase B was acetonitrile (ACN) solution containing 0.1% FA;

[0063] The chromatographic column was a reverse phase column with a diameter of 21 mm, a length of 50 mm, and a particle size of 4 μm;

[0064] The gradient elution conditions for high-resolution mass spectrometry detection of the complete molecular weight of the dual antibody are as follows:

[0065] 0min: starting from 10% B;

[0066] 3min-7min: from 10% B to 90% B;

[0067] 7min-8min: maintain 90% B;

[0068] 8min-8.1min: from 90%B to 10%B;

[0069] 8.1min-10min: maintain 10% B;

[0070] The flow rate was maintained at 0.3 mL / min.

[0071] Furthermore, before the high-resolution mass spectrometry is used to detect and analyze the intact molecular weight of the bispecific antibody, the bispecific antibody sample is pretreated as follows: the sample is diluted to 0.2-1 mg / ml using a diluent, preferably 0.5 mg / ml.

[0072] Furthermore, the diluent is one of water, 50 mM ammonium bicarbonate solution, and 0.1% formic acid aqueous solution.

[0073] Furthermore, before the high-resolution mass spectrometry was used to detect the reduced molecular weight of the bispecific antibody, the bispecific antibody sample was pretreated as follows: 50-100 μg of the sample was taken, 4-6 M guanidine hydrochloride, 10-50 mM dithiothreitol (DTT) were added, and the mixture was incubated at 60° C. for 30-60 min. The sample was replaced into a 50 mM NH 4 HCO 3 solution using a desalting column, and the supernatant was taken after high-speed centrifugation and added to a sample tube for detection;

[0074] The sample is preferably 100 μg, the guanidine hydrochloride is preferably 6 M, the DTT is preferably 30 Mm, and the incubation is preferably 40 min.

[0075] Furthermore, when the high-resolution mass spectrometry is used to detect the reduced molecular weight of the dual antibody, the gradient elution conditions of the chromatographic part are as follows:

[0076] 0min: Start from 25% B;

[0077] 1min-13min: from 25% B to 32% B;

[0078] 13min-14min: from 32% B to 80% B;

[0079] 14min-16min: maintain 80% B;

[0080] 16min-16.1min: from 80%B to 25%B;

[0081] 16.1min-20min: maintain 25% B;

[0082] The flow rate was maintained at 0.25 mL / min.

[0083] Furthermore, the mass spectrometry detection conditions are as follows:

[0084] Use positive ion Full MS-SIM scan mode, Microscans set to 10, Resolution set to 17500, AGC target set to 3e6, Maximum IT set to 200ms, Scan range set to 500-4000, Sheath gas flow (arb) set to 30, Aux gas flow rate (arb) set to 10, Sweep gas flow rate (arb) set to 0, Spray voltage (kV) set to 3.8, Capillary temperature (℃) set to 300, S-lens RF level set to 60, Aux gas heater temperature (℃) set to 300.

[0085] Furthermore, the capillary electrophoresis technique is non-reducing CE-SDS capillary electrophoresis detection;

[0086] Before the non-reduced CE-SDS capillary electrophoresis analysis, the sample was pretreated as follows:

[0087] Dilute the sample with water to 2 mg / mL, add 35.5 μL 1X Sample Buffer and 2 μL 25X molecular weight standard into the tube; add 2.5 μL 250 mM iodoacetamide solution; add 10 μL diluted sample, vortex to mix, the total volume is 50 μL; after centrifugation, heat in a 70°C water bath for 10 min; place at 5±3°C for 5 min, vortex to mix, and centrifuge at 12000 rpm for 5 min; take out 45 μL sample and add to a 96-well plate; centrifuge at 2000 rpm for 5 min.

