LC-MS / MS detection method for palbozuril monoclonal antibody drug in blood sample

Abstract

The invention discloses an LC-MS / MS detection method for a palbozuril monoclonal antibody drug in a blood sample, which comprises the following steps: enriching and purifying the palbozuril monoclonal antibody drug in serum and plasma by coupling an enrichment reagent I with magnetic beads, magnetically separating the enriched blood sample, discarding the supernatant, adding a buffer solution I for leaching, and adding a buffer solution II for diluting after leaching; the method comprises the following steps: carrying out high-temperature denaturation on a diluted blood sample, sequentially adding an internal standard and protease for enzymolysis, and then adopting a high performance liquid chromatography-tandem mass spectrometry to realize high-sensitivity rapid detection of the drug concentration of the palbozuril monoclonal antibody in the blood sample. In the pretreatment process, the steps of protein reduction and alkylation are removed, the proteolysis time is shortened, the whole process is simple, convenient and rapid, and multiple requirements of clinical detection on timeliness, accuracy and low detection cost can be met at the same time.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an LC-MS / MS detection method for a pambrolizumab monoclonal antibody drug in a blood sample. Background Art

[0002] Therapeutic monoclonal antibodies (mAb) are a class of antibody drugs with highly uniform pharmacological effects targeting a single antigen epitope obtained through molecular biological methods. They have been widely used in the targeted treatment of diseases such as tumors, autoimmune diseases, and infections.

[0003] With the in-depth research and development and clinical use of monoclonal antibody drugs, clinical problems have gradually emerged. There are significant differences in the degree of response of different individuals to monoclonal antibody drugs. The initial treatment response rate is 40% to 90%. There are also some patients who respond to the initial treatment but lose the response during the treatment, leading to disease progression. Individual differences in treatment response and loss of response are believed to be related to factors such as the concentration of monoclonal antibody drugs in the blood and the immunogenicity of the drug after use. Therefore, dose optimization based on therapeutic drug monitoring can provide an effective means for the precision treatment of monoclonal antibody drugs.

[0004] At present, there are two main methods for detecting monoclonal antibody drug concentration: liquid chromatography-mass spectrometry and immunoassay.

[0005] Immunoassay is currently a commonly used method in clinical practice, but it has many disadvantages in therapeutic drug monitoring of monoclonal antibodies: narrow quantitative range (about 2 orders of magnitude); long development time and difficulty in obtaining highly specific antibodies; cross-immunity; nonspecific binding; poor specificity; and only the free form of antibodies can be measured.

[0006] In recent years, liquid chromatography-mass spectrometry (LC-MS / MS) has gradually become one of the indispensable means for monoclonal antibody drug research due to its advantages of high separation, high specificity and accurate quantification. In May 2020, the "Pharmacy Expert Consensus on Therapeutic Drug Monitoring of Anti-tumor Biosimilars" initiated by the Chinese Pharmacological Society and the China-Japan Friendship Hospital strongly recommended the use of LC-MS for therapeutic drug monitoring. The LC-MS / MS methods reported for the determination of monoclonal antibody drug concentrations have complex pretreatment processes, time-consuming enzymatic hydrolysis, or are too expensive to meet clinical testing needs.

[0007] Pambrolizumab is a commonly used anti-tumor monoclonal antibody in clinical practice. It is increasingly used in clinical tumor treatment because of its broad-spectrum anti-tumor efficacy (melanoma, non-small cell lung cancer and dozens of different types of cancer). At present, there is a lack of efficient, simple and rapid liquid chromatography-tandem mass spectrometry method to detect its blood drug concentration.

