A monoclonal antibody drug quantitative detection method based on a multiple capture strategy

Abstract

The application discloses a kind of monoclonal antibody drug quantitative detection methods based on multiple capture strategy, comprising the following steps: (1) dissociation;(2) renaturation-capture: then join renaturation reagent, the non-magnetic carrier fluid coupled with target monoclonal antibody and the magnetic carrier fluid coupled with target antigen, 10-25 min are oscillated at room temperature, and the primary capture liquid is obtained;(3) cycle capture: the primary capture liquid is captured 1-3 times in cycle;(4) separation.The application significantly reduces the consumption of target antigen, improves the enrichment efficiency and purity of target mAb, and has the advantages of high cost efficiency, efficient operation and wide application range.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to a method for the quantitative detection of monoclonal antibody drugs based on a multiple capture strategy. Background Technology

[0002] Monoclonal antibodies, as a class of highly targeted biological agents with well-defined mechanisms of action, have been widely used in the treatment of tumors, autoimmune diseases (such as rheumatoid arthritis, Crohn's disease, and psoriasis), and various chronic inflammatory diseases. However, their pharmacokinetics and pharmacodynamics vary significantly among individuals. This heterogeneity is mainly influenced by factors such as body weight, inflammatory state, concomitant medications, immunogenicity (i.e., anti-drug antibodies, ADA), and genetic polymorphism. Therefore, therapeutic drug monitoring (TDM) plays a crucial role in optimizing clinical efficacy and achieving personalized medication. TDM provides a scientific basis for dose adjustment by dynamically and quantitatively detecting drug concentrations and ADA levels in patients' serum. Prospective TDM can identify subtherapeutic concentrations before clinical symptoms worsen, thereby allowing for early intervention and optimization of dosing regimens; reactive TDM is used to elucidate the underlying mechanisms of poor efficacy or adverse reactions (such as infusion reactions, increased risk of infection, etc.). For example, in cases of secondary loss of response in patients, TDM can help distinguish whether the cause is low drug concentration, suggesting the need to increase the dose or shorten the dosing interval; or accelerated drug clearance mediated by ADA, which may require switching to a drug with a different mechanism of action.

[0003] Currently, among methods for monitoring the blood concentration of monoclonal antibody drugs, liquid chromatography-tandem mass spectrometry (LC-MS / MS) is gradually replacing traditional ligand binding methods (such as ELISA) and becoming the mainstream quantitative analysis method due to its high specificity, high sensitivity, and ability to simultaneously detect multiple components. The core principle of this method is: monoclonal antibodies (mAbs) in serum or plasma samples are enzymatically digested with trypsin to generate a series of peptides; subsequently, highly specific "specific peptides" are screened out—peptides whose amino acid sequences are not present in endogenous human proteins and are derived solely from exogenous monoclonal antibody drugs—as quantitative targets, which are then accurately detected using LC-MS / MS in multiple reaction monitoring mode.

[0004] However, blood samples are highly complex biological matrices, containing up to 60-80 mg / mL of total protein, primarily albumin and immunoglobulins. This high protein background not only leads to column contamination and salt buildup in the mass spectrometry ion source, significantly increasing baseline noise, but also triggers a "matrix effect," inhibiting the ionization efficiency of target peptides during electrospray ionization (ESI), thereby reducing detection sensitivity and quantitative accuracy. Therefore, effective enrichment and purification of the target monoclonal antibody in the sample are essential before enzymatic digestion to minimize the co-extraction of interfering substances.

[0005] Because therapeutic monoclonal antibodies and human endogenous IgG are highly homologous in their Fc region structure (sequence similarity >90%), conventional protein A / G affinity chromatography is insufficient for selective separation. Therefore, researchers commonly employ affinity enrichment strategies based on antigen-antibody specific recognition: the target antigen corresponding to the monoclonal antibody drug (e.g., TNF-α for infliximab, CD20 peptide for rituximab) is covalently coupled to the surface of a solid-phase carrier (e.g., magnetic microbeads, agarose gel, carboxylate microspheres, porous glass beads, etc.) to form an antigen-capturing probe. Upon addition of a serum sample, the free monoclonal antibody drug can specifically bind to the solid-phase antigen through its Fab variable region. After washing to remove unbound contaminating proteins, the captured antibody is then subjected to enzymatic digestion and mass spectrometry analysis.

[0006] However, this method faces a key challenge in clinical application: some patients develop anti-drug antibodies (ADA) after receiving monoclonal antibody therapy, or have high levels of soluble target antigens (sAg) in their bodies. These molecules can bind to monoclonal antibody drugs, forming large molecular complexes—namely, monoclonal antibody-antibody (mAb-ADA) immune complexes or monoclonal antibody-antigen (mAb-sAg) complexes. Because the antigen-binding sites of the monoclonal antibody in these complexes are already occupied, they can no longer interact with the antigen on the solid-phase carrier, leading to their omission in the enrichment step and resulting in an underestimation of blood drug concentration.

[0007] To address this issue, some studies have attempted to introduce a "reversible denaturation-competitive enrichment" strategy. The basic procedure is as follows: First, serum samples are briefly acidified (e.g., pH 1.5-3.5, 5-15 minutes). The low pH disrupts non-covalent protein interactions, causing the mAb-ADA or mAb-sAg complex to dissociate and release free monoclonal antibodies. Subsequently, the system pH is rapidly neutralized, and a large excess of antigen probes immobilized on magnetic beads is added simultaneously. This allows the dissociated monoclonal antibodies to preferentially bind to the solid-phase antigen, thus achieving effective capture. Theoretically, this method can restore the ability to detect "blocked" drugs and improve the accuracy of the results.