[0088] Further, the capillary electrophoresis technique is reduced CE-SDS capillary electrophoresis detection;

[0089] Before the reduced CE-SDS capillary electrophoresis analysis, the sample was pretreated as follows:

[0090] Dilute the sample with water to 2 mg / mL. Add 35.5 μL 1X Sample Buffer and 2 μL 25X molecular weight standard to the tube; add 2.5 μL mercaptoethanol solution; add 10 μL diluted sample, vortex to mix, total volume 50 μL; after centrifugation, heat in 70°C water bath for 10 min; place at 5±3°C for 5 min, vortex to mix, centrifuge at 12000 rpm for 5 min; take out 45 μL sample and add to 96-well plate; centrifuge at 2000 rpm for 5 min.

[0091] Furthermore, the injection voltage of the CE-SDS capillary electrophoresis detection is set to 4600 V, the injection time is 20 s, and the separation voltage is set to 5750 V, and the separation time is 30 min-40 min.

[0092] Furthermore, the bispecific antibody sample is a bispecific antibody with a non-IgG structure or an IgG-like structure; for example, the light chain sequence of the bispecific antibody sample is shown in SEQ ID NO: 1, one heavy chain sequence is shown in SEQ ID NO: 2, and the other heavy chain sequence is shown in SEQ ID NO: 3.

[0093] Example

[0094] In the following Examples 1 and 2, the detection conditions and detection reagents, instruments, etc. described below were used.

[0095] When high-resolution mass spectrometry is used for detection, the bispecific antibody sample pretreatment method and detection process are as follows:

[0096] Sample pretreatment for intact molecular weight detection: Use 0.1% FA (formic acid) / H2O (mobile phase A) to dilute the sample to about 0.5 mg / ml, centrifuge at high speed, and add the supernatant to the sample tube for detection.

[0097] Sample pretreatment for reduction molecular weight detection: Take 50 μg of sample, add 6M guanidine hydrochloride (Thermo, item number 24115), 30mM dithiothreitol (DTT) (Sigma, item number D0632), incubate at 60°C for 30 minutes, use a desalting column (Thermo, item number 89883) to replace the sample into a 50mM NH4HCO3 (Merck, item number 5.33005) solution (the solution volume is adjusted according to the actual situation, and the final sample concentration can be adjusted to 1mg / ml), and take the supernatant after high-speed centrifugation and add it to the sample tube for detection. Among them, the concentrations of guanidine hydrochloride and dithiothreitol are the final concentrations of guanidine hydrochloride and dithiothreitol after adding both guanidine hydrochloride and dithiothreitol.

[0098] The detection conditions of the intact molecular weight detection chromatographic portion are as follows:

[0099] Mobile phase A was an aqueous solution containing 0.1% FA (formic acid) (Fisher, product number A117);

[0100] Mobile phase B was acetonitrile (ACN) solution containing 0.1% FA (formic acid) (Thermo, product number A955);

[0101] The chromatographic column was a reverse phase column with a diameter of 21 mm, a length of 50 mm, and a particle size of 4 μm (Thermo, catalog number 088648);

[0102] The gradient elution conditions were as follows:

[0103] 0min: starting from 10% B;

[0104] 3min-7min: from 10% B to 90% B;

[0105] 7min-8min: maintain 90% B;

[0106] 8min-8.1min: from 90%B to 10%B;

[0107] 8.1min-10min: maintain 10% B;

[0108] The flow rate was maintained at 0.3 mL / min;

[0109] The detection conditions of the reduced molecular weight detection chromatographic part are as follows:

[0110] Mobile phase A was an aqueous solution containing 0.1% FA;

[0111] Mobile phase B was acetonitrile (ACN) solution containing 0.1% FA;

[0112] The chromatographic column was a reverse phase column with a diameter of 21 mm, a length of 50 mm, and a particle size of 4 μm;

[0113] The gradient elution conditions were as follows:

[0114] 0min: Start from 25% B;

[0115] 1min-13min: from 25% B to 32% B;

[0116] 13min-14min: from 32% B to 80% B;

[0117] 14min-16min: maintain 80% B;

[0118] 16min-16.1min: from 80%B to 25%B;

[0119] 16.1min-20min: maintain 25% B;

[0120] The flow rate was maintained at 0.25 mL / min;