[0008] Chinese patent document CN 113804804 A discloses "a monitoring method for rapid determination of monoclonal antibody drugs in plasma by ultra-high performance liquid chromatography tandem mass spectrometry". This method can simultaneously detect three monoclonal antibody drugs, rituximab, trastuzumab and cetuximab, in plasma. However, its pretreatment process (taking about 6 hours) and chromatographic analysis process (analysis time for each sample is 11 minutes) are relatively time-consuming, and a high-sensitivity mass spectrometer (Waters-TQS) is required for detection, so it is relatively limited in clinical application. Summary of the invention

[0009] The technical problem to be solved by the present invention is to provide an LC-MS / MS detection method for the pembrolizumab monoclonal antibody drug in a blood sample, providing a technical means for the detection of the pembrolizumab monoclonal antibody drug concentration with high accuracy, good stability, strong sensitivity, simple and fast pre-treatment process and low detection cost.

[0010] In order to solve the above technical problems, the present invention provides an LC-MS / MS detection method for a pembrolizumab monoclonal antibody drug in a blood sample, which comprises the following steps:

[0011] S1. Take a blood sample, add enrichment reagent I for enrichment, and use the pre-washed magnetic beads to capture the target monoclonal antibody drug;

[0012] S2. After the enriched blood sample is separated by magnetic absorption, the supernatant is discarded, and buffer I is added for elution. After elution, buffer II is added for dilution;

[0013] S3. The diluted blood sample of step S2 is denatured at high temperature, and then an internal standard solution and a protease are added to the sample;

[0014] S4. After the enzymatic hydrolysis is completed, an enzymatic hydrolysis stop solution is added to terminate the enzymatic hydrolysis reaction to obtain a peptide solution to be tested;

[0015] S5. The peptide solution to be tested is subjected to liquid chromatography tandem mass spectrometry (LC-MS / MS) analysis, and the content of pembrolizumab monoclonal antibody drug in the blood sample is obtained by respectively detecting the characteristic peptides of pembrolizumab.

[0016] Preferably, the blood sample is plasma or serum.

[0017] Preferably, in step S1, the enrichment reagent is PD-1 protein with a concentration of 5 to 30 μg / ml.

[0018] Preferably, in step S1, the magnetic beads used are one of Protein A, Protein G or streptavidin, and the pre-washing uses one of phosphate buffer solution or Tris buffer solution, pH 6 to 9; the enrichment conditions are: temperature 8°C to 40°C, time 10min to 1.5hr, and oscillation speed 500rpm to 2000rpm.

[0019] Preferably, in step S2, the buffer I is a phosphate buffer solution or a Tris buffer solution, with a pH of 6-9.

[0020] Preferably, in step S2, the magnetic separation is to separate the magnetic beads from the supernatant using a magnetic frame, and the supernatant is discarded;

[0021] In step S2, the buffer II is one of ammonium bicarbonate solution or Tris buffer solution, with a concentration of 20 mM to 200 mM and a pH of 6 to 8;

[0022] In step S3, the high temperature denaturation conditions are: temperature 60°C to 100°C; time 5min to 20min; shaking speed 500rpm to 2000rpm;

[0023] In step S3, the internal standard solution used contains the isotopic peptide fragment of Pablolide beads at a concentration of 0.1 to 20 μg / mL;

[0024] In step S3, the protease is one of trypsin or lysine protease (Lys-C), and the enzymatic hydrolysis conditions are: enzymatic hydrolysis ratio: 1:10-1:200; enzymatic hydrolysis time: 20min-16hr; enzymatic hydrolysis temperature: 15°C-60°C; oscillation speed: 500rpm-2000rpm;

[0025] In step S4, the enzymatic hydrolysis termination solution is one of a formic acid aqueous solution or a formic acid acetonitrile solution, and the volume percentage of formic acid is 5% to 25%.

[0026] Preferably, in step S5, the liquid chromatography conditions are:

[0027] Chromatographic column model: one of C4, C8, C12 and C18 column types;

[0028] Mobile phase A: aqueous solution containing formic acid (0.01% to 0.5%); Mobile phase B: acetonitrile or methanol solution containing formic acid (0.01% to 0.5%); Column temperature: 30°C to 60°C;

[0029] The mobile phase adopts gradient elution, and the elution procedure is: mobile phase A+mobile phase B=100%; 0-0.5min, the volume of mobile phase B is maintained at 2-20%; 3.0-3.5min, the volume of mobile phase B is increased to 20-95%; 3.5-4.5min, the volume of mobile phase B is gradually increased to 70-95%; 4.5-5.0min, the volume of mobile phase B is reduced to 2-20%;

[0030] Flow rate: 0.3~1.0mL / min;

[0031] Injection volume: 2~50μL.

