A method for quantitative analysis of lencanezumab concentration in blood based on liquid chromatography-tandem mass spectrometry

Abstract

The present invention relates to a method for quantitatively analyzing the concentration of lencanezumab in blood based on liquid chromatography-tandem mass spectrometry, wherein a serum sample is pretreated to obtain a characteristic peptide segment, and the characteristic peptide segment is quantified by an internal standard method combined with liquid chromatography-tandem mass spectrometry technology, thereby quantifying the content of lencanezumab in blood. The method of the present invention detects the concentration of lencanezumab with high sensitivity and high specificity, and the accuracy and precision meet the requirements. It can be used for the quantitative analysis of lencanezumab in serum, and provides a reliable detection method for monitoring the concentration of lencanezumab, thereby guiding its clinical use.

Description

Technical Field

[0001] The present invention belongs to the field of detection methods, and particularly relates to a method for quantitatively analyzing the concentration of lencanezumab in blood based on liquid chromatography-tandem mass spectrometry. Background Art

[0002] Lecanemab is a humanized immunoglobulin IgG1 monoclonal antibody that can directly antagonize aggregated soluble and insoluble amyloid β protein (Aβ) in the brain, thereby reducing Aβ deposition in the brains of Alzheimer's patients and alleviating disease progression. The U.S. Food and Drug Administration approved its marketing on January 6, 2023 for the treatment of early Alzheimer's disease (AD) accompanied by mild cognitive impairment or mild dementia.

[0003] Most of the currently reported methods for determining monoclonal antibody drugs in human blood samples are immunological methods such as enzyme-linked immunosorbent assay, which have low sensitivity, poor specificity, poor precision, and narrow analysis range compared to liquid chromatography-tandem chromatography technology, and cannot meet the actual needs of clinical testing; and there are currently no relevant publicly reported methods for lencanezumab, while this method can accurately and precisely detect lencanezumab, which is more in line with the actual clinical needs for drug concentration monitoring. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for quantitatively analyzing the concentration of lencanezumab in blood based on liquid chromatography-tandem mass spectrometry. The present invention is an accurate, rapid, highly sensitive and low-cost quantitative analysis method based on liquid chromatography-tandem chromatography technology to determine lencanezumab in blood samples.

[0005] The present invention provides a method for quantitatively analyzing the concentration of lencanezumab in blood based on liquid chromatography-tandem mass spectrometry, comprising:

[0006] The serum samples were pretreated to obtain characteristic peptides, and the characteristic peptides were quantified by the internal standard method combined with liquid chromatography-tandem mass spectrometry to quantify the content of lencanezumab in the blood;

[0007] Among them, infliximab was used as the internal standard;

[0008] The characteristic peptide segment of the lencanerumab: EGGYYYGR, as shown in SEQ ID NO.1;

[0009] The characteristic peptide segment of infliximab is: YASESMSGIPSR, as shown in SEQ ID NO.2.

[0010] Preferably, the pretreatment of the serum sample includes magnetic bead enrichment, denaturation, and enzymatic hydrolysis of the lencanezumab monoclonal antibody in the serum sample.

[0011] Preferably, the conditions of the liquid chromatography are:

[0012] Chromatographic column: PEPTIDE XB-C18 column, 100×2.1 mm, particle size 1.7 μm;

[0013] Mobile phase A: water containing 0.1% formic acid;

[0014] Mobile phase B: acetonitrile containing 0.1% formic acid;

[0015] Needle washing solution: 50% methanol;

[0016] Gradient elution was used with a flow rate of 0.3 mL / min, a column temperature of 45 °C, and an injection of 20 μL.

[0017] Preferably, the elution gradient of the gradient elution is as follows: 0-1.0min, the volume fraction of mobile phase A is maintained at 90%, and the volume fraction of mobile phase B is maintained at 10%; 1.0-7.5min, the volume fraction of mobile phase A decreases from 90% to 65%, and the volume fraction of mobile phase B increases from 10% to 35%; 7.5-8.0min, the volume fraction of mobile phase A decreases from 65% to 10%, and the volume fraction of mobile phase B increases from 35% to 90%; 8.0-10.0min, the volume fraction of mobile phase A is maintained at 10%, and the volume fraction of mobile phase B is maintained at 90%; 10.0-10.1min, the volume fraction of mobile phase A increases from 10% to 90%, and the volume fraction of mobile phase B decreases from 90% to 10%; the volume fractions of mobile phase A and mobile phase B are maintained until 12.0min and stopped.