[0121] The mass spectrometry detection conditions for intact and reduced molecular weight detection were as follows: positive ion Full MS-SIM scan mode was used, Microscans was set to 10, Resolution was set to 17500, AGC target was set to 3e6, Maximum IT was set to 200ms, Scan range was set to 500-4000, Sheath gas flow (arb) was set to 30, Aux gas flow rate (arb) was set to 10, Sweep gas flow rate (arb) was set to 0, Spray voltage (kV) was set to 3.8, Capillary temperature (°C) was set to 300, S-lens RF level was set to 60, and Aux gas heater temperature (°C) was set to 300;

[0122] The non-reduced sample capillary electrophoresis detection was pretreated as follows: the sample was diluted to 2 mg / mL with ultrapure water, and 35.5 μL 1X Sample Buffer (Protein Simple, Catalog No. 046-567) was added to a microcentrifuge tube, and 2 μL 25X molecular weight standard 10KD (10KD internal standard) (Yongtuo Bio, Catalog No. R300112) was added; 2.5 μL 250 mM iodoacetamide solution (Sigma, Catalog No. I1149) was added; 10 μL of the diluted sample was added, and vortexed to mix, and the total volume was 50 μL. After centrifugation, it was heated in a 70°C water bath for 10 min; it was placed at 5±3°C for 5 min, vortexed to mix, and centrifuged at 12000 rpm for 5 min; 45 μL of the sample was taken out and added to a 96-well plate; it was centrifuged at 2000 rpm for 5 min; if there were bubbles, they were punctured with a clean pipette tip.

[0123] The reduced sample capillary electrophoresis detection was pretreated as follows: the sample was diluted to 2 mg / mL with ultrapure water, and 35.5 μL 1X Sample Buffer and 2 μL 25X molecular weight standard 10KD were added to a microcentrifuge tube; 2.5 μL mercaptoethanol solution (Solarbio, catalog number M8210) were added; 10 μL of the diluted sample was added, and vortexed to mix, with a total volume of 50 μL. After centrifugation, the sample was heated in a 70°C water bath for 10 min; placed at 5±3°C for 5 min, vortexed to mix, and centrifuged at 12000 rpm for 5 min; 45 μL of the sample was taken out and added to a 96-well plate; centrifuged at 2000 rpm for 5 min; if there were bubbles, they were punctured with a clean pipette tip.

[0124] The instrument used for capillary electrophoresis (CE) detection was Protein simple (Bio-Techne), and the capillary cartridge used was Maurice CE-SDS PLUS Cartridge (Bio-Techne). The reagents and consumables used in the detection process were all related to the instrument. For non-reduction detection, the injection voltage was set to 4600 V, the injection time was 20 s, the separation voltage was set to 5750 V, and the separation time was 40 min; for reduction detection, the injection voltage was set to 4600 V, the injection time was 20 s, the separation voltage was set to 5750 V, and the separation time was 30 min.

[0125] Embodiment 1:

[0126] The above-mentioned high-resolution mass spectrometry and capillary electrophoresis technology combined detection method was used to analyze the assembly of the non-IgG structured bispecific antibody molecules. In this embodiment, the sequence information of the antibody is as follows. The possible assembly forms of the antibody include: HC1HC1_LC (i.e., composed of two identical heavy chains 1+1 light chain), HC2HC2_LC (i.e., composed of two identical heavy chains 2+1 light chain), and HC1HC2_LC (i.e., composed of heavy chain 1+heavy chain 2+1 light chain).

[0127] The sequence of the bispecific antibody molecule LC (light chain) (SEQ ID NO: 1):

[0128] SYELTQPPSVSVSPGQTARITCSGDALPKKYAYWYQQKPGQAPVLVISEDAKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYCYSTDASGNHRVFGGGTKLTV LGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0129] The sequence of the bispecific antibody molecule HC1 (heavy chain 1) (SEQ ID NO: 2):

[0130] EVQLVESGGSVVRPGGSLRLSCAASGFTVDDYSMSWVRQVPGKGLEWVSGINWNAGRTRYADAVKGRFTISRDSAKNSLYLQMNSLRAEDTALYYCAREFNNFESNWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGQGQDRHMIEMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKAKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