[0032] Preferably, in step S5, the mass spectrometry conditions are:

[0033] Ion source: electrospray ion source; Ion mode: positive ion mode;

[0034] Monitoring mode: Multiple reaction monitoring;

[0035] The ion source temperature is 500℃~750℃;

[0036] The spray voltage of positive ions is 4500V~5500V;

[0037] The air curtain gas is 25-40 psi;

[0038] The pressure of the atomizing gas Gas1 and the auxiliary heating gas pressure Gas2 is 30-60 psi.

[0039] The LC-MS / MS detection method of the pembrolizumab monoclonal antibody drug in the blood sample of the present invention comprises the following steps: enriching and purifying the pembrolizumab monoclonal antibody drug in serum and plasma by coupling magnetic beads with an enrichment reagent I, separating the enriched blood sample by magnetic absorption, discarding the supernatant, adding buffer solution I for elution, and adding buffer solution II for dilution after elution; denaturing the diluted blood sample by high temperature, successively adding an internal standard and protease hydrolysis, optimizing the operation steps of the traditional operation (the protein reduction and alkylation steps are removed from the pretreatment process, and the protease hydrolysis time is shortened to 1 hour, the overall process is simple and fast, and all pretreatment operations can be completed in only 3 hours), and then adopting high performance liquid chromatography-tandem mass spectrometry to realize high-sensitivity rapid detection of the pembrolizumab monoclonal antibody drug concentration in the blood sample, providing a technical means with high accuracy, good stability, strong sensitivity, simple and fast pretreatment process and low detection cost for the detection of the pembrolizumab monoclonal antibody drug concentration, and meeting the multiple requirements of clinical detection for timeliness, accuracy and low detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings required for use in the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0041] Figure 1 is a total chromatogram of pembrolizumab and internal standard in an embodiment of the LC-MS / MS detection method of the pembrolizumab monoclonal antibody drug in a blood sample of the present invention;

[0042] Figure 2 is a chromatogram of pembrolizumab in an embodiment of the LC-MS / MS detection method of the pembrolizumab monoclonal antibody drug in a blood sample of the present invention;

[0043] Figure 3 is a qualitative chromatogram of pembrolizumab in an embodiment of the LC-MS / MS detection method of the pembrolizumab monoclonal antibody drug in a blood sample of the present invention;

[0044] Figure 4 is a chromatogram of the internal standard of pembrolizumab in a blood sample according to an embodiment of the LC-MS / MS detection method of the pembrolizumab monoclonal antibody drug in the present invention;

[0045] Figure 5 is a pembrolizumab standard curve of an embodiment of the LC-MS / MS detection method of the pembrolizumab monoclonal antibody drug in a blood sample of the present invention;

[0046] Figure 6 It is a flow chart of an embodiment of the LC-MS / MS detection method of the pembrolizumab monoclonal antibody drug in a blood sample of the present invention. DETAILED DESCRIPTION

[0047] The following will be combined with the accompanying drawings to clearly and completely describe the technical solutions in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] Embodiment 1

[0049] A LC-MS / MS method for detecting pembrolizumab monoclonal antibody drug in blood samples, such as Figure 6 As shown, the following steps are included:

[0050] S1. Take a blood sample, add enrichment reagent I for enrichment, and use the pre-washed magnetic beads to capture the target monoclonal antibody drug;

[0051] S2. After the enriched blood sample is separated by magnetic absorption, the supernatant is discarded, and buffer I is added for elution. After elution, buffer II is added for dilution;