[0018] The mass spectrometry conditions are as follows: ion source: electrospray ion source, positive ion mode; capillary voltage: 5500 V; ion source temperature (TEM): 500° C.; ion source nebulizer gas (GS1): 55 psi; ion source heating auxiliary gas (GS2): 50 psi; curtain gas (CUR): 30 psi; collision gas (CAD): 8 psi; scanning mode: MRM;

[0019] Preferably, the compound MRM scanning parameters of the mass spectrometry are as follows:

[0020] Analytes Characteristic peptides Precursor ion (m / z) Product ion (m / z) CE(V) DP(V) Lencanelumab EGGYYYGR 482.712 558.247 28.0 60 Infliximab YASESMSGIPSR 642.798 834.414 33.5 80

[0021] Preferably, the method comprises:

[0022] (1) Prepare standard working solution; prepare quality control working solution; prepare internal standard solution;

[0023] (2) Take Protein G magnetic beads according to the amount of standard working solution, quality control working solution, and sample to be tested;

[0024] (3) Add PBS solution containing Tween 20 to the corresponding magnetic beads, then add the standard working solution, quality control working solution, and the sample to be tested, and then add the internal standard solution and mix well;

[0025] (4) incubation and washing;

[0026] (5) eluting the monoclonal antibody from the magnetic beads by vortexing an aqueous solution containing formic acid;

[0027] (6) Take the eluate, add ammonium bicarbonate solution, and denature at 95°C with shaking for 30 min;

[0028] (7) Allow the solution to cool to room temperature, add pancreatic enzyme solution to the solution, and perform enzymatic hydrolysis at 37°C for 2 h;

[0029] (8) Add acetonitrile-water solution containing formic acid to the enzymatic hydrolyzate, vortex mix and centrifuge, and take the supernatant for detection by liquid chromatography-tandem mass spectrometry.

[0030] Preferably, the concentration of the standard working solution is 0.5-200 μg / mL; the concentration of the quality control working solution is 0.5-150 μg / mL.

[0031] Preferably, the concentration of the internal standard solution is 50 μg / mL; the internal standard solution is a PBS solution of infliximab.

[0032] Furthermore, the detection method also includes the step of obtaining a sample to be tested:

[0033] (1) Take 60 μL of ProteinG magnetic beads and add 500 μL of PBS solution to wash twice;

[0034] (2) Add 250 μL of PBS solution containing 0.05% Tween 20 to the washed magnetic beads, then add 20 μL of biological sample, and then add 10 μL of internal standard solution (50 μg / mL infliximab PBS solution) and mix at low speed;

[0035] (3) Incubate on a roller mixer for 1 h, wash twice with 500 μL PBS solution containing 0.1% BSA, and wash twice with deionized water;

[0036] (4) Use 100 μL of 0.25% formic acid aqueous solution to vortex for 10 min to elute the monoclonal antibody from the magnetic beads;

[0037] (5) Take 90 μL of the eluate, add 17 μL of 1 mol / L ammonium bicarbonate solution, and denature at 95°C with shaking for 30 min;

[0038] (6) Allow the solution to cool to room temperature, add 15 μL of 0.5 mg / mL pancreatic enzyme solution to the solution, and perform enzymatic hydrolysis at 37°C for 2 h;

[0039] (7) Add 23.3 μL of 50% acetonitrile-water solution (containing 10% formic acid) to the enzymatic hydrolyzate, vortex mix, centrifuge at 15,000 rpm for 5 min, and take the supernatant for analysis.

[0040] The biological sample is selected from human serum samples.

[0041] The detection method also includes the steps of obtaining standard curve samples and quality control products and performing detection: the standard curve samples and quality control products are first obtained by diluting a mixed standard stock solution S0 with a blank matrix; the mixed standard stock solution S0 contains 1500 μg / mL of lencanezumab.