[0131] Sequence of bispecific molecule HC2 (heavy chain 2) (SEQ ID NO: 3):

[0132] EVQLVESGGSVVRPGGSLRLSCAASGFTVDDYSMSWVRQVPGKGLEWVSGINWNAGRTRYADAVKGRFTISRDSAKNSLYLQMNSLRAEDTALYYCAREFNNFESNWFDPWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0133] The analysis results are shown in Figures 1 to 4 middle. Figure 1 It is a mass spectrum of the complete molecular weight of the bispecific antibody described in Example 1 of the present invention. Figure 2 It is a mass spectrum of the reduced molecular weight of the bispecific antibody described in Example 1 of the present invention. Figure 3 This is the non-reduced CE-SDS capillary electrophoresis diagram of the dual antibody described in Example 1 of the present invention. Figure 4 This is the reduced CE-SDS capillary electrophoresis diagram of the dual antibody described in Example 1 of the present invention.

[0134] Depend on Figure 1-Figure 4 The results show that the bispecific antibody molecule is a non-IgG structured bispecific antibody, consisting of a light chain LC and two different heavy chains HC1 and HC2. That is, the complete form of the bispecific antibody molecule assembly (intact) is HC1HC2_LC. The complete molecular weight mass spectrometry results are shown in Figure 1 . Figure 1 In the example, intact Reference indicates the complete form of the molecular assembly. Figure 1 In addition to the target form, there is also an LC light chain form and an HC2_LC form. The reduced molecular mass spectrometry results are shown in Figure 2 . Figure 2 In the formula, Reduce Reference refers to the reduced form of the light chain and heavy chain. Figure 2 The results of non-reduced CE-SDS are shown in Figure 2. Figure 3 show, Figure 3 IS indicates 10KD internal standard. Figure 3 The relative time 1.362min corresponds to LC, accounting for about 1.89%, the relative time 1.815min corresponds to HC2_LC form, accounting for about 7.51%, and the relative time 2.365min corresponds to the intact form, accounting for about 87.11%. The results of reduced CE-SDS are as follows Figure 4 It shows that the relative time 1.313min corresponds to LC, accounting for about 27.48%, the relative time 1.555min corresponds to HC2, accounting for about 34.79%, and the relative time 1.682min corresponds to HC1, accounting for about 35.43%.

[0135] Embodiment 2:

[0136] The above-mentioned high-resolution mass spectrometry and capillary electrophoresis technology combined detection method was used to analyze the assembly of bispecific antibody molecules with similar IgG structures. The possible assembly forms of the antibody include: HC1LC1_HC1LC1 (i.e., composed of two identical heavy chains 1 + two identical light chains 1), HC2LC2_HC2LC2 (i.e., composed of two identical heavy chains 2 + two identical light chains 2), HC1LC2_HC1LC1 (i.e., composed of two heavy chains 1 + two different light chains), HC1LC2_HC2LC1 (i.e., composed of heavy chain 1 + heavy chain 2 + light chain 1 + light chain 2), etc.

[0137] The analysis results are shown in Figures 5 to Figure 8 Among them, Figure 5A This is the mass spectrum of the complete molecular weight result of the dual antibody described in Example 2 of the present invention, Figure 5B It is the enlarged spectrum of the main peak. Fig. 6A is a mass spectrum of the reduced molecular weight result of the dual antibody described in Example 2 of the present invention, Figure 6B This is an enlarged spectrum of the light chain. Figure 7 This is the non-reduced CE-SDS capillary electrophoresis diagram of the dual antibody described in Example 2 of the present invention. Figure 8 This is the reduced CE-SDS capillary electrophoresis diagram of the dual antibody described in Example 2 of the present invention.