[0052] S3. The diluted blood sample of step S2 is denatured at high temperature, and then an internal standard solution and a protease are added to the sample;

[0053] S4. After the enzymatic hydrolysis is completed, an enzymatic hydrolysis stop solution is added to terminate the enzymatic hydrolysis reaction to obtain a peptide solution to be tested;

[0054] S5. The peptide solution to be tested is subjected to liquid chromatography-mass spectrometry (LC-MS / MS) analysis, and the content of pembrolizumab monoclonal antibody drug in the blood sample is obtained by detecting the characteristic peptides of pembrolizumab respectively.

[0055] Preferably, the blood sample is plasma or serum.

[0056] The LC-MS / MS detection method of the monoclonal antibody drug of Pablolide in the blood sample of Example 1 is to enrich the monoclonal antibody drug of Pablolide in the purified serum and plasma by coupling magnetic beads with enrichment reagent I, and the enriched blood sample is separated by magnetic absorption, the supernatant is discarded, and buffer solution I is added for elution. After elution, buffer solution II is added for dilution; the diluted blood sample is denatured by high temperature, and internal standard and protease hydrolysis are added successively, and the operation steps of the traditional operation are optimized (the pretreatment process removes the protein reduction and alkylation steps, and the protease hydrolysis time is shortened to 1 hour. The overall process is simple and fast, and all pretreatment operations can be completed in only 3 hours). Then, high performance liquid chromatography-tandem mass spectrometry is used to achieve high sensitivity and rapid detection of the concentration of the monoclonal antibody drug of Pablolide in the blood sample, which provides a technical means with high accuracy, good stability, strong sensitivity, simple and fast pretreatment process and low detection cost for the detection of the monoclonal antibody drug concentration of Pablolide, and at the same time meets the multiple requirements of clinical detection for timeliness, accuracy and low detection cost.

[0057] Embodiment 2

[0058] Based on the LC-MS / MS detection method of the pembrolizumab monoclonal antibody drug in the blood sample of Example 1, in step S1, the enrichment reagent is PD-1 protein with a concentration of 5 to 30 μg / ml.

[0059] Preferably, in step S1, the magnetic beads used are one of Protein A or Protein G or streptavidin, and the pre-washing uses one of phosphate buffer solution or Tris buffer solution, pH 6 to 9; the enrichment conditions are: temperature 8°C to 40°C, time 10min to 1.5hr, and oscillation speed 500rpm to 2000rpm.

[0060] Preferably, in step S2, the buffer I is a phosphate buffer solution or a Tris buffer solution, with a pH of 6-9.

[0061] Preferably, in step S2, the magnetic separation is to separate the magnetic beads from the supernatant using a magnetic frame, and the supernatant is discarded.

[0062] Preferably, in step S2, the buffer II is an ammonium bicarbonate solution or a Tris buffer solution, with a concentration of 20 mM to 200 mM and a pH of 6 to 8.

[0063] Preferably, in step S3, the high temperature denaturation conditions are: temperature 60°C to 100°C; time 5min to 20min; oscillation speed 500rpm to 2000rpm.

[0064] Preferably, in step S3, the internal standard solution used contains Pabloxil isotope peptide fragments at a concentration of 0.1 to 20 μg / mL.

[0065] Preferably, in step S3, the protease is one of trypsin or lysine protease (Lys-C), and the enzymatic hydrolysis conditions are: enzymatic hydrolysis ratio: 1:10 to 1:200; enzymatic hydrolysis time: 20 min to 16 hr; enzymatic hydrolysis temperature: 15°C to 60°C; oscillation speed: 500rpm to 2000rpm.