[0008] However, the implementation of this strategy relies on the effectiveness of the "competitive enrichment" mechanism. This means the local concentration of the solid-phase antigen must be significantly higher than the concentration of natural ADA or sAg in the sample to ensure that almost all dissociated monoclonal antibodies bind to the magnetic beads rather than reforming into complexes. For example, with infliximab, if the sTNF-α concentration in a patient's serum reaches 100 ng / mL (approximately 4.4 nM), the effective binding capacity of the solid-phase antigen needs to reach at least a 50-100-fold excess (i.e., >200-400 nM) to achieve efficient competition. However, many target antigens (such as extracellular domains of membrane proteins and cytokines) are difficult to express, have high purification costs, are expensive (up to several thousand yuan / mg), and have limited solubility in aqueous phase (often <1 mg / mL), making high-density conjugation on solid-phase carriers difficult. Therefore, although this method shows potential at the research level, it has not yet been translated into a mature methodology or commercial kit suitable for large-scale clinical testing due to limitations in reagent cost, process stability, and standardization difficulties. Summary of the Invention

[0009] The purpose of this invention is to provide a quantitative detection method for monoclonal antibody drugs based on a multiple capture strategy, which aims to solve the problem of multiple interferences encountered when detecting monoclonal antibody drugs in complex biological matrices.

[0010] The technical solution adopted by this invention to solve its technical problem is: A method for quantitative detection of monoclonal antibody drugs based on a multiple capture strategy includes the following steps: (1) Dissociation: Add acidic dissociation solution to blood samples and shake at room temperature for 5-15 min; perform reversible denaturation treatment on samples containing ADA–mAb and sAg–mAb complexes to dissociate the complexes and release free mAbs; (2) Renaturation-capture: Then add renaturation reagent, non-magnetic carrier solution conjugated with target monoclonal antibody and magnetic carrier solution conjugated with target antigen, shake at room temperature for 10-25 min to obtain primary capture solution; During the refolding process, a non-magnetic carrier conjugated to the target monoclonal antibody (mAb) is used to capture any ADA and sAg that may be present; another magnetic carrier conjugated to the target antigen is used to specifically bind to the refolded mAb. During the refolding process, the mAb being tested recovers its conformation and binds to the magnetic carrier conjugated to the target antigen, while any ADA and sAg that may be present in the sample are captured by the non-magnetic carrier conjugated to the target monoclonal antibody (mAb). (3) Cyclic capture: The primary capture solution is subjected to 1-3 cycles of capture. The operation of each cycle is as follows: centrifugation, adding acidic dissociation solution to the supernatant after centrifugation and shaking at room temperature for 5-15 min to obtain the re-dissociation solution, adding refolding reagent to the precipitate (i.e. the solid support) after centrifugation to obtain the refolding solution, then mixing the re-dissociation solution and the refolding solution, shaking at room temperature for 10-25 min to obtain the recapture solution; In response to the possibility that some mAbs may re-form complexes with ADA or sAg during the refolding process, this invention employs a cyclic capture method to repeatedly dissociate and refold the supernatant of the primary capture solution. By using an appropriate number of cyclic captures, the detection accuracy can be improved. (4) Separation: The magnetic carrier of the coupled target antigen in the capture liquid obtained in step (3) is separated by magnetic force. Then the magnetic carrier of the coupled target antigen obtained by separation is cleaned and the capture on the magnetic carrier is quantitatively detected.

[0011] In traditional one-way capture systems, endogenous free antigens and anti-drug antibodies in the sample directly compete with the carrier antigen added to capture the monoclonal antibody. According to the law of mass action in chemical equilibrium, at equilibrium, the ratio of monoclonal antibody captured by the carrier to that bound by the interfering substance is directly equal to the molar concentration ratio of the carrier antigen to the interfering substance. Therefore, we can assume the molar ratio of carrier antigen to interfering substance is N, and the probability (P_loss) of capture failure due to monoclonal antibody binding to the interfering substance in any independent binding event is... For a system requiring n capture cycles, the probability that a monoclonal antibody molecule will ultimately not be captured is the probability of capture failure occurring in n consecutive cycles, i.e., (P_loss). n Therefore, the theoretical overall recovery rate of the entire system is as described in Equation 1: Equation 1: Overall recovery rate (%) = 1 - (P_loss) n =1-( ) n For example, when N=1 (i.e., the molar ratio of carrier antigen to interferon is 1:1), the recovery rate is 1-(0.5). n The recovery rate can only reach 99% when the number of cycles reaches 7; when N=4 (i.e., the molar ratio of carrier antigen to interferon = 4:1), the recovery rate is 1 - (0.2). n The number of cycles required to achieve the same recovery rate is significantly reduced. To achieve a 99% recovery rate with a single capture, i.e., 1 – ( Since ) = 0.99, we get N = 99. To achieve a 99% single-shot capture recovery rate, we need to add a carrier antigen equivalent to 99 times the amount of interfering substances in the sample.

[0012] This invention introduces a non-magnetic carrier (a second type of solid-phase carrier) to conjugate the target monoclonal antibody, actively capturing and removing interfering substances in the system. This significantly reduces the concentration of competitors, effectively shifting the chemical equilibrium towards the formation of the monoclonal antibody-carrier antigen complex. This allows for a high carrier antigen-to-interfering substance ratio with only a small amount of carrier antigen added, resulting in high recovery rates in single or very few cycles. This fundamentally optimizes the capture process and is key to achieving efficient and low-cost capture. This principle has been fully validated in experimental results.

[0013] Previous studies have shown that the recovery rate of traditional single-capture strategies is significantly reduced in samples containing endogenous interfering proteins. For example, in the detection of ustekinumab (UST) in serum samples, when both UST (20 μg / mL) and IL (32 μg / mL) are present in the sample (i.e., the molar ratio of UST to endogenous IL is 1:4), the recovery rate using a single capture method with biotinylated IL-bound magnetic beads is only 13.97%. However, the method of this invention, which specifically captures UST by coupling IL with magnetic microparticles and simultaneously uses non-magnetic microparticles to couple UST to remove endogenous IL interference in the sample, achieves a recovery rate significantly higher than 94.8% in a single sample (200 μL) pretreatment process, requiring only 200 μL of solid-phase carrier and consuming only 6 μg of target antigen. Therefore, this invention significantly reduces the consumption of target antigen.