[0138] The bispecific antibody is a bispecific antibody with a similar structure to IgG, consisting of two different light chains LC1 and LC2, and two different heavy chains HC1 and HC2. The target assembly form is LC1HC1_LC2HC2 (intact). The mass spectrometry results of the intact molecular weight are shown in Figure 5A and Figure 5B . Figure 5A and Figure 5BIn the figure, intact-EA indicates the complete form with amino acid truncated form (EA missing), and intact+Hex indicates the complete form with Hex modification. Figure 5 shows that in addition to the target form, there are also intact truncated forms, LC1LC2 double light chain forms, and HC1HC2 double chain forms. The reduced molecular mass spectrometry results are shown in Fig. 6A and Figure 6B . Fig. 6A and Figure 6B In the figure, LC2-EA indicates the amino acid truncated form of LC2 (EA missing). Figure 6 shows that LC2 has two N-terminal amino acid truncations, and LC1, LC2, HC1, and HC2 can all be confirmed. The non-reducing CE-SDS results are shown in Figure 7 . Figure 7 It shows that there are other peaks on the left side of the main peak, which makes the main peak asymmetric, corresponding to the truncated form of intact in the full molecular weight mass spectrum. The relative time 1.547min corresponds to the LC1LC2 part, accounting for about 1.14%, the relative time 1.896min corresponds to the HC1HC2 part, accounting for about 2.55%, and the relative time 2.218min corresponds to intact and intact truncated forms, accounting for about 93.81%. The results of reduced CE-SDS are shown in Figure 8 . Figure 8 It shows that the relative time 1.231min corresponds to LC1, accounting for about 14.79%, the relative time 1.288min corresponds to the truncated form of LC2, accounting for 2.74%, the relative time 1.318min corresponds to LC2, accounting for about 14.65%, the relative time 1.491min corresponds to HC2, accounting for about 20.88%, and the relative time 1.531min corresponds to HC1, accounting for about 39.46%.

[0139] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for analyzing bispecific antibody assembly by combining high-resolution mass spectrometry and capillary electrophoresis, characterized in that: High-resolution mass spectrometry was used to analyze the intact molecular weight and reduced molecular weight of the double antibody samples, and capillary electrophoresis was used to analyze the double antibody samples by non-reduced CE-SDS and reduced CE-SDS samples. Wherein, in the high-resolution mass spectrometry detection, the detection conditions of the chromatographic part are as follows: Mobile phase A was an aqueous solution containing 0.1% formic acid; Mobile phase B was acetonitrile (ACN) solution containing 0.1% formic acid; The chromatographic column was a reverse phase column with a diameter of 21 mm, a length of 50 mm, and a particle size of 4 μm; The gradient elution conditions for high-resolution mass spectrometry detection of the complete molecular weight of the dual antibody are as follows: 0min: Start from 10% B; 3min-7min: from 10%B to 90%B; 7min-8min: maintain 90% B; 8min-8.1min: from 90%B to 10%B; 8.1min-10min: maintain 10% B; The flow rate was maintained at 0.3 mL / min.

2. The method for analyzing bispecific antibody assembly by combining high-resolution mass spectrometry and capillary electrophoresis technology according to claim 1, characterized in that: Before the high-resolution mass spectrometry is used to analyze the intact molecular weight of the bispecific antibody, the bispecific antibody sample is pretreated as follows: the sample is diluted to 0.2-1 mg / ml using a diluent, preferably 0.5 mg / ml.

3. The method for analyzing bispecific antibody assembly by combining high-resolution mass spectrometry and capillary electrophoresis according to claim 2, characterized in that: The diluent is one of water, 50 mM ammonium bicarbonate solution, and 0.1% formic acid aqueous solution.

4. The method for analyzing bispecific antibody assembly by combining high-resolution mass spectrometry and capillary electrophoresis according to claim 1, characterized in that: Before the high-resolution mass spectrometry was used to detect the reduced molecular weight of the bispecific antibody, the bispecific antibody sample was pretreated as follows: 50-100 μg of the sample was taken, 4-6 M guanidine hydrochloride and 10-50 mM dithiothreitol (DTT) were added, and the mixture was incubated at 60° C. for 30-60 min. The sample was replaced with a 50 mM NH 4 HCO 3 solution using a desalting column, and the supernatant was added to a sample tube for detection after high-speed centrifugation. The sample is preferably 100 μg, the guanidine hydrochloride is preferably 6 M, the DTT is preferably 30 Mm, and the incubation is preferably 40 min.