[0066] Preferably, in step S4, the enzymatic hydrolysis termination solution is one of a formic acid aqueous solution or a formic acid acetonitrile solution, and the volume percentage of formic acid is 5% to 25%;

[0067] Programmed death 1 (PD-1), also known as CD279, is a transmembrane glycoprotein with a molecular weight of 50-55kD. It is mainly expressed on the surface of activated immune cells such as T lymphocytes, B lymphocytes, and monocytes. In a healthy state, its normal function is to downregulate unnecessary or excessive immune responses, including autoimmune responses. PD-1 is a member of the immunoglobulin (Ig) superfamily. It has been confirmed that PD-1 can negatively regulate antigen receptor signals when bound to its ligands (PD-L1 and / or PD-L2). As a negative regulator of T cells, PD-1 and its ligands PD-L1 / PD-L2 participate in the T cell immune process. A large number of studies have confirmed that PD-1 plays an important role in the process of inflammation and tumor immunity in the body. The PD-1 / PD-L1 regulatory axis plays a key role in the control of human T cell activation and the maintenance of the body's immune tolerance, and is therefore also used by tumor cells and chronic viral infections. Although normal organs can only express a small amount of PD-1, studies have shown that various tumor cells can express a large amount of this T cell inhibitor. The PD-1 / PD-L1 regulatory axis has been shown to promote T cell exhaustion, and the tumor- or virus-induced PD-1 / PD-L1 signaling pathway can also achieve escape from host immune surveillance through a variety of mechanisms, including promoting T cell inactivation, exhaustion, unresponsiveness and apoptosis, inducing Treg cell expansion, and enhancing the tumor's intrinsic ability to resist killing and apoptosis.

[0068] The LC-MS / MS detection method of the monoclonal antibody drug of Pablolide in the blood sample of Example 2 enriches and purifies the monoclonal antibody drug of Pablolide in the blood sample by PD-1 coupled magnetic beads, and optimizes the operating steps of the traditional operation (omitting the reduction and alkylation steps, the treatment process before enzymatic hydrolysis only takes 3 hours, omitting the reduction and alkylation steps, and achieving ultra-fast enzymatic hydrolysis in 1 hour).

[0069] Embodiment 3

[0070] Based on the LC-MS / MS detection method of the pembrolizumab monoclonal antibody drug in the blood sample of Example 1, in step S5, the liquid chromatography conditions are:

[0071] Chromatographic column model: one of C4, C8, C12 and C18 column types;

[0072] Mobile phase A: aqueous solution containing formic acid (0.01% to 0.5%); Mobile phase B: acetonitrile or methanol solution containing formic acid (0.01% to 0.5%); Column temperature: 30°C to 60°C;

[0073] The mobile phase adopts gradient elution, and the elution procedure is: mobile phase A+mobile phase B=100%; 0-0.5min, the volume of mobile phase B is maintained at 2-20%; 3.0-3.5min, the volume of mobile phase B is increased to 20-95%; 3.5-4.5min, the volume of mobile phase B is gradually increased to 70-95%; 4.5-5.0min, the volume of mobile phase B is reduced to 2-20%;

[0074] Flow rate: 0.3~1.0mL / min;

[0075] Injection volume: 2~50μL.

[0076] Preferably, in step S5, the mass spectrometry conditions are:

[0077] Ion source: electrospray ion source; Ion mode: positive ion mode;

[0078] Monitoring mode: Multiple reaction monitoring;

[0079] The ion source temperature is 500°C to 750°C, preferably, the ion source temperature is 600°C;

[0080] The spray voltage of positive ions is 4500V to 5500V, preferably, the spray voltage of positive ions is 5500V;

[0081] The air curtain gas is 25-40 psi, preferably, the air curtain gas is 35 psi;

[0082] The pressure of the atomizing gas Gas1 and the auxiliary heating gas pressure Gas2 is 30-60 psi, preferably 55 psi.

[0083] Preferably, in step S5, the characteristic peptide information of the target monoclonal antibody drug is shown in Table 1.