[0014] Preferably, in step (1), the pH of the acidic dissociation solution is 1.5-3.5 (preferably 2-3), and the acidic dissociation solution is selected from at least one of glycine-hydrochloric acid buffer, citrate-citrate buffer, formic acid-formate buffer, acetic acid-acetate buffer, maleic acid-maleate buffer, tartaric acid-tartrate buffer, hydrogen phthalate-hydrochloric acid buffer, phthalate buffer, benzoate buffer, succinate buffer, malate buffer, and phosphate-phosphate buffer.

[0015] Preferably, in step (2), the pH of the refolding reagent is 6.0-8.5 (preferably 7-8), and the refolding reagent is selected from at least one of PB, PBS, borate-borax buffer, Tris-HCl, TBS buffer, glycine buffer, HEPES buffer, MOPS buffer, PIPES buffer, BES buffer, and TES buffer.

[0016] Preferably, in step (2), the non-magnetic carrier is selected from one of agarose gel, cross-linked agarose gel, dextran gel, polystyrene microspheres, polyacrylate microspheres, porous glass microspheres, titanium dioxide particles, zirconium oxide particles, alumina particles, carbon nanotubes, graphene oxide, silver nanoparticles, and gold nanoparticles, and the average particle size of the non-magnetic carrier is 5 nm-500 μm. The magnetic carrier is a magnetic bead with an average particle size of 5 nm-300 μm.

[0017] Preferably, in step (2), when the carrier is particulate, the concentrations of the non-magnetic carrier solution conjugated with the target monoclonal antibody and the magnetic carrier solution conjugated with the target antigen are both 10 μg / mL to 1200 mg / mL.

[0018] Preferably, in step (2), the target monoclonal antibody is selected from one of adalimumab, infliximab, ustekinumab, and eculizumab; the target antigen is selected from one or more of TNF-α, IL-12, IL-23, and complement protein C5.

[0019] Preferably, in steps (1) and (2), the volume ratio of blood sample: acidic dissociation solution: refolding reagent: non-magnetic carrier solution conjugated with target monoclonal antibody: magnetic carrier solution conjugated with target antigen is 1:0.2-20:0.2-20:0.1-30:0.1-30.

[0020] Preferably, in step (3), during each cycle of capture, the amount of acidic dissociation solution used is the same as in step (1), and the amount of refolding reagent used is the same as in step (2).

[0021] Preferably, in step (4), the quantitative detection is performed using mass spectrometry, ELISA, or immunoturbidimetry.

[0022] The beneficial effects of this invention are: This invention significantly reduces the consumption of target antigens, improves the enrichment efficiency and purity of target mAbs, and offers advantages such as high cost-effectiveness, high operational efficiency, and wide applicability. It is particularly suitable for long-term monitoring of therapeutic antibody drugs with high immunogenicity risks and for precise quantitative analysis of low-concentration samples. Furthermore, this strategy is compatible with automated liquid handling platforms, meeting the current clinical testing requirements for high-throughput and automated operation. Attached Figure Description

[0023] Figure 1 This is a graph showing the optimized results of adding magnetic and non-magnetic particles; Figure 2 This is a graph showing the results of optimizing the number of loop captures; Figure 3 This is a graph showing the comparison of recovery rates between single competitive capture and dual competitive capture. Detailed Implementation

[0024] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0025] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0026] Example 1: Preparation of magnetic carrier solutions for conjugating target antigens Place the entire bottle of magnetic microparticle liquid (particle size 5 nm~300 μm) in a shaker and shake for 10 minutes to fully disperse it; Take 0.2 mL of the microparticle suspension into a 2.0 mL EP tube, wash with MES (0.015 M, pH = 5.5 ± 0.5), repeat the washing step twice, and finally adjust the volume to 0.2 mL with MES. Add 20 μL of 10 mg / mL EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 20 μL of 10 mg / mL NHS (N-hydroxysuccinimide), mix well, and incubate in a shaker at 37°C for 30 min.

[0027] Magnetic separation was performed using a magnetic rack, the supernatant was removed, the mixture was washed once with MES and resuspended to 0.2 mL. 40 μg of target antigen was added, and the mixture was incubated in a shaker at 37°C for 4 h.

[0028] Magnetic separation was performed using a magnetic rack, and the supernatant was collected for measurement of the coupling rate.

[0029] The magnetic microparticles were washed twice with PBS buffer (pH 7.4±0.5), magnetically separated using a magnetic rack, and then 0.2 mL of 1% BSA (prepared with PBS buffer) was added. The mixture was then incubated in a shaker at 37 °C for 1 h to block the microparticles.

[0030] Magnetic separation was performed using a magnetic rack. The supernatant was discarded, and the mixture was brought to a final volume with PBS buffer (pH 7.4±0.5) to achieve a magnetic carrier concentration of 10 μg / mL to 40 mg / mL (solid phase carrier concentration).

[0031] Preparation of non-magnetic carrier solutions for conjugating target monoclonal antibodies Place the entire bottle of non-magnetic microparticle liquid (particle size 5 nm~500 μm) in a shaker and shake for 10 minutes to fully disperse it; Take 0.2 mL of the microparticle suspension into a 2.0 mL EP tube, wash with MES (0.015 M, pH=5.5), repeat the washing step twice, and finally adjust the volume to 0.2 mL with MES. Add 20 μL of 10 mg / mL EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 20 μL of 10 mg / mL NHS (N-hydroxysuccinimide), mix well, and incubate in a shaker at 37°C for 30 min.

[0032] Centrifuge to remove supernatant, wash once with MES, resuspend to 0.2 mL, add 40 μg monoclonal antibody, and incubate at 37°C on a shaker for 4 h.

[0033] Centrifuge, collect the supernatant, and measure the coupling rate.

[0034] Wash twice with PBS buffer (pH 7.4±0.5), centrifuge, add 0.2 mL of 1% BSA (prepared with PBS buffer), and incubate at 37 ℃ on a shaker for 1 h to block.

[0035] Centrifuge, discard the supernatant, and adjust the volume with PBS buffer (pH 7.4±0.5) to a non-magnetic carrier concentration of 10 μg / mL-1200 mg / mL (solid phase carrier concentration).

