A method for detecting HCP antibody coverage by automated immunomagnetic bead capture combined with liquid chromatography-mass spectrometry and application thereof

Abstract

The application belongs to the field of biological product quality control detection, and particularly relates to a method for detecting HCP antibody coverage by using automatic immunomagnetic bead capture and liquid chromatography-mass spectrometry and application thereof, which adopts automatic immunomagnetic bead capture, and is suitable for replacing HCP antibody buffer with 50-200 mmol / L sodium bicarbonate solution; pre-activated magnetic beads are coupled with antibody working solution after being activated by an acidic activation solution; after being blocked by a blocking solution, the magnetic beads are incubated with HCP antigens for capture; the magnetic beads are sequentially washed with TBST solution containing 290-320 mmol / L sodium chloride, PBS solution and ultrapure water; then, elution, liquid chromatography-mass spectrometry analysis and calculation of HCP antibody coverage are performed. The application significantly improves the antibody coupling efficiency and the repeatability and accuracy of HCP coverage detection by using sodium bicarbonate buffer system, phased gradient washing and gradient strength increasing elution and the like.

Description

Technical Field

[0001] This invention belongs to the field of quality control and testing of biological products, specifically relating to an automated method and application for detecting HCP antibody coverage using immunomagnetic bead capture combined with liquid chromatography-mass spectrometry. Background Technology

[0002] Host cell proteins (HCPs) are unavoidable process-related impurities introduced into biopharmaceuticals based on cell matrix manufacturing processes. They may affect the safety and efficacy of the products, making HCP detection a critical quality control requirement in the biopharmaceutical manufacturing process.

[0003] Enzyme-linked immunosorbent assay (ELISA) is a commonly used method for detecting HCP in biopharmaceutical development and quality control. Due to its simplicity, high throughput, and controllable cost, it is widely used in biopharmaceutical process development, process validation, and routine release testing. HCP polyclonal antibodies are the core raw material determining ELISA performance, and their coverage assessment is crucial.

[0004] Currently, commonly used methods for assessing HCP antibody coverage include:

[0005] (1) Two-dimensional electrophoresis combined with protein immunoblotting (2D-WB): This method has limitations such as easy deletion of low molecular weight proteins, difficulty in analysis due to signal ambiguity, and deviation between the recognition spectrum and the actual binding behavior of ELISA due to antigen denaturation and loss of natural conformation.

[0006] (2) Immunoaffinity chromatography combined with two-dimensional differential gel electrophoresis (IAC / 2D-DIGE): Although proteins in the antigen capture stage can maintain their native conformation, they are still limited by problems such as poor separation by 2D electrophoresis, complicated operation, large antibody consumption and non-specific interference.

[0007] (3) Immunomagnetic Bead Capture Separation (IMBS) combined with liquid chromatography-tandem mass spectrometry (LC-MS / MS): IMBS technology can reflect the true HCP antibody coverage level to the greatest extent under non-denaturing conditions, similar to ELISA experiments. However, existing IMBS methods are mostly manual, which has problems such as large human error, high batch-to-batch variability, and low throughput. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide an automated immunomagnetic bead capture combined liquid chromatography-mass spectrometry (IMBS) method for analyzing host cell protein antibody coverage. This method can overcome the technical defects of existing manual IMBS methods, such as large human operation error, high batch-to-batch variability, and low throughput, and realize the standardization, automation, and high throughput of HCP antibody coverage detection.

[0009] To address the aforementioned technical problems, this invention provides an automated method for detecting HCP antibody coverage using immunomagnetic bead capture combined with liquid chromatography-mass spectrometry, comprising the following steps:

[0010] (a) Antibody solution replacement and concentration: The HCP antibody was replaced and concentrated by replacing the antibody buffer with an 80-120 mmol / L sodium bicarbonate solution and adjusting the HCP antibody concentration to 1-5 mg / mL as the antibody working solution.

[0011] (b) Antibody-magnetic bead coupling: Take the pre-activated magnetic beads, activate them with acidic activation solution, and then transfer them to the antibody working solution obtained in step (a) to covalently couple the antibody with the magnetic beads to obtain the antibody-magnetic bead complex.

[0012] (c) Blocking: The antibody-magnetic bead complex obtained in step (b) is placed in a blocking solution for blocking treatment.

[0013] (d) HCP antigen capture: The antibody-magnetic bead complex blocked in step (c) is mixed with the HCP antigen solution and incubated to allow HCP to be specifically captured by the antibody, resulting in an HCP-antibody-magnetic bead ternary complex.

[0014] (e) Washing: The ternary complex obtained in step (d) was washed sequentially with washing solution I, washing solution II, and ultrapure water. Washing solution I was a 0.05%-0.08% TBST solution containing 290-320 mmol / L sodium chloride. Washing solution II was a PBS solution.

[0015] (f) Elution and neutralization: The ternary complex washed in step (e) is eluted with acid at least twice with gradient intensity, and the eluent is collected each time; the eluents are combined and neutralized with neutralizing solution.