[0084] Table 1

[0085]

[0086] In high performance liquid chromatography (HPLC) technology, the performance of the chromatographic column as a core component directly affects the accuracy and efficiency of the analysis. C18, C8, and C4 columns are commonly used reversed-phase columns, each with its own characteristics, and are suitable for the analysis of different types of compounds. The C18 column is named after its octadecyl carbon chain, which has high hydrophobicity and a long carbon chain. This structure makes the C18 column have excellent separation performance in reversed-phase chromatography, especially for the analysis of compounds such as long-chain fatty acids and drug components. The alkane chain of the C8 column contains eight carbon atoms and has moderate hydrophobicity. Due to its shorter carbon chain, the retention time of the C8 column is relatively short, the peak shape is sharper, and it is suitable for the analysis of small organic compounds. The carbon chain length of the C4 column is four carbon atoms, which has strong hydrophilicity. This characteristic makes the C4 column perform well in separating polar compounds, especially for some polar acids and bases that are difficult to separate. The C18 column has the longest carbon chain and the highest hydrophobicity, and is suitable for the separation of non-polar and weakly polar compounds. The C8 column has moderate hydrophobicity and can be used for a wide range of compound analysis. The C4 column has strong hydrophilicity and is suitable for the separation of polar compounds. The C18 column has excellent separation performance and can effectively separate compounds such as long-chain fatty acids and drug components. Although the separation performance of the C8 column is slightly inferior to that of the C18 column, it performs well in the analysis of certain specific compounds. The C4 column has unique advantages in separating polar compounds, especially for some polar acids and bases that are difficult to separate. Since the carbon chain of the C18 column is relatively long, its retention time is relatively long, which makes the C18 column have higher resolution when separating complex samples. The retention time of the C8 column is moderate and is suitable for the analysis of most compounds. The retention time of the C4 column is short and is suitable for rapid analysis. The C18, C8, and C4 columns all have good compatibility and can be used in different solvent systems. However, in strong polar solvents, the separation performance of the C18 column may be affected to a certain extent. The C8 column and the C4 column have good stability in polar solvents and are suitable for a wide range of solvent systems. C18, C8, and C4 columns all have high stability and durability, and can withstand high pressure and multiple uses without being easily damaged. However, due to the longer carbon chain of the C18 column, its stability under extreme conditions may be slightly inferior to that of the C8 and C4 columns.

[0087] Embodiment 4

[0088] Based on the LC-MS / MS detection method of the pembrolizumab monoclonal antibody drug in the blood sample of Example 1, step S1 comprises:

[0089] S11. Accurately pipette 20.0 μL of enrichment reagent I (self-prepared), 200.0 μL of buffer I working solution and 5.0 μL of calibration, quality control, or blood sample to be tested into a 1.5 mL low-absorption centrifuge tube and incubate at room temperature for 1 hour;

[0090] S12. Accurately pipette 50.0 μL of magnetic beads (self-prepared) and 100.0 μL of buffer I working solution into all low-adsorption centrifuge tubes in step S11 in sequence, incubate at 30°C for 30 min, and then place under magnetic suction. When the solution becomes clear, discard the supernatant to complete the capture of the target monoclonal antibody drug;

[0091] Step S2 includes:

[0092] S21. Accurately transfer 1000.0 μL of buffer I working solution to all low-adsorption centrifuge tubes in step S12, incubate at 30°C for 2 min, place under magnetic suction, and discard the supernatant after the solution becomes clear;

[0093] S22. Accurately transfer 125.0 μL of buffer II solution to all low-adsorption centrifuge tubes in step S21, incubate at 100°C for 5 min, return to room temperature, and centrifuge at 1000 rpm for 30 sec at room temperature;

[0094] Step S3 includes:

[0095] S31. Accurately transfer 10.0 μL of internal standard and 3.0 μL of protease solution (prepared by yourself) to all low-adsorption centrifuge tubes in step S22, incubate at 37°C, 1100 rpm for 1 hr, and then centrifuge at 1000.0 rpm for 30 sec at room temperature for enzymatic hydrolysis;

[0096] Step S4 includes:

[0097] S41. Accurately transfer 7.0 μL of the enzymatic hydrolysis stop solution to all low-adsorption centrifuge tubes in step S31, and shake at 1000 rpm for 5 min at room temperature to terminate the enzymatic hydrolysis reaction;

[0098] S42. After terminating the enzyme digestion reaction, all centrifuge tubes were magnetically aspirated and allowed to stand. After the solution became clear, 120.0 μL of the supernatant was transferred to a new 96-well plate. After shaking at 1300.0 rpm for 2 min at room temperature, the plate was centrifuged at 4000.0 rpm for 5 min at 4°C to obtain a peptide solution to be tested.