[0036] Example 2: NHS-activated agarose gel 4FF resin coupling with mAb Place the entire bottle of resin (NHS activated agarose gel 4FF resin) in a shaker and shake for 10 minutes to fully disperse it; Take 1 mL of resin suspension into a 15 mL EP tube, centrifuge at 5000 rcf for 3 min, discard the supernatant, wash once with 10 times the resin volume of medium washing buffer (1 mM hydrochloric acid, 4℃), and then wash once with 2-3 times the resin volume of coupling buffer (PBS buffer pH 7.4±0.5). ③ Add 1 mL of ligand coupling buffer (containing 1 mg / mL of the protein to be coupled, PBS buffer pH 7.4±0.5, resin volume: ligand coupling buffer volume = 1:1) to the resin, mix well, and react with shaking at room temperature for 3 h. ④ Centrifuge at 1000 g for 3 min and collect the supernatant; wash the resin with 3 times the resin volume of coupling buffer (PBS buffer pH 7.4 ± 0.5), collect the washing solution and mix it with the supernatant after coupling, and then measure the coupling rate. ⑤ Add 2 times the volume of the resin blocking solution (Tris-HCl solution, 0.1 M, pH 8.5), and block in a water bath at 28°C for 1 hour, inverting the container occasionally. ⑥ Discard the supernatant and wash the resin alternately with high pH washing buffer (Tris-HCl buffer, 0.1 M, pH 8.5±0.5) and low pH washing buffer (acetic acid-sodium acetate buffer, 0.1 M, 0.5 M NaCl, pH 4.5±0.5), repeating the cycle 3-6 times. ⑦ Adjust the coupling buffer (PBS buffer pH 7.4±0.5) to the required concentration.

[0037] Example 3: Acutelimumab capture pretreatment – ​​non-magnetic microparticles: PEGylated gold nanoparticles (carboxyl terminus) TNF-α-coated magnetic microparticles (carboxyl magnetic beads, particle size: 1 μm, concentration: 10 mg / mL, manufacturer: Nanjing Dongna Biotechnology Co., Ltd.) and adalimumab-coated non-magnetic microparticles (PEGylated spherical gold nanoparticles (carboxyl terminus), average particle size: 40 nm, concentration: 0.05 mg / mL, manufacturer: Nanjing Dongna Biotechnology Co., Ltd.) were prepared according to the method in Example 1.

[0038] Serum spiked samples were prepared (adalimumab (AbbVie, USA) 32 μg / mL, TNF-α (Jiangsu Dongkang Biopharmaceutical Technology Co., Ltd.) 15 μg / mL).

[0039] Take 200 μL of serum spiked sample into a 2.0 mL EP tube, add 200 μL of glycine-hydrochloric acid buffer (pH 2.3±0.5), mix well, and shake continuously for 10 min.

[0040] Add 40 μL of magnetic microparticles (with TNF-α conjugated to the microparticles at a concentration of 200 μg / mL) and 280 μL of non-magnetic microparticles (with adalimumab conjugated to the microparticles at a concentration of 200 μg / mL), add 100 μL of PB buffer (pH 7.4±0.5) to renature, mix well, and vortex for 20 min.

[0041] Centrifuge at 10000 g for 5 min, transfer all supernatant to a new EP tube, add 200 μL of glycine-hydrochloric acid buffer (pH 2.3±0.5) to re-acidify, mix well, and vortex for 10 min. Add 100 μL of PB buffer (pH 7.4±0.5) to the original tube to reconstitute the solid support, then add the acidified supernatant, mix well, and vortex for 20 min.

[0042] Magnetic separation is performed, and the supernatant and non-magnetic particles are discarded.

[0043] The separated magnetic particles were cleaned, and the trapped material on the magnetic particles was eluted with glycine-hydrochloric acid (pH 2.3 ± 0.5) buffer for quantitative detection.

[0044] Example 4: Capture pretreatment of infliximab – Non-magnetic microparticles: PEGylated silver (carboxyl terminus) TNF-α-coated magnetic microparticles (carboxyl magnetic beads, particle size: 1 μm, concentration: 10 mg / mL, manufacturer: Nanjing Dongna Biotechnology Co., Ltd.) and infliximab-coated non-magnetic microparticles (PEGylated spherical silver nanoparticles (carboxyl terminus), average particle size: 80 nm, concentration: 0.1 mg / mL, manufacturer: Nanjing Dongna Biotechnology Co., Ltd.) were prepared according to the method in Example 1.

[0045] Take 100 μL of serum spiked sample (infliximab (Janssen Biotech, Inc.) 100 μg / mL, TNF-α 50 μg / mL) into a 2.0 mL EP tube, add 200 μL of glycine-hydrochloric acid buffer (pH 2.3±0.5), mix well, and vortex continuously for 10 min.

[0046] Add 10 μL of magnetic microparticles (TNF-α conjugated on the microparticles at a concentration of 200 μg / mL) and 260 μL of non-magnetic microparticles (infliximab conjugated on the microparticles at a concentration of 200 μg / mL), add 100 μL of PB buffer (pH 7.4±0.5) for renaturation, mix well, and vortex for 20 min.

[0047] Centrifuge at 10000 g for 5 min, transfer all supernatant to a new EP tube, add 200 μL of glycine-hydrochloric acid buffer (pH 2.3±0.5) to re-acidify, mix well, and vortex for 10 min. Add 100 μL of PB (pH 7.4±0.5) buffer to the original tube to reconstitute the solid support, then add the acidified supernatant, mix well, and vortex for 20 min.

[0048] Magnetic separation is performed, and the supernatant and non-magnetic particles are discarded.

[0049] The separated magnetic particles were cleaned, and the trapped material on the magnetic particles was eluted with glycine-hydrochloric acid (pH 2.3 ± 0.5) buffer for quantitative detection.

[0050] Example 5: Capture Pretreatment of Ustekinumab – Non-magnetic Microparticles: Carboxylate Microspheres IL-12 or IL-23 coated magnetic microparticles (carboxylated magnetic beads, particle size: 1 μm, concentration: 10 mg / mL, manufacturer: Nanjing Dongna Biotechnology Co., Ltd.) and ustekinumab coated non-magnetic microparticles (carboxylated latex microspheres, average particle size: 400 nm, concentration: 1.05 g / cm³) were prepared according to the method in Example 1. 3 Manufacturer: Changzhou Boyi Biotechnology Co., Ltd.