5. The method for analyzing bispecific antibody assembly by combining high-resolution mass spectrometry and capillary electrophoresis technology according to claim 4, characterized in that: When the high-resolution mass spectrometry is used to detect the reduced molecular weight of the dual antibody, the gradient elution conditions of the chromatographic part are as follows: 0min: Start from 25% B; 1min-13min: from 25%B to 32%B; 13min-14min: from 32%B to 80%B; 14min-16min: maintain 80% B; 16min-16.1min: from 80%B to 25%B; 16.1min-20min: maintain 25% B; The flow rate was maintained at 0.25 mL / min.

6. The method for analyzing bispecific antibody assembly by combining high-resolution mass spectrometry and capillary electrophoresis according to any of the above claims, characterized in that: The mass spectrometry detection conditions are as follows: Use positive ion Full MS-SIM scanning mode, Microscans is set to 10, Resolution is set to 17500, AGC target is set to 3e6, Maximum IT is set to 200ms, Scan range is set to 500-4000, Sheath gasflow (arb) is set to 30, Aux gas flow rate (arb) is set to 10, Sweep gas flow rate (arb) is set to 0, Spray voltage (kV) is set to 3.8, Capillary temperature (℃) is set to 300, S-lens RFlevel is set to 60, and Aux gas heater temperature (℃) is set to 300.

7. The method for analyzing bispecific antibody assembly by combining high-resolution mass spectrometry and capillary electrophoresis according to claim 1, characterized in that: The capillary electrophoresis technique is non-reducing CE-SDS capillary electrophoresis detection; Before the non-reducing CE-SDS capillary electrophoresis analysis, the sample was pretreated as follows: Dilute the sample with water to 2 mg / mL, add 35.5 μL 1X Sample Buffer and 2 μL 25X molecular weight standard into the tube; add 2.5 μL 250 mM iodoacetamide solution; add 10 μL diluted sample, vortex to mix, the total volume is 50 μL; after centrifugation, heat in a 70°C water bath for 10 min; place at 5±3°C for 5 min, vortex to mix, and centrifuge at 12000 rpm for 5 min; take out 45 μL sample and add to a 96-well plate; centrifuge at 2000 rpm for 5 min.

8. The method for analyzing bispecific antibody assembly by combining high-resolution mass spectrometry and capillary electrophoresis according to claim 1, characterized in that: The capillary electrophoresis technique is reduced CE-SDS capillary electrophoresis detection; Before the reduced CE-SDS capillary electrophoresis analysis, the sample was pretreated as follows: Dilute the sample with water to 2 mg / mL. Add 35.5 μL 1X Sample Buffer and 2 μL 25X molecular weight standard to the tube; add 2.5 μL mercaptoethanol solution; add 10 μL diluted sample, vortex to mix, total volume 50 μL; after centrifugation, heat in 70°C water bath for 10 min; place at 5±3°C for 5 min, vortex to mix, centrifuge at 12000 rpm for 5 min; take out 45 μL sample and add to 96-well plate; centrifuge at 2000 rpm for 5 min.

9. The method for analyzing bispecific antibody assembly by combining high-resolution mass spectrometry and capillary electrophoresis according to claim 7 or 8, characterized in that: The CE-SDS capillary electrophoresis detection injection voltage was set to 4600 V, injection was 20 s, and the separation voltage was set to 5750 V, separation was 30 min-40 min.

10. The method for analyzing bispecific antibody assembly by combining high-resolution mass spectrometry and capillary electrophoresis technology according to claim 1, characterized in that: The bispecific antibody sample is a bispecific antibody with a non-IgG structure or an IgG-like structure; for example, the light chain sequence of the bispecific antibody sample is shown in SEQ ID NO: 1, one heavy chain sequence is shown in SEQ ID NO: 2, and the other heavy chain sequence is shown in SEQ ID NO: 3.

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