[0099] Step S5 includes:

[0100] S51. Place the 96-well plate in the automatic sampler of the liquid chromatography-tandem mass spectrometer for sample injection analysis with an injection volume of 5.0 μL. The content of the pembrolizumab monoclonal antibody drug in the blood sample is obtained by detecting the characteristic peptide segments of pembrolizumab respectively.

[0101] Embodiment 5

[0102] Based on the LC-MS / MS detection method of the pembrolizumab monoclonal antibody drug in the blood sample of Example 1, in step S5, the peptide solution to be tested is analyzed by liquid chromatography-mass spectrometry (LC-MS / MS):

[0103] Liquid chromatography conditions: AB SCIEX 4500MD;

[0104] Chromatographic column model: C18;

[0105] Column temperature: 45°C;

[0106] Mobile phase A: aqueous solution containing formic acid (0.1%); Mobile phase B: acetonitrile solution containing formic acid (0.1%);

[0107] The mobile phase adopts gradient elution, and the elution procedure is: mobile phase A+mobile phase B=100%; 0-0.5min, the volume of mobile phase B is maintained at 5%; 0.5-3.0min, the volume of mobile phase B is increased from 5% to 35%; 3.0-3.5min, the volume of mobile phase B is increased from 35% to 95%; 3.5-4.5min, the volume of mobile phase B is maintained at 95%; 4.50-4.51min, the volume of mobile phase B is reduced from 95% to 5%; 4.51-5.0min, the volume of mobile phase B is maintained at 5%;

[0108] Flow rate: 0.5 mL / min;

[0109] Injection volume: 5 μL.

[0110] Mass spectrometry parameters: AB SCIEX 4500MD was used;

[0111] Ion source: electrospray ion source; ion mode: positive ion mode; monitoring mode: multiple reaction monitoring (parameters are shown in Table 2 for multiple reaction monitoring mass spectrometry parameters of pembrolizumab monoclonal antibody drug);

[0112] Table 2

[0113] Compound Precursor ion (Da) Product ion (Da) Dwell Time CE(volts) DP(volts) 1 553.400 439.200 105 50.000 21 2 553.400 667.200 105 50.000 32 3 558.400 444.200 105 50.000 21

[0114] The ion source temperature was 600°C; the spray voltage for positive ions was 5500 V;

[0115] The pressure of the curtain gas is 35psi; the pressure of the atomizing gas Gas1 is 55psi; the pressure of the auxiliary heating gas Gas2 is 55psi.

[0116] The LC-MS / MS detection method of the pembrolizumab monoclonal antibody drug in the blood sample of Example 5 is to obtain the content of the pembrolizumab monoclonal antibody drug in the blood sample by respectively detecting the characteristic peptide segments of the pembrolizumab. The total chromatogram of the monoclonal antibody and the internal standard is shown in FIG. Figure 1 As shown, the chromatogram of pembrolizumab is Figure 2 As shown in the figure, the qualitative chromatogram of pembrolizumab is as follows Figure 3 As shown, the internal standard chromatogram of pembrolizumab is as follows Figure 4 As shown; it can be seen that the chromatograms of the pembrolizumab drug and the internal standard obtained by the detection method of the pembrolizumab monoclonal antibody drug in serum of Example 5 have good peak shapes, the detection baseline is stable, and there is no obvious interference. Through the labeled concentration of each calibrator (X), the peak area ratio of each monoclonal antibody drug characteristic peptide segment in the series of calibrators to the characteristic peptide segment of the internal standard (Y), the calibration curve is drawn and the calibration curve equation is fitted to obtain the linear regression equation and correlation coefficient, as shown Figure 5 As shown in Table 3, the drug concentrations of pembrolizumab obtained by this detection method all have a good linear relationship, and the linear correlation coefficient r 2 The values ​​were all greater than >0.995.