[0051] Take 200 μL of serum spiked sample (ustekinumab, Janssen Biotech, 20 μg / mL), IL-12 or IL-23 (Jiangsu Dongkang Biopharmaceutical Technology Co., Ltd., 32 μg / mL) into a 2.0 mL EP tube, add 200 μL of glycine-hydrochloric acid (pH 2.3±0.5) buffer, mix well, and vortex continuously for 10 min.

[0052] Add 40 μL of magnetic microparticles (with IL-12 conjugated to the microparticles at a concentration of 200 μg / mL) and 160 μL of non-magnetic microparticles (with ustekinumab conjugated to the microparticles at a concentration of 200 μg / mL), add 100 μL of PB buffer (pH 7.4±0.5) for renaturation, mix well, and vortex for 20 min.

[0053] Centrifuge at 8000 g for 5 min, transfer all supernatant to a new EP tube, add 200 μL of glycine-hydrochloric acid buffer (pH 2.3±0.5) to re-acidify, mix well, and vortex for 10 min. Add 100 μL of PB buffer to the original tube to reconstitute the solid carrier, then add the acidified supernatant, mix well, and vortex for 20 min.

[0054] Magnetic separation is performed, and the supernatant and non-magnetic particles are discarded.

[0055] The separated magnetic particles were cleaned, and the trapped material on the magnetic particles was eluted with glycine-hydrochloric acid (pH 2.3 ± 0.5) buffer for quantitative detection.

[0056] Example 6: Pretreatment for capture of eculizumab – non-magnetic microparticles: NHS activated agarose gel 4FF Complement protein C5-coated magnetic microparticles (carboxylated magnetic beads, particle size: 1 μm, concentration: 10 mg / mL, manufacturer: Nanjing Dongna Biotechnology Co., Ltd.) were prepared according to the method of Example 1. Eculizumab-coated non-magnetic microparticles (NHS activated agarose gel 4FF, average particle size: 90 μm, Shanghai Yuanye Biotechnology Co., Ltd.) were prepared according to the method of Example 2. Take 50 μL of serum spiked sample (eculizumab, Alexion Pharmaceuticals, USA, 200 μg / mL) and complement protein C5 (Beijing Baipusai Biotechnology Co., Ltd., 550 μg / mL) into a 2.0 mL EP tube, add 200 μL of glycine-hydrochloric acid buffer (pH 2.3±0.5), mix well, and vortex continuously for 10 min.

[0057] Add 150 μL of magnetic microparticles (with complement protein C5 conjugated to the microparticles at a concentration of 200 μg / mL), 50 μL of non-magnetic microparticles (with eculizumab conjugated to the microparticles at a concentration of 1 mg / mL), add 100 μL of PB buffer for renaturation, mix well, and vortex for 20 min.

[0058] Centrifuge at 5000 g for 3 min, transfer all supernatant to a new EP tube, add 200 μL of glycine-hydrochloric acid buffer to re-acidify, mix well, and vortex for 10 min. Add 100 μL of PB buffer to the original tube to reconstitute the solid-phase carrier, then add the acidified supernatant, mix well, and vortex for 20 min.

[0059] Magnetic separation is performed, and the supernatant and non-magnetic particles are discarded.

[0060] The separated magnetic particles were cleaned, and the trapped material on the magnetic particles was eluted with glycine-hydrochloric acid (pH 2.3 ± 0.5) buffer for quantitative detection.

[0061] Example 7: Enzymatic digestion of eluent for mass spectrometry detection Add 90 μL of NH4HCO3 (300 mM, pH 8.0±0.5) solution and 10 μL of DTT (150 mM) solution to the sample to be enzymatically digested (the capture eluent of Examples 3-6), mix well, and incubate in a water bath at 75°C for 30 min.

[0062] Add 10 μL of IAA (450 mM) solution, mix well, and incubate in the dark for 30 min.

[0063] Add 60 μL of NH4HCO3 (300 mM, pH 8.0±0.5) solution and 10 μL of trypsin (1.5 mg / mL) solution, mix well, and incubate in a water bath at 37°C for 8 h.

[0064] Add 10 μL of formic acid-water (40 / 60, v / v) solution to terminate the reaction.

[0065] Add 10 μL of internal standard solution (adalimumab: APYTF*GQGTK, 1.6 μg / mL; infliximab: SINSATHYAESVK*, 22 μg / mL; ustekinumab: GLDWIGIMSPVDSDIR*, 2 μg / mL; eculizumab: LLIYGATNLADGVPSR*, 4 μg / mL).

[0066] General chromatographic conditions Chromatographic column: ProChrom C18 reversed-phase column (2.1×100 mm, 3μm, 300 Å, Spectrum Technology); Mobile phase: 0.1% formic acid aqueous solution (A) - acetonitrile (B); Gradient elution: 0~3.5 min, 5%→40%B; 3.5~3.6 min, 40%→100%B; 3.6~4.4 min, 100%→100%B; 4.4~4.5 min, 100%→5%B; 4.5~6.5 min, 5%B; Flow rate: 0.3 mL·min -1 Column temperature: 40 ℃; Injection volume: 10 μL.

[0067] General mass spectrometry conditions Electrospray ionization source with positive ion detection mode, multi-ion reaction monitoring for quantitative analysis, capillary voltage 3.0 kV, source temperature 150 ℃, desolventizing temperature 550 ℃, desolventizing gas flow rate 900 L·Hr -1 The backflush air flow rate at the conical orifice is 150 L·Hr. -1 .

[0068] MRM List

[0069] # These are quantitative ions.