[0016] (g) Liquid chromatography-tandem mass spectrometry detection: The eluent after neutralization in step (f) is analyzed by liquid chromatography-tandem mass spectrometry to identify the type and quantity of HCP.

[0017] (h) Coverage calculation: Compare the mass spectrometry identification results of step (g) with the total HCP library to calculate the HCP antibody coverage.

[0018] Steps (b) to (f) are performed on an automated magnetic bead processing instrument.

[0019] The shift from manual to automated methods is naturally what the field desires, but in reality, this shift is not a simple replacement. The inventors encountered the following technical problems in automating the manual immunomagnetic bead capture method:

[0020] (1) Dispersion problem of magnetic beads in automated systems: In manual operation, operators can disperse magnetic beads by vortexing, inverting and other methods; however, in automated systems, magnetic beads rely on magnetic rods to transfer between well plates, and magnetic beads tend to accumulate at the bottom of the wells, especially at the V-shaped or U-shaped bottom of deep well plates, which reduces the effective surface area of ​​the magnetic beads and thus affects antibody conjugation efficiency and HCP capture efficiency.

[0021] (2) Non-specific adhesion between magnetic beads and plastic surfaces: During the transfer process, the magnetic beads repeatedly come into contact with the surfaces of the magnetic rod and the deep-hole plate, resulting in non-specific adhesion and a decrease in the magnetic bead recovery rate. Unlike manual operation, which uses centrifuge tubes and magnetic force to fix the magnetic beads and remove the liquid, the automated system relies on the magnetic rod to adsorb and fix the magnetic beads, and then removes the liquid by transferring the magnetic beads. The relative movement between the magnetic beads and the liquid surface exacerbates the adhesion loss.

[0022] (3) Compatibility issues of the coupling buffer system with automated processes: In the manual IMBS method, PBS is usually used as the antibody solvent. However, in the automated system, the phosphate ions in PBS may interact undesirably with the active groups on the surface of the magnetic beads, resulting in a decrease in antibody coupling efficiency and failing to meet the requirements of high coupling rate for automated high-throughput detection.

[0023] (4) The contradiction between the removal of non-specific HCPs and the retention of specific HCPs: Under automated conditions, since washing parameters cannot be flexibly adjusted based on real-time observation as in manual operation, two situations are prone to occur: First, insufficient washing leads to the residue of non-specific HCPs, resulting in an inflated coverage calculation result; second, excessive washing leads to the removal of low-affinity specific HCPs, resulting in an underestimation of the coverage calculation result. This contradiction is more prominent in automated systems.

[0024] To address the technical problems existing in the process of converting manual labor to automation, the technical solution of this invention has been systematically optimized in the following aspects:

[0025] (1) In step (a) of this invention, an 80-120 mmol / L sodium bicarbonate solution is used as the antibody solvent, and the antibody concentration is adjusted to 1-5 mg / mL. The sodium bicarbonate solution can enhance the stability of the antibody in automated operation to a certain extent; at the same time, the appropriately increased antibody concentration increases the protein density of the solution, which helps to maintain the binding efficacy of the antibody and antigen during automated oscillation.

[0026] (2) In step (e), the washing solution 1 of this invention uses a TBST solution containing 0.05%-0.08% Tween-20. Tween-20, as a nonionic surfactant, can form a protective film on the surface of the magnetic beads, reducing the hydrophobic interaction between the magnetic beads and the plastic surface and reducing nonspecific adhesion. At the same time, in step (c), the blocking solution uses a Tris buffer or phosphate buffer containing 50-200 mmol / L inorganic salts. Through the physical adsorption of BSA or Tris molecules on the surface of the magnetic beads, the sites that may cause adhesion are further blocked.

[0027] (3) In step (e), this invention designs a unique phased gradient washing scheme: first, washing is performed with TBST solution containing a high concentration of salt (290-320 mmol / L sodium chloride) to fully remove non-specifically bound HCPs; then, washing is performed with PBS solution to gradually reduce the salt concentration; finally, washing is performed with ultrapure water to completely remove residual salt ions. This gradient design ensures a decreasing trend in salt concentration, avoiding the impact of abrupt changes from high to low salt on the state of the magnetic beads, while standardized program parameters ensure batch-to-batch consistency.

[0028] (4) In step (f) of this invention, at least two gradient-intensity acidic elutions are employed, preferably three (pH 3.0, 3.0, 2.5), each lasting 15 minutes, with separate collection. The advantages are: the first two elutions use a milder pH 3.0 condition to elute HCPs bound to high-affinity antibodies; the third elution uses a stronger pH 2.5 condition to ensure complete recovery. This gradient-increasing strategy avoids HCP denaturation that may result from direct strong acid elution, while separate collection allows for evaluation of the efficiency of each elution. Furthermore, a pre-mixing step before elution of the magnetic beads is incorporated into the automated program to restore the dispersion of the magnetic beads and improve elution efficiency.