[0117] Table 3

[0118] Serial number Monoclonal antibody drug name Linear range Linear regression equation <![CDATA[Coefficient of correlation r 2 > 1 Pembrolizumab 2–80 μg / mL y=0.9575x+0.7063 0.9984

[0119] Example 6, accuracy of detection method:

[0120] Mixed human serum and plasma (obtained by mixing a certain number of human serum samples and plasma samples to reflect the common biochemical properties of human serum and plasma and exclude the influence of individual differences, and the specific amount can be determined according to experimental requirements) are selected as routine samples, and a solvent without the substance to be tested is added to the routine sample (mixed human serum and plasma) in an amount equal to that of the recovered sample to prepare a basic sample; different amounts of the standard substance to be tested are added to the routine sample (mixed human serum and plasma) (the volume of the added standard solution does not exceed 5% of the total volume of the sample) to prepare three recovered samples with different added concentrations (low, medium and high concentrations), and the recovered samples and the basic samples are measured using the detection method of the monoclonal antibody drug of pembrolizumab in serum of Example 1. The sample processing method is the same as that of Example 4, the chromatographic conditions and mass spectrometry parameters are the same as those of Example 5, and each concentration level sample is analyzed three times in the same analysis batch, and the average is taken for calculation. The detection results of the LC-MS / MS detection method of the pembrolizumab monoclonal antibody drug in the blood samples of the present invention are shown in Tables 4 and 5. The recovery rates of the pembrolizumab monoclonal antibody drug are all in the range of 85% to 115%, which meets the technical requirements, indicating that the detection results of the detection method of the present invention are accurate and reliable.

[0121] Table 4 Pembrolizumab plasma spike recovery

[0122]

[0123] Table 5 Recovery of spiked serum of pembrolizumab

[0124]

[0125] Example 7, intra-batch and inter-batch precision of the detection method:

[0126] A standard substance to be tested is added to a mixed human serum and plasma sample (the same as the mixed human serum and plasma sample of Example 6) to prepare a quality control sample of a certain concentration: low, medium and high concentrations. Each concentration level is measured 6 times a day, and the measurement is continued for 3 days, i.e., 3 batches. The sample processing method is the same as that of Example 4, and the chromatographic conditions and mass spectrometry parameters are the same as those of Example 5. The coefficient of variation (CV%) of each concentration level is calculated, and the CV% is used to represent the corresponding intra-batch and inter-batch precision. The test results and calculation results are shown in Table 6. The intra-batch and inter-batch precision of the monoclonal antibody drug of Pambrolizumab in serum and plasma samples are both within 15.0%, indicating that the detection method of the present invention has good precision.

[0127] Table 6 Intra-batch and inter-batch precision of pembrolizumab

[0128]

[0129] Example 8, carryover contamination of the detection method:

[0130] A high concentration sample was prepared by adding a standard substance of the substance to be tested to a mixed human serum and plasma sample (the same as the mixed human serum and plasma sample in Example 3).

[0131] After the high concentration sample was injected, 5 injections of blank sample were injected continuously for evaluation; the measurement was continued for 3 days, the sample processing method was the same as in Example 4, the chromatographic conditions and mass spectrometry parameters were the same as in Example 5, and the test results were shown in Table 7. The peak area residues of the blank sample compounds of the five monoclonal antibodies were all less than 20% of the peak area of ​​the quantitative lower limit compound. It met the technical requirements (the peak area of ​​the blank solution was less than 20% of the C1 peak area), and the peak area residue of the internal standard in the blank sample was less than