[0070] Example 8: ELISA method (using adalimumab as an example) Materials and reagents ELISA plate: High-binding 96-well plate (such as Nunc MaxiSorp); Capture antibody: TNF-α; Detection antibody: HRP-labeled anti-human IgG Fc antibody (Sigma-Aldrich, St. Louis, MO, USA) (with a different epitope than the capture antibody); Coating buffer: carbonate-bicarbonate buffer (pH 9.6); Blocking solution: 1% BSA dissolved in PBST (PBS 10 mM pH 7.4±0.5, 0.05% Tween-20); Washing buffer: PBST (PBS 10 mM pH 7.4±0.5, 0.05% Tween-20); Standard diluent: PBST containing 1% BSA (simulating the neutralized sample matrix) Sample neutralization solution: 1 M Tris-HCl (pH 8.5±0.5); Developing substrate: TMB developer (Beijing Solarbio Science & Technology Co., Ltd.); Termination solution: 2 M H2SO4; Standard: Purified target mAb (known concentration); Microplate reader: Reads up to 450 nm (reference 630 nm).

[0071] Experimental steps Coating and blocking capture antibodies Add TNF-α diluted with coating buffer (2 μg / mL, 100 μL / well) to the microplate and incubate at 37°C for 2 hours, or at 4°C overnight.

[0072] Discard the liquid in the microplate, add 300 μL of PBST to each well, and wash 3 times.

[0073] Add 200 μL of blocking solution to each well and incubate at 37°C for 1 h.

[0074] Discard the liquid in the microplate, add 300 μL of PBST to each well, and wash 3 times.

[0075] Sampling and testing Take 200 μL of the sample to be tested (the eluent after the final capture in Example 3) into a new EP tube, add 400 μL of sample neutralization solution, and neutralize the pH of the system to 7.1 ± 0.3. Take 15 μL of the neutralized supernatant and dilute it with 485 μL of diluent.

[0076] Add 100 μL of the diluted sample solution to the prepared microplate wells and incubate at 37°C in a shaker for 1 hour.

[0077] Discard the liquid in the microplate, add 300 μL of PBST to each well, and wash 3 times.

[0078] Add 100 μL of HRP-labeled anti-human IgG Fc antibody (diluted to 1:5000 with blocking buffer) to each well and incubate at 37°C for 1 hour.

[0079] Discard the liquid in the microplate, add 300 μL of PBST to each well, and wash 3 times.

[0080] Add 100 μL of chromogenic substrate to each well, incubate at room temperature in the dark for 20 minutes, and observe carefully. When the solution in the well gradually turns blue, add 50 μL of H2SO4 (2 M), mix well, and terminate the reaction. The solution will then turn yellow.

[0081] Use an ELISA reader to read the OD value at 450 nm, and use a reference wavelength of 630 nm to correct the background. Perform three replicates for each sample.

[0082] Data Analysis OD was measured for each well. 450 nm Deduct OD630 nm After blank absorbance, the average OD difference of the three measurement results was calculated.

[0083] Use four-parameter logistic fitting or log-log linear regression to plot a standard curve, and substitute the average OD difference of the test samples to calculate the concentration.

[0084] Example 9: Immunoturbidimetry (using adalimumab as an example) Experimental steps TNF-α-coated nonmagnetic microparticles were prepared according to the method in Example 1.

[0085] Take 20 μL of the sample to be tested (the eluent after the final capture in Example 3), add 180 μL of PB (pH 7.4±0.5) buffer, and mix well.

[0086] Take 100 μL of the diluted sample to be tested and add it to a 96-well microplate. Then add 100 μL of TNF-α-coated non-magnetic microparticles, mix well, and repeat the process in triplicate for each sample.

[0087] After incubating the microplate at 37°C for 10 min, the absorbance was measured at a wavelength of 340 nm or 570 nm.

[0088] Data Analysis The absorbance difference is obtained by subtracting the absorbance of the blank control from the absorbance of the sample to be tested, and the average absorbance difference of the three parallel tests is calculated.

[0089] A standard curve was plotted with the concentration of the standard (μg / mL) on the x-axis and the difference in absorbance on the y-axis. A linear regression equation was then fitted, and the average difference in absorbance of the test samples was substituted into the calculated concentration.

[0090] Example 10: Optimization of the ratio of magnetic microparticles to non-magnetic microparticles (using ustekinumab as an example) 1. Optimization of the ratio of magnetic particles to non-magnetic particles Following the method in Example 1, magnetic microparticles coated with IL-12 (with an IL-12 concentration of 200 μg / mL coupled to the microparticles) and non-magnetic microparticles coated with ustekinumab (with an ustekinumab concentration of 200 μg / mL coupled to the microparticles) were prepared.

[0091] Prepare serum spiked samples (containing ustekinumab 20 μg / mL and IL-12 32 μg / mL).

[0092] Take 200 μL of serum spiked sample into a 2.0 mL EP tube, add 200 μL of glycine-hydrochloric acid buffer (pH 2.3±0.5), and shake for 10 min.

[0093] Immediately add 40 μL of magnetic microparticles, then add 40 / 80 / 120 / 160 / 200 μL of non-magnetic microparticles according to the volume ratios of magnetic microparticle solution to non-magnetic microparticle solution of 1:1, 1:2, 1:3, 1:4, and 1:5, respectively; add 100 μL of PB (pH 7.4±0.5) buffer for refolding, mix well, and shake for 20 min.

[0094] Magnetic separation was performed, the supernatant and non-magnetic particles were discarded, and 200 μL of glycine-hydrochloric acid (2.3±0.5) buffer was added to the remaining magnetic particles to elute the captured material.

[0095] The enzymatic digestion and subsequent sample injection and detection operations were performed as described in Example 7.

[0096] The recovery rate of each group was calculated with the theoretical concentration as 100%.

[0097] 2. Results Analysis like Figure 1 As shown, when the volume ratio of magnetic particles to non-magnetic particles is 1:1, the recovery rate is only 23.68%. With the increase of the proportion of non-magnetic particles, the recovery rate significantly improves, reaching 80.02% when the volume of non-magnetic particles is four times that of magnetic particles. Further increasing the proportion of non-magnetic particles leads to a stable recovery rate, maintaining within the range of 79.05%–80.02%. Therefore, the optimal experimental conditions are determined to be a volume ratio of magnetic particles to non-magnetic particles of 1:4.