[0029] (5) In steps (a) and (b), this invention innovatively uses sodium bicarbonate solution instead of PBS as the antibody solvent. The weakly alkaline environment of sodium bicarbonate solution is conducive to the covalent coupling reaction between NHS-activated magnetic beads and antibody amino groups, and bicarbonate ions do not interfere with the active groups on the surface of the magnetic beads. Experimental results show that the antibody-magnetic bead coupling rate of the 2 mg / mL antibody working solution prepared with 100 mmol / L sodium bicarbonate can reach 98.5%, which is significantly higher than that of the PBS system.

[0030] (6) In step (a), this invention controls the antibody density on the magnetic bead surface by optimizing the antibody concentration to avoid non-specific adsorption caused by excessive density; in step (e), it gradually removes HCPs with different strengths of non-specific binding through a gradient washing process from high salt to no salt, while retaining specific binding; in step (f), it ensures that HCPs with different affinity levels are fully recovered through a gradient elution process with increasing gradient intensity. This synergistic optimization makes the coverage calculation results more accurate and reliable. This invention solves the contradiction between specific retention and non-specific removal through the synergistic design of the entire process from steps (a) to (f).

[0031] Preferably, the concentration of the sodium bicarbonate solution in step (a) is 80-120 mmol / L, and the concentration of the HCP antibody is 1.5-2.5 mg / mL. More preferably, the concentration of the sodium bicarbonate solution is 100 mmol / L, and the concentration of the HCP antibody is 2 mg / mL.

[0032] Preferably, the pre-activated magnetic beads in step (b) are NHS-activated agarose magnetic beads or toluenesulfonyl-activated magnetic beads. More preferably, the pre-activated magnetic beads are NHS-activated agarose magnetic beads.

[0033] Preferably, the acidic activation solution in step (b) is pre-cooled dilute hydrochloric acid, and the activation treatment time is 10-30 seconds.

[0034] Preferably, the coupling time between the antibody and the magnetic bead in step (b) is 1.5-3 hours.

[0035] Preferably, the blocking solution in step (c) is a Tris buffer or ethanolamine containing 50-200 mmol / L inorganic salts, with a pH of 9.0. More preferably, the blocking solution is a 100 mmol / L Tris buffer containing 100-150 mmol / L sodium chloride, with a pH of 8.0.

[0036] Preferably, the blocking process in step (c) adopts a two-step blocking method: first, the antibody-magnetic bead complex is placed in the blocking solution and mixed for 1.5-3 hours, and then transferred to fresh blocking solution and mixed for 0.5-1.5 hours.

[0037] Preferably, the concentration of the HCP antigen solution in step (d) is 2-6 mg / mL, and the incubation time is 12-20 hours.

[0038] Preferably, the number of times the washing is performed in step (e) is: washing with washing solution I 2-4 times, washing with washing solution II 2-4 times, and washing with ultrapure water 2-4 times. More preferably, washing with washing solution I 3 times, washing with washing solution II 3 times, and washing with ultrapure water 3 times.

[0039] Preferably, the acidic elution in step (f) is performed 2-4 times, using glycine-hydrochloric acid buffer as the elution solution. More preferably, the acidic elution is performed 3 times, wherein the pH of the glycine-hydrochloric acid buffer used for the first and second elutions is 2.8-3.2; and the pH of the glycine-hydrochloric acid buffer used for the third elution is 2.3-2.7.

[0040] Preferably, the neutralizing solution in step (f) is a Tris buffer solution, and the volume ratio of the elution solution to the neutralizing solution is (8-12):1. More preferably, the neutralizing solution is a 1 mol / L Tris buffer solution with a pH of 7.5-8.5.

[0041] Preferably, the liquid chromatography-tandem mass spectrometry analysis in step (g) uses a nanoliter liquid chromatography-ultra-high resolution mass spectrometry system.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] This invention introduces an automated magnetic bead processing instrument, completing antibody conjugation, blocking, antigen capture, washing, and elution steps on automated equipment. This significantly reduces human error, ensures high reproducibility and traceability of experimental data, and greatly shortens detection time. Addressing the shortcomings of automated detection, this invention optimizes the conjugation buffer system by using sodium bicarbonate solution instead of traditional PBS as the antibody solvent, significantly improving the antibody-magnetic bead conjugation efficiency to over 95%. A staged gradient washing scheme is employed, sequentially using TBST solution with high salt concentration, PBS solution, and ultrapure water, forming a gradient washing system from high salt to salt-free. This effectively removes non-specifically bound HCPs while retaining specifically bound HCPs. A multi-stage acid elution strategy with progressively increasing gradient intensity ensures that HCPs of different affinity levels are fully eluted and recovered, improving the comprehensiveness and accuracy of HCP identification coverage. Furthermore, this invention significantly improves sample preparation throughput through the parallel processing capabilities of automated processes, enabling large-scale antibody screening and multi-batch coverage assessment, and providing real-time data-driven decision support for rapid iterative optimization of downstream processes. Attached Figure Description

[0044] Figure 1 The image shows the total ion chromatogram of an HCP sample obtained using the method of this invention; wherein, A. antibody sample; B. antibody-captured HCP sample; C. blank control sample.