[0042] The blank matrix is ​​blank human plasma.

[0043] The standard curve working solution is prepared from S0 and a blank matrix; S0 is fully mixed, and S1, S2, S3, S4, S5, S6, S7 and S8 are obtained by dissolving with the blank matrix; wherein S8 is composed of 100 μL S0 and 650 μL of blank matrix; S7 is composed of 80 μL S0 and 670 μL of blank matrix; S6 is composed of 50 μL S0 and 700 μL of blank matrix; S5 is composed of 50 μL S8 and 150 μL of blank matrix; S4 is composed of 20 μL S8 and 180 μL of blank matrix; S3 is composed of 50 μL S6 and 950 μL of blank matrix; S2 is composed of 100 μL S3 and 400 μL of blank matrix; and S1 is composed of 20 μL S3 and 180 μL of blank matrix.

[0044] The quality control product S0 is prepared with a blank matrix; S0 is fully mixed and diluted with the blank matrix to obtain HQC, MQC, LQC and LLOQ; wherein HQC is composed of 20 μL S0 and 180 μL blank matrix; MQC is composed of 20 μL HQC and 180 μL blank matrix; LQC is composed of 20 μL MQC and 180 μL blank matrix; LLOQ is composed of 50 μL LQC and 100 μL blank matrix.

[0045] Beneficial Effects

[0046] The method of the present invention is used to detect the concentration of lencanezumab with high sensitivity and specificity, and the accuracy and precision meet the requirements. It can be used for quantitative analysis of lencanezumab in serum, and provides a reliable detection method for monitoring the concentration of lencanezumab, thereby guiding its clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 MRM spectra of characteristic peptides of lencanezumab and internal standard infliximab;

[0048] Figure 2 Standard curve of characteristic peptides of lencanezumab. DETAILED DESCRIPTION

[0049] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0050] Example 1

[0051] The instruments and equipment used in the present invention include:

[0052] AB SCIEX Triple Quad 4500MD high performance liquid chromatography tandem triple quadrupole mass spectrometer (ABSCIEX, USA);

[0053] KS-060AL ultrasonic cleaning machine (Shenzhen Kejie Ultrasonic Technology Co., Ltd., China);

[0054] Eppendorf Centrifuge 5430R desktop high-speed refrigerated centrifuge (Eppendorf, Germany);

[0055] EVortex-Genie2 vortex mixer (Scientific Industries, USA);

[0056] Synergy UV ultrapure water preparation system (MerckMillipore, Germany);

[0057] MB100-4A Microplate Constant Temperature Oscillator (Shanghai Luodanlun Scientific Instrument Co., Ltd., China)

[0058] XSR204 electronic balance (METTLER TOLEDO, Switzerland).

[0059] The reagents and consumables used in the present invention include: methanol: purity ≥99.97%, LCMS grade (item number 1.06007.4008, Merk, Germany); acetonitrile: purity ≥99.97%, LCMS grade (item number 1.00030.4008, Merk, Germany); 300μl 96-well plate and sealing pad (Thermo Fisher, USA); 1.5mL centrifuge tube (Axygen, USA); pipette (Eppendorf, Germany).

[0060] The detection method of lencanezumab based on LC-MS / MS comprises the following steps:

[0061] (1) Preparation of internal standard solution

[0062] The infliximab stock solution was dissolved and diluted with PBS to a concentration of 1 mg / L. An appropriate amount of the above stock solution was accurately pipetted and diluted with PBS solution to obtain an internal standard working solution with a concentration of 50 μg / mL.

[0063] (2) Preparation of standard curve working solution

[0064] Prepared from S0 and blank matrix; S0 was thoroughly mixed, and diluted with blank matrix to obtain S1, S2, S3, S4, S5, S6, S7 and S8. The preparation method and concentration are shown in Table 1, and sealed and stored in a -80℃ refrigerator. (All units are μg / mL).