[0098] Example 11: Optimization of Cycle Capture Count (Ustekinumab as an Example) 1. Optimized loop capture count Following the method in Example 1, magnetic microparticles coated with IL-12 (with IL-12 conjugated to the microparticles at a concentration of 200 μg / mL) and non-magnetic microparticles coated with ustekinumab (with ustekinumab conjugated to the microparticles at a concentration of 200 μg / mL) were prepared.

[0099] Prepare serum spiked samples (containing ustekinumab 20 μg / mL and IL-12 32 μg / mL).

[0100] Take 200 μL of serum spiked sample into a 2.0 mL EP tube, add 200 μL of glycine-HCl buffer (pH 2.3±0.5), and shake for 10 min.

[0101] Add 40 μL of magnetic microparticles and 160 μL of non-magnetic microparticles, then add 100 μL of PB (pH 7.4±0.5) buffer, mix well, and vortex for 20 min.

[0102] This is a basic capture.

[0103] To increase the number of capture cycles, the primary capture solution should be treated as follows: After primary capture, centrifuge at 8000 g for 5 min, collect the supernatant in a new EP tube, add 200 μL of glycine-hydrochloric acid buffer (pH 2.3±0.5) to acidify again, and shake for 10 min; add 100 μL of PB (pH 7.4±0.5) buffer to the original EP tube to reconstitute the solid carrier, then add the re-acidified supernatant, mix well, and shake for 20 min. This constitutes one capture cycle.

[0104] After repeating the capture cycle 1, 2, and 3 times, magnetic separation was performed. The supernatant and non-magnetic particles were discarded, and 200 μL of glycine-hydrochloric acid (pH 2.3±0.5) buffer was added to the remaining magnetic particles to elute the captured material.

[0105] The enzymatic digestion and subsequent sample injection and detection operations were performed as described in Example 7.

[0106] The recovery rate of each group was calculated with the theoretical concentration as 100%.

[0107] 2. Results Analysis Optimize the number of loop captures, and the results are as follows: Figure 2 As shown, the recovery rate was 82.00% during the initial capture; after one capture cycle, the recovery rate increased to 95.32%; however, the recovery rate showed a slight decreasing trend as the number of cycles increased further. The results indicate that one capture cycle is sufficient for efficient recovery, and excessive cycles lead to reduced recovery efficiency due to losses during the operation. Therefore, the optimal number of capture cycles is one.

[0108] Comparative Example 1: Sample comparison demonstrates the ability to monitor "inactive" monoclonal antibodies. Comparison of recovery rates between traditional methods and dual competitive capture (1) Traditional methods and dual competitive capture IL-12-coated magnetic microparticles (with IL-12 conjugated at a concentration of 200 μg / mL) and ustekinumab-coated non-magnetic microparticles (with ustekinumab conjugated at a concentration of 200 μg / mL) were prepared as described in Example 1.

[0109] Serum spiked samples were prepared (ustekinumab 20 μg / mL, IL-12 32 μg / mL).

[0110] Take 200 μL of serum spiked sample into a 2.0 mL EP tube, add 200 μL of glycine-hydrochloric acid (pH 2.3) buffer, mix well, and shake continuously for 10 min.

[0111] Add 40 μL of magnetic microparticles and 100 μL of PB (pH 7.4±0.5) buffer to form the conventional method group; add 40 μL of magnetic microparticles, 160 μL of non-magnetic microparticles and 100 μL of PB (pH 7.4±0.5) buffer to form the dual competitive capture group; mix well and vortex for 20 min.

[0112] The samples were magnetically separated using the traditional method, the supernatant was discarded, and the samples were ready for further processing.

[0113] The dual competitive capture group samples were centrifuged at 8000 g for 5 min, and the supernatant was transferred to a new EP tube. 200 μL of glycine-HCl buffer (pH 2.3±0.5) was added to acidify the sample again, and the tube was shaken for 10 min. 200 μL of PB buffer (pH 7.4±0.5) was added to the original EP tube to reconstitute the solid-phase carrier, and the acidified supernatant was added. The tube was mixed and shaken for 20 min.

[0114] Magnetic separation was performed, and the supernatant and non-magnetic particles were discarded. 200 μL of glycine-hydrochloric acid (pH 2.3±0.5) buffer was added to the remaining magnetic particles to elute the captured material.

[0115] Enzymatic hydrolysis and detection were performed as described in Example 7, and the recovery rate of each group was calculated with the theoretical concentration as 100%.

[0116] (2) Results Analysis The results are as follows Figure 3 As shown, the recovery rate of the traditional method using only magnetic microparticles for competitive capture was 13.97%; however, after introducing non-magnetic microparticles coated with ustekinumab to achieve dual competition, the recovery rate significantly increased to 94.80%. This indicates that the ustekinumab on the surface of the non-magnetic microparticles can effectively bind to interfering substances such as IL-12 or anti-drug antibodies in the sample, reducing their occupation of the target monoclonal antibody binding sites, thereby improving the capture efficiency of the magnetic microparticles for the target analyte. This strategy can reduce the consumption of target antigen while ensuring a high recovery rate.

[0117] Clinical sample testing IL-12-coated magnetic microparticles and ustekinumab-coated non-magnetic microparticles were prepared according to Example 1.

[0118] Serum samples were collected from 6 Crohn's disease patients who received ustekinumab treatment.

[0119] Traditional detection steps: Take 200 μL of serum spiked sample into a 2.0 mL EP tube, add 200 μL of glycine-hydrochloric acid (pH 2.3±0.5) buffer, mix well, and shake continuously for 10 min.

[0120] Add 40 μL of magnetic microparticles and 100 μL of PB (pH 7.4±0.5) buffer, mix well, and vortex for 20 min.

[0121] Magnetic separation was performed, the supernatant was discarded, and 200 μL of glycine-hydrochloric acid (pH 2.3 ± 0.5) buffer was added to the remaining magnetic particles to elute the captured material.

[0122] Subsequent enzymatic digestion and detection were performed as described in Example 7.