[0045] Figure 2 To improve the reproducibility of HCP sample proteins captured by the antibody.

[0046] Figure 3Venn diagram showing the types of antigen proteins captured by the two batches of antibodies.

[0047] Figure 4 The comparison is between automated and manual methods for antibody coverage. A. Automated antibody capture HCP versus antigen protein Venn diagram; B. Manual antibody capture HCP versus antigen protein Venn diagram; C. Automated antibody capture HCP versus antigen protein bubble diagram; D. Manual antibody capture HCP versus antigen protein bubble diagram. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0049] Example 1

[0050] This embodiment provides a method for detecting HCP antibody coverage using automated immunomagnetic bead capture combined with liquid chromatography-mass spectrometry.

[0051] 1. Instruments, reagents, and materials

[0052] 1.1 Instruments

[0053] The following instruments were used: Orbitrap nano-liquid chromatography-ultra-high resolution mass spectrometry system (equipped with a Vanquish neo nano-liquid chromatograph and a Q Exactive HF mass spectrometer), and SPD2030 SpeedVac dry evaporator, both from Thermo Fisher Scientific; NPA-16H fully automated nucleic acid extraction and purification system from Hangzhou Bori Technology Co., Ltd.; QB-128 rotary mixer from Qilin Bell Instrument Manufacturing Co., Ltd.; 5427R benchtop low-temperature centrifuge from Eppendorf AG, Germany; SpectraMax M2 multi-functional microplate reader from Meigu Molecular Instruments Co., Ltd.; and HM100-Pro shaking metal bath from Dalong Xingchuang Experimental Instruments Co., Ltd.

[0054] 1.2 Reagents and Materials

[0055] Mass spectrometry-grade trypsin (batch number 20241024C) was purchased from Beijing Shengxia Protein Technology Co., Ltd.; sodium chloride (batch number 20210701) was purchased from Jiangsu Qiangsheng Functional Chemical Co., Ltd.; phosphate-buffered saline (PBS, batch number L515FC0600), Tris buffered saline (TBS, batch number M113KA2753), and Tween-20 (batch number J323BA0013) were all from Sangon Biotech Co., Ltd.; chloroacetamide (CAA, batch number SLCL4705), sodium deoxycholate (batch number BCCK0642), trifluoroacetic acid (TFA, batch number STBK6505), sodium bicarbonate (NaHCO3, batch number BCCH3549), ammonium bicarbonate (NH4HCO3, batch number BCCH2391), and glycine (batch number WXBD9391V) were all purchased from Sigma-Aldrich. 1 mol·L -1 Tris buffer (lot number 01151684, pH 8.0), tri-(2-carboxyethyl)phosphine hydrochloride (TCEP, lot number 3272027), mass spectrometry grade methanol (lot number F25P5D204), mass spectrometry grade acetonitrile (lot number F25P57212), and mass spectrometry grade formic acid (lot number 244337) were all purchased from Thermo Fisher Scientific.

[0056] Magrose NHS agarose magnetic beads (lot number RTBXTTE0101), purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.; 3 kD ultrafiltration tubes (Merck Millipore, lot number 0000249796), SOLAμ HRP desalination plates (Waters, lot number C56130A1A), Acclaim PepMap RSLC C 18 Chromatographic column (batch number 2765105), Acclaim PepMap 100 C 18 Trap columns (batch number 2726780), Bradford protein assay kits (batch number ZK396273A), and quantitative peptide assay kits (batch number YI373426) were purchased from Thermo Fisher Scientific; 4-tube sheaths (batch number H462210002) and 7-well single-tube deep-well plates (batch number H502212101) were purchased from Hangzhou Borui Technology Co., Ltd.

[0057] The Escherichia coli expression antigen (batch number 24030701) and antibody (batch numbers 23112301 and 26041401) were sourced from Huzhou Shenke Biotechnology Co., Ltd.

[0058] The preparation process for 0.05% PBST, 0.05% TBST, and 0.05% TBST (containing 300 mmol / L sodium chloride) is as follows:

[0059] Dilute 10×PBS with ultrapure water to obtain 1×PBS, add 0.05% Tween-20, and mix well to obtain 0.05% PBST.

[0060] Dilute 20×TBS with ultrapure water to obtain 1×TBS, add 0.05% Tween-20, and mix thoroughly to obtain 0.05% TBST. Accurately weigh sodium chloride and dissolve it completely in 0.05% TBST to obtain a sodium chloride concentration of 300 mmol / L.