[0065] Table 1 Preparation of standard curve working solution

[0066] Working fluid Volume (μL) Dilution volume (μL) Concentration (μg / mL) mark S0 100 650 200 S8 S0 80 670 160 S7 S0 50 700 100 S6 S8 50 150 50 S5 S8 20 180 20 S4 S6 50 950 5 S3 S3 100 400 1 S2 S3 20 180 0.5 S1

[0067] (3) Preparation of quality control working solution

[0068] Table 2 Preparation of quality control working solution

[0069]

[0070]

[0071] (4) Preparation of test samples

[0072] a. Take 60μL ProteinG magnetic beads and add 500μL PBS solution to wash twice;

[0073] b. Add 250 μL of PBS solution containing 0.05% Tween 20 to the washed magnetic beads, then add 20 μL of biological sample, and then add 10 μL of internal standard solution (50 μg / mL infliximab PBS solution), and mix at low speed;

[0074] c. Incubate on a roller mixer for 1 h, wash twice with 500 μL PBS solution containing 0.1% BSA, and wash twice with deionized water;

[0075] d. Use 100 μL of 0.25% formic acid aqueous solution to vortex for 10 min to elute the monoclonal antibody from the magnetic beads;

[0076] e. Take 90 μL of the eluate, add 17 μL of 1 mol / L ammonium bicarbonate solution, and denature at 95°C with shaking for 30 min;

[0077] f. Allow the solution to cool to room temperature, add 15 μL of 0.5 mg / mL pancreatic enzyme solution to the solution, and perform enzymatic hydrolysis at 37°C for 2 h;

[0078] g. Add 23.3 μL of 50% acetonitrile-water solution (containing 10% formic acid) to the enzymatic hydrolyzate, vortex mix, centrifuge at 15,000 rpm for 5 min, and take the supernatant for analysis.

[0079] The standard products and quality control products were treated in the same way as above.

[0080] (5) Instrument testing

[0081] Chromatographic column: PEPTIDE XB-C18 column, 100×2.1 mm, particle size 1.7 μm;

[0082] Mobile phase A: water (containing 0.1% formic acid);

[0083] Mobile phase B: acetonitrile (containing 0.1% formic acid);

[0084] Needle washing solution: 50% methanol;

[0085] Gradient elution was used with a flow rate of 0.3 mL / min, a column temperature of 45 °C, and an injection of 20 μL.

[0086] The elution gradient of the gradient elution is as follows: from 0 to 1.0 min, the volume fraction of mobile phase A is maintained at 90%, and the volume fraction of mobile phase B is maintained at 10%; from 1.0 to 7.5 min, the volume fraction of mobile phase A decreases from 90% to 65%, and the volume fraction of mobile phase B increases from 10% to 35%; from 7.5 to 8.0 min, the volume fraction of mobile phase A decreases from 65% to 10%, and the volume fraction of mobile phase B increases from 35% to 90%; from 8.0 to 10.0 min, the volume fraction of mobile phase A is maintained at 10%, and the volume fraction of mobile phase B is maintained at 90%; from 10.0 to 10.1 min, the volume fraction of mobile phase A increases from 10% to 90%, and the volume fraction of mobile phase B decreases from 90% to 10%; the volume fractions of mobile phase A and mobile phase B are maintained until 12.0 min and then stopped.

[0087] Preferably, the conditions of the mass spectrometry are as follows: ion source: electrospray ion source, positive ion mode; capillary voltage: 5500 V; ion source temperature (TEM): 500° C.; ion source nebulizer gas (GS1): 55 psi; ion source heating auxiliary gas (GS2): 50 psi; curtain gas (CUR): 30 psi; collision gas (CAD): 8 psi; scanning mode: MRM.

[0088] The mass spectrometry parameters were optimized by injecting the diluted standard solution. The conditions are shown in Table 3 below:

[0089] Table 3 Quantitative ion pair information

[0090]

[0091]

[0092] like Figure 1 As shown, under this detection condition, the peak times of the lencanezumab quantitative characteristic peptide and the internal standard infliximab characteristic peptide were 1.55 min and 3.01 min, respectively.

[0093] (6) Quantitative calculation

[0094] The standard sample after detection was used to draw the standard curve equation, with the concentration ratio of the standard sample to the internal standard as the X-axis and the peak area ratio as the Y-axis, and linear regression analysis was performed to obtain the standard curve and the standard curve equation. The standard curve equation is shown in Table 4 below; Figure 2 shown.