[0123] Detection steps of the present invention Take 200 μL of serum spiked sample into a 2.0 mL EP tube, add 200 μL of glycine-hydrochloric acid (pH 2.3±0.5) buffer, mix well, and shake continuously for 10 min.

[0124] Add 40 μL of magnetic microparticles, 160 μL of non-magnetic microparticles and 100 μL of PB (pH 7.4±0.5) buffer, mix well and shake for 20 min.

[0125] Centrifuge at 8000 g for 5 min, transfer all supernatant to a new EP tube, add 200 μL of glycine-hydrochloric acid (pH 2.3±0.5) buffer to re-acidify, mix well, and vortex for 10 min. Add 200 μL of PB (pH 7.4±0.5) buffer to the original tube to reconstitute the solid support, then add the re-acidified supernatant, mix well, and vortex for 20 min.

[0126] Magnetic separation was performed, and the supernatant and non-magnetic particles were discarded. 200 μL of glycine-hydrochloric acid (pH 2.3±0.5) buffer was added to the remaining magnetic particles to elute the captured material.

[0127] Subsequent enzymatic digestion and detection were performed as described in Example 7.

[0128] Results Analysis The results are shown in the table below. The drug concentrations obtained from clinical samples using traditional methods were significantly lower than those obtained using this invention. Considering the clinical manifestations of drug inresponsiveness in these patients, it is speculated that although acidic buffer solution can temporarily dissociate interfering substances such as anti-drug antibodies or IL-12 that bind to monoclonal antibody drugs in the sample, after refolding and with only the target antigen added, most of the binding sites of the target monoclonal antibody may still be reoccupied by interfering substances, leading to significantly lower detection results. However, by introducing non-magnetic microparticles coated with ustekinumab and performing two repeated capture cycles, the influence of interfering substances can be effectively reduced, allowing most of the monoclonal antibody drugs in the sample to be successfully captured by the target antigen.

[0129] Comparison of detection results between traditional methods and the present invention .

[0130] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A method for quantitative detection of monoclonal antibody drugs based on a multiple capture strategy, characterized in that, Includes the following steps: (1) Dissociation: Add acidic dissociation solution to the blood sample and shake at room temperature for 5-15 min; (2) Renaturation-capture: Then add renaturation reagent, non-magnetic carrier solution conjugated with target monoclonal antibody and magnetic carrier solution conjugated with target antigen, shake at room temperature for 10-25 min to obtain primary capture solution; (3) Cyclic capture: The primary capture solution is subjected to 1-3 cycles of capture. The operation of each cycle is as follows: centrifugation, adding acidic dissociation solution to the supernatant after centrifugation and shaking at room temperature for 5-15 min to obtain the re-dissociation solution, adding refolding reagent to the precipitate after centrifugation to obtain the refolding solution, then mixing the re-dissociation solution and the refolding solution, shaking at room temperature for 10-25 min to obtain the recapture solution; (4) Separation: The magnetic carrier of the coupled target antigen in the capture liquid obtained in step (3) is separated by magnetic force. Then the magnetic carrier of the coupled target antigen obtained by separation is cleaned and the capture on the magnetic carrier is quantitatively detected.

2. The method for quantitative detection of monoclonal antibody drugs according to claim 1, characterized in that, In step (1), the pH of the acidic dissociation solution is 1.5-3.5, and the acidic dissociation solution is selected from at least one of glycine-hydrochloric acid buffer, citrate-citrate buffer, formic acid-formate buffer, acetic acid-acetate buffer, maleic acid-maleate buffer, tartaric acid-tartrate buffer, hydrogen phthalate-hydrochloric acid buffer, phthalate buffer, benzoate buffer, succinate buffer, malate buffer, and phosphate-phosphate buffer.

3. The method for quantitative detection of monoclonal antibody drugs according to claim 1, characterized in that, In step (2), the pH of the refolding reagent is 6.0-8.5, and the refolding reagent is selected from at least one of PB, PBS, borate-borax buffer, Tris-HCl, TBS buffer, glycine buffer, HEPES buffer, MOPS buffer, PIPES buffer, BES buffer, and TES buffer.

4. The method for quantitative detection of monoclonal antibody drugs according to claim 1, characterized in that, In step (2), the non-magnetic carrier is selected from one of the following: agarose gel, cross-linked agarose gel, dextran gel, polystyrene microspheres, polyacrylate microspheres, porous glass microspheres, titanium dioxide particles, zirconium oxide particles, alumina particles, carbon nanotubes, graphene oxide, silver nanoparticles, and gold nanoparticles. The average particle size of the non-magnetic carrier is 5 nm-500 μm. The magnetic carrier is a magnetic bead with an average particle size of 5 nm-300 μm.

5. The method for quantitative detection of monoclonal antibody drugs according to claim 1, characterized in that, In step (2), when the carrier is particulate, the concentrations of the non-magnetic carrier solution conjugated with the target monoclonal antibody and the magnetic carrier solution conjugated with the target antigen are both 10 μg / mL to 1200 mg / mL.

6. The method for quantitative detection of monoclonal antibody drugs according to claim 1, characterized in that, In step (2), the target monoclonal antibody is selected from one of adalimumab, infliximab, ustekinumab, and eculizumab; the target antigen is selected from one or more of TNF-α, IL-12, IL-23, and complement protein C5.

7. The method for quantitative detection of monoclonal antibody drugs according to claim 1, characterized in that, In steps (1) and (2), the volume ratio of blood sample: acidic dissociation solution: refolding reagent: non-magnetic carrier solution conjugated with target monoclonal antibody: magnetic carrier solution conjugated with target antigen is 1:0.2-20:0.2-20:0.1-30:0.1-30.

8. The method for quantitative detection of monoclonal antibody drugs according to claim 1, characterized in that, In step (3), during each cycle of capture: the amount of acidic dissociation solution used is the same as in step (1), and the amount of refolding reagent used is the same as in step (2).

9. The method for quantitative detection of monoclonal antibody drugs according to claim 1, characterized in that, In step (4), quantitative detection is performed using mass spectrometry, ELISA, or immunoturbidimetry.

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