[0061] 2. Antibody solution replacement and concentration

[0062] Add ultrapure water to a 3 kD ultrafiltration tube, centrifuge at 14,000 g for 10 min to remove any small amount of glycerol; add HCP antibody, centrifuge at 14,000 g for 30 min; add 100 mmol / L sodium bicarbonate, centrifuge at 14,000 g for 30 min for displacement and concentration, and quantify the protein in the displaced antibody stock solution. Before antibody coupling with magnetic beads, dilute the antibody stock solution to 2 mg / mL with 100 mmol / L sodium bicarbonate aqueous solution as the antibody working solution.

[0063] 3. Antibody-magnetic bead conjugation

[0064] Take 500 μL of the well-mixed NHS-activated agarose magnetic bead suspension, use magnetic adsorption to remove the supernatant. Transfer the magnetic beads to pre-cooled 1 mM dilute hydrochloric acid and wash for 15 seconds, then immediately transfer to 0.25 mL of the antibody working solution obtained in step 2 and mix for 2 hours.

[0065] To verify the effect of sodium bicarbonate buffer on improving coupling efficiency, a control experiment was also set up in this embodiment: PBS solution and 100 mmol / L sodium bicarbonate solution were used as solvents to prepare antibody working solutions of 1 mg / mL and 2 mg / mL, respectively. After coupling with magnetic beads, the amount of residual antibody in the flow-through solution was measured, and the coupling rate was calculated. The results are shown in Table 1.

[0066] Table 1 Antibody-Magnetic Bead Coupling Efficiency

[0067] PBS 998.7±18.9 499.4±9.4 245.3±15.4 122.6±7.7 75.4%±2.0% <![CDATA[NaHCO3]]> 1071.1±40.9 535.5±20.4 163.2±5.1 81.6±2.6 84.7%±1.1% <![CDATA[NaHCO3]]> 2133.2±62.4 533.3±15.6 32.3±0.3 8.1±0.1 98.5%±0.0%

[0068] The results showed that, at the same antibody concentration, the antibody-magnetic bead conjugation rate (84.7%-98.5%) was significantly higher when using 100 mmol / L sodium bicarbonate as the solvent than when using PBS as the solvent (75.4%). When the solvent was the same (100 mmol / L sodium bicarbonate), the efficiency of antibody-magnetic bead conjugation increased from 84.7% to 98.5% as the antibody working solution concentration increased from 1 mg / mL to 2 mg / mL.

[0069] 4. Sealing treatment

[0070] In step 3, the antibody-magnetic bead complex obtained using 100 mmol / L sodium bicarbonate as solvent and an antibody working solution concentration of 2 mg / mL was transferred to 1.5 mL of 100 mmol / L Tris buffer (containing 150 mmol / L sodium chloride, pH 8.0) and mixed for 2 hours for blocking. Then, it was transferred to fresh 1.5 mL of 100 mmol / L Tris buffer blocking solution and mixed for 1 hour.

[0071] 5. HCP antigen capture

[0072] The blocked magnetic beads were washed with ultrapure water and transferred to 0.5 mL of HCP antigen at a concentration of 4 mg / mL. The mixture was then mixed for 16 hours to allow HCP to be specifically captured by the antibody.

[0073] 6. Staged gradient washing

[0074] After completing the antigen-antibody binding in step 5, wash the magnetic beads three times with 10 mL, 1.5 mL and 1.5 mL of 0.05% TBST (containing 300 mmol / L sodium chloride), then wash the magnetic beads three times with 1.5 mL of PBS, and finally wash the magnetic beads three times with 1.5 mL of ultrapure water.

[0075] The above washing process forms a gradient washing system of high salt (300 mmol / L NaCl), medium salt (approximately 137 mmol / L NaCl), and no salt, which effectively removes non-specifically bound HCPs.

[0076] To verify the effect of washing solutions on washing efficiency, a control experiment was also set up in this embodiment: washing was performed with 0.05% PBST, 0.05% TBST, and 0.05% TBST (containing 300 mmol / L sodium chloride), respectively. The effect of washing solutions in removing non-specific binding was evaluated by quantifying the protein in the final washing solution before elution. The results are shown in Table 2.

[0077] Table 2 Quantification of Non-specific Adsorbed Proteins

[0078] 0.05% PBST 15.8±1.5 23.7±2.3 0.05% TBST 10.8±0.8 16.2±1.2 <![CDATA[0.05% TBST (containing 300 mmol·L -1 sodium chloride)]]> ND ND

[0079] Note: ND (not detected) means not detected.

[0080] The results showed that the highest amount of residual protein was found when using 0.05% PBST as the washing buffer. Even after changing the washing buffer from 0.05% PBST to 0.05% TBST, a small amount of protein still remained in the final washing buffer. The protein concentration was adjusted by increasing the sodium chloride content in the 0.05% TBST to 300 mmol·L⁻¹. -1 This method is used to clean non-specifically adsorbed proteins. Under these conditions, no protein residue was detected in the final washing solution, resulting in the best washing effect.