[0095] Table 4 Standard curve equation

[0096] Curve concentration μg / mL Standard curve equation Linear coefficient r Quantitative characteristic peptides of Lencanermab 0.5-200 y=0.24517x+0.04013 0.99739

[0097] The precision and accuracy of the detection method of the present invention were tested, and the results are shown in Table 5:

[0098] Table 5 Precision and accuracy

[0099]

Claims

1. A method for quantitatively analyzing the concentration of lencanezumab in blood based on liquid chromatography-tandem mass spectrometry, comprising: The serum samples were pretreated to obtain characteristic peptides, and the characteristic peptides were quantified by the internal standard method combined with liquid chromatography-tandem mass spectrometry to quantify the content of lencanezumab in the blood; Among them, infliximab was used as the internal standard; The characteristic peptide segment of the lencanerumab: EGGYYYGR; The characteristic peptide segment of infliximab: YASESMSGIPSR; The pretreatment of the serum sample includes magnetic bead enrichment, denaturation, and enzymatic hydrolysis of lencanerumab in the serum sample; The conditions of the liquid chromatography are: Chromatographic column: PEPTIDE XB-C18 column, 100×2.1 mm, particle size 1.7 μm; Mobile phase A: water containing 0.1% formic acid; Mobile phase B: acetonitrile containing 0.1% formic acid; Needle washing solution: 50% methanol; Gradient elution was used with a flow rate of 0.3 mL / min, a column temperature of 45 °C, and an injection of 20 μL; The mass spectrometry conditions are as follows: ion source: electrospray ion source, positive ion mode; capillary voltage: 5500 V; ion source temperature (TEM): 500° C.; ion source nebulizer gas (GS1): 55 psi; ion source heating auxiliary gas (GS2): 50 psi; curtain gas (CUR): 30 psi; collision gas (CAD): 8 psi; scanning mode: MRM; The compound MRM scanning parameters of the mass spectrometry are as follows: The method comprises: (1) preparing a standard working solution; preparing a quality control working solution; preparing an internal standard solution; (2) Take Protein G magnetic beads according to the amount of standard working solution, quality control working solution, and sample to be tested; (3) Add PBS solution containing Tween 20 to the corresponding magnetic beads, then add the standard working solution, quality control working solution, and the sample to be tested, and then add the internal standard solution and mix well; (4) incubation and washing; (5) The monoclonal antibody is eluted from the magnetic beads; (6) Take the eluate, add ammonium bicarbonate solution, and oscillate to denature; (7) cooling the solution to room temperature, adding pancreatic enzyme solution to the solution for enzymatic hydrolysis; (8) Add acetonitrile-water solution containing formic acid to the enzymatic hydrolyzate, vortex mix and centrifuge, and take the supernatant for detection by liquid chromatography-tandem mass spectrometry.

2. The method according to claim 1, characterized in that: The elution gradient of the gradient elution is as follows: 0-1.0min, the volume fraction of mobile phase A is maintained at 90%, and the volume fraction of mobile phase B is maintained at 10%; 1.0-7.5min, the volume fraction of mobile phase A decreases from 90% to 65%, and the volume fraction of mobile phase B increases from 10% to 35%; 7.5-8.0min, the volume fraction of mobile phase A decreased from 65% to 10%, and the volume fraction of mobile phase B increased from 35% to 90%; From 8.0 to 10.0 min, the volume fraction of mobile phase A was maintained at 10%, and the volume fraction of mobile phase B was maintained at 90%; from 10.0 to 10.1 min, the volume fraction of mobile phase A increased from 10% to 90%, and the volume fraction of mobile phase B decreased from 90% to 10%; the volume fractions of mobile phase A and mobile phase B were maintained until 12.0 min.

3. The method according to claim 1, characterized in that: The concentration of the standard working solution is 0.5-200 μg / mL; the concentration of the quality control working solution is 0.5-150 μg / mL.

4. The method according to claim 1, characterized in that: The concentration of the internal standard solution is 50 μg / mL; the internal standard solution is a PBS solution of infliximab.

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