[0081] 7. Gradient intensity elution

[0082] After washing away nonspecific HCPs, the magnetic beads were transferred to the eluent. Eluting was performed sequentially using 0.3 mL each of glycine-hydrochloric acid (pH 3.0), glycine-hydrochloric acid (pH 3.0), and glycine-hydrochloric acid (pH 2.5), mixing for 15 minutes each time. The three eluents were combined and neutralized with 1 mol / L Tris buffer (pH 8.0) at a ratio of eluent to neutralization buffer of 10:1 (v / v) to obtain the HCP sample.

[0083] By employing a gradient elution strategy with increasing intensity (pH 3.0, pH 3.0, pH 2.5), it was ensured that HCPs of different affinity levels could be fully eluted and recovered, thus improving the comprehensiveness of HCP identification.

[0084] 8. Mass spectrometry analysis

[0085] 8.1 Sample Preparation

[0086] Accurately weigh ammonium bicarbonate and prepare a 100 mmol / L solution with ultrapure water. Accurately weigh sodium deoxycholate and prepare a 100 mg / mL stock solution (10% (g / mL) of sodium deoxycholate) with a 100 mmol / L ammonium bicarbonate aqueous solution. Accurately weigh chloroacetamide and prepare a 250 mmol / L solution with a 100 mmol / L ammonium bicarbonate aqueous solution.

[0087] Add ultrapure water to a 3 kD ultrafiltration tube, centrifuge at 14,000 g for 10 min to remove a small amount of glycerol from the ultrafiltration tube; add the HCP sample obtained from the automated immunomagnetic bead capture and elution in step 7, centrifuge at 14,000 g for 30 min; add 100 mmol L... -1 The ammonium bicarbonate aqueous solution was centrifuged at 14,000 g for 30 min to displace and concentrate the solution. The HCP antigen sample was subjected to the same solution displacement process. Protein quantification of the displaced sample was performed using a Bradford protein assay kit.

[0088] Add 100 mmol / L ammonium bicarbonate aqueous solution to the sample to a final volume of 200 μL and mix well. Add 10% (g / mL) sodium deoxycholate to a final concentration of 1%. Add TCEP to a final concentration of 10 mmol / L, react at 95 ℃ for 3 min, and cool to room temperature. Add CAA to a final concentration of 25 mmol / L, react at 95 ℃ for 7 min, and cool to room temperature. Add mass spectrometry grade trypsin at an enzyme:protein ratio of 1:20 (w / w), digest overnight at 37 ℃ for 17 hours. Add 10% formic acid to a final concentration of 1%, mix well, incubate at 4 ℃ for 10 min, centrifuge at 14,000 g for 15 min, and collect the supernatant. Desalt using a SOLA μHRP desalting plate, and then evaporate the solvent. Reconstitute with 20 μL of 0.1% formic acid water, centrifuge at 14,000 g for 10 min, collect the supernatant, quantify the peptides using a quantitative peptide detection kit, and transfer to a sample vial.

[0089] 8.2 Chromatographic conditions

[0090] Samples were detected using nanoliter liquid chromatography-tandem high-resolution mass spectrometry. Acclaim PepMap RSLC C18 (75 µm × 25 cm, 2 µm) columns and Acclaim PepMap 100 C18 (100 Å, 75 µm × 15 cm, 3 µm) trap columns were used. The mobile phase consisted of 0.1% formic acid-water (A) – 0.1% formic acid-acetonitrile (B). The column temperature was 55 °C. The elution gradients are shown in Table 3.

[0091] Table 3 Liquid phase elution gradient

[0092] 0~3.0 0.7→0.3 3 3.0~80.0 0.3 3→30 80.0~85.0 0.3 30→50 85.0~85.5 0.3→0.7 50→99 85.5~90.0 0.7 99

[0093] 8.3 Mass Spectrometry Conditions

[0094] Ion source spray voltage: 2.0 kV; capillary temperature: 275 ℃. Mass spectrometry scanning acquisition mode: data-dependent acquisition (DDA). First-order resolution: 60,000 at m / z 200, scan range m / z 350~1600, AGC target: 3e 6 MaxIT: 50 ms; Secondary resolution: 15,000 at m / z 200; Scan range starting at m / z 110; AGC target: 2e 5 MaxIT: 50 ms; Dynamic exclusion: 40 s, TopN=20.

[0095] 8.4 Proteomics Data Analysis Methods

[0096] Data analysis was performed using the proteomics data processing software Proteome Discoverer 3.0. Database source: UniProt, taxonomy: Escherichia coli (E. coli, Taxonomy ID: 562).

[0097] Specific analytical parameters are as follows: Immobilized modification: carbamidomethyl (C); Enzyme: trypsin; Missed cleavage site: 2; First-order mass spectrometry error: 1×10⁻⁶ -5 Secondary mass spectrometry error: 0.02 Da.

[0098] Comparative Example 1

[0099] This comparative example provides a manual detection method, and the specific operation steps are as follows (the parts not described in detail are the same as those in Example 1).

[0100] Transfer the mixed magnetic beads to centrifuge tubes and enrich them using a magnetic rack, discarding the supernatant. Wash with 1 mmol / L hydrochloric acid, discarding the acid, and add 0.5 mg of antibody, mixing for 2 h. After antibody-conjugated magnetic beads, enrich them again using a magnetic rack, removing the antibody flow-through. Block the magnetic beads with 100 mmol / L Tris buffer for 2 h, then wash with water. Discard the supernatant, add 2 mg of antigen to each tube of magnetic beads, mix, and incubate overnight for HCP antigen incubation. After incubation, fractionate the magnetic beads using a magnetic rack, remove the antigen flow-through, and wash the beads three times with 1 mL of 0.05% PBST. After washing, elute each tube of magnetic beads sequentially with 0.3 mL each of glycine-hydrochloric acid (pH 3.0), glycine-hydrochloric acid (pH 3.0), and glycine-hydrochloric acid (pH 2.5), mixing for 15 minutes each time, and combine the eluents.

[0101] Example 1 (Automated): Validation of the antibody coverage analysis method and comparison with the Comparative Example 1 (Manual) method.

[0102] 1. Specificity

[0103] E. coli expression bacteria antibody and antigen were prepared using an automated method in Example 1 via immunomagnetic bead capture, followed by elution to obtain antibody-captured HCP. Additionally, uncoupled antibodies were taken from the magnetic beads and captured using the same method, followed by elution to obtain a blank control sample. The captured HCP and antigen samples were detected using high-resolution mass spectrometry according to step 8 of Example 1, and their characteristic total ion chromatograms are shown below. Figure 1 (In the figure, A. Antibody sample; B. Antibody-captured HCP sample; C. Blank control sample).

[0104] The results showed that the chromatographic peak shapes and responses of the antigen and captured HCP samples were good, the retention times of the identified peptides generally exhibited a normal distribution, and the deviations between the relative molecular masses of the peptides and the theoretical molecular masses were basically within 1×10⁻⁶. -5 Within this range, the high-resolution mass spectrometry mass axis is stable, and the identification results are good and reliable. Only a few chromatographic peaks were observed in the blank control sample, indicating almost no non-specific adsorption interference.

[0105] 2. Antibody capture of HCP reproducibility

[0106] E. coli expression bacteria antibody and antigen were used to prepare three independent samples in parallel using the automated method described in Example 1, via immunomagnetic bead capture. Three antibody-captured HCPs were obtained after elution. The captured HCP samples were detected using high-resolution mass spectrometry as described in step 8 of Example 1. The acquired data were analyzed using Proteome Discoverer 3.0 proteomics data processing software. The Escherichia coli protein database was selected, and proteins identified in the three parallel samples were compared using a selection criterion of at least two characteristic peptides. The results are shown in [link to results]. Figure 2 The number of HCPs captured by the antibody was identified in three independent samples: 1375, 1361, and 1368, respectively. Among them, 1222 HCPs were identified in all three samples, and the protein identification reproducibility was 82.0%. The results indicate that the proteins prepared and analyzed by this method have good reproducibility.

[0107] 3. Comparison of antibody capture proteins from different batches

[0108] Two batches of E. coli expression bacteria antibodies and a single batch of antigen were collected and samples were prepared using the automated method described in Example 1, via immunomagnetic bead capture. The HCPs captured by the antibodies were then eluted. The captured HCP samples were detected using high-resolution mass spectrometry as described in step 8 of Example 1. The collected data were analyzed using Proteome Discoverer 3.0 proteomics data processing software. The Escherichia coli protein database was selected, and proteins identified in the HCP samples captured from the two batches of antigen were compared using a screening criterion of at least two characteristic peptides. The number of HCPs captured by the two batches of antibodies was 1375 and 1367, respectively. 1271 HCPs were identified in both batches, and 86.4% of the identified proteins were identical. (See [link to relevant documentation]). Figure 3 The results showed that HCPs captured by this method were reproducible across different batches of antibodies.

[0109] 4. Comparison of antibody coverage between automated and manual methods

[0110] E. coli expression bacteria antibody and antigen were used, and samples were prepared by immunomagnetic bead capture using the automated method described in Example 1. The samples were then eluted to obtain automatically captured HCPs. Alternatively, the same antigen and antibody were used, and magnetic bead capture was performed using the manual method described in Comparative Example 1. The manually captured HCPs were then eluted to obtain manually captured HCPs. The captured HCP samples were detected using high-resolution mass spectrometry as described in step 8 of Example 1. The collected data were analyzed using Proteome Discoverer 3.0 proteomics data processing software. The Escherichia coli protein database was selected, and a minimum of 2 characteristic peptides was used as the screening criterion. The total number of proteins identified in the antibody-captured HCP samples and the number of proteins contained in the union of the proteins identified in the antibody-captured HCP samples and the antigen samples were calculated. The ratio of these two values ​​is the coverage of the antibody, as shown in Formula (1).

[0111] The antibody coverage rates of automated and manual methods were compared; the results are shown in [link to results]. Figure 4 , Figure 4 In the diagram, A and B represent the Venn diagrams of antibody-captured HCPs and antigen proteins prepared by automated and manual methods, respectively, after pretreatment and mass spectrometry analysis. The antibody coverage rates for automated and manual methods were calculated to be 86.1% and 86.7%, respectively, using formula (1). Figure 4 Figures C and D show the superimposed bubble diagrams of the identified antibody-captured HCPs and antigen proteins processed by automated and manual methods, respectively. The identified antibody-captured HCP group and antigen protein group have a high degree of overlap in isoelectric point and molecular weight space, indicating that there is little difference in the types of proteins between the two groups. Figure 4 The similarity in the overall bubble outlines of C and D indicates that the automated and manual methods have similar capabilities in capturing HCPs. These results suggest no significant difference in antibody coverage determined by the two methods.

[0112] Formula 1.

Claims

1. A method for automated detection of HCP antibody coverage using immunomagnetic bead capture combined with liquid chromatography-mass spectrometry, characterized in that, Includes the following steps: (a) Antibody solution replacement and concentration: The HCP antibody was replaced and concentrated by replacing the antibody buffer with an 80-120 mmol / L sodium bicarbonate solution and adjusting the HCP antibody concentration to 1-5 mg / mL. -1 , as an antibody working solution; (b) Antibody-magnetic bead coupling: Take the pre-activated magnetic beads, activate them with acidic activation solution, and then transfer them to the antibody working solution obtained in step (a) to covalently couple the antibody with the magnetic beads to obtain the antibody-magnetic bead complex. (c) Blocking: The antibody-magnetic bead complex obtained in step (b) is placed in a blocking solution for blocking treatment; (d) HCP antigen capture: The antibody-magnetic bead complex blocked in step (c) is mixed with the HCP antigen solution and incubated to allow HCP to be specifically captured by the antibody, resulting in an HCP-antibody-magnetic bead ternary complex. (e) Washing: The ternary complex obtained in step (d) is washed sequentially with washing solution I, washing solution II, and ultrapure water. Washing solution I is a 0.05%-0.08% TBST solution containing 290-320 mmol·L⁻¹. -1 Sodium chloride; Washing solution II is PBS solution; (f) Elution and neutralization: The ternary complex washed in step (e) is eluted at least twice with acidic gradient intensities, and the eluent is collected each time; the eluents are combined and neutralized with a neutralizing solution; (g) Liquid chromatography-tandem mass spectrometry detection: The eluent after neutralization in step (f) is analyzed by liquid chromatography-tandem mass spectrometry to identify the type and quantity of HCPs; (h) Coverage calculation: Compare the mass spectrometry identification results of step (g) with the total HCP library to calculate the HCP antibody coverage; Steps (b) to (f) are performed on an automated magnetic bead processing instrument.

2. The method according to claim 1, characterized in that, The specific operation of solvent replacement in step (a) is as follows: add ultrapure water to the ultrafiltration tube, centrifuge at 13,000 g - 14,000 g for 8 min - 10 min to remove glycerol from the ultrafiltration tube; add HCP antibody, centrifuge at 13,500 g - 14,000 g for 25 min - 30 min; then add 80-120 mmol·L⁻¹ -1 Centrifuge at 13500 g - 14000 g for 25 min - 30 min with sodium bicarbonate to complete the replacement and concentration of the antibody solution.

3. The method according to claim 1, characterized in that, The concentration of the sodium bicarbonate solution in step (a) is 100 mmol·L⁻¹. -1 The HCP antibody concentration is 2 mg / mL. -1 .

4. The method according to claim 1, characterized in that, The acidic activation solution in step (b) is pre-cooled dilute hydrochloric acid, and the activation treatment time is 10-30 seconds.

5. The method according to claim 1, characterized in that, The blocking solution in step (c) contains 100-150 mmol·L⁻¹ -1 100 mmol·L of sodium chloride -1 Tris buffer, pH 8.

0.

6. The method according to claim 1, characterized in that, The acid elution in step (f) is performed 2-4 times, using glycine-hydrochloric acid buffer as the elution solution, and the elution time is 10-20 minutes.

7. The method according to claim 6, characterized in that, The acid elution in step (f) is performed three times, wherein: The pH of the glycine-hydrochloric acid buffer used for the first and second elutions was 2.8-3.

2. The third elution used a glycine-hydrochloric acid buffer solution with a pH of 2.3-2.

7.

8. The method according to claim 6, characterized in that, The neutralizing solution in step (f) is Tris buffer, and the volume ratio of the elution solution to the neutralizing solution is (8-12):

1.

9. The application of the method according to any one of claims 1-8 in the evaluation of host cell protein antibody coverage of biological products.

10. The application according to claim 9, characterized in that, The biological products mentioned are monoclonal antibodies, recombinant proteins, vaccines, cell therapy products, or gene therapy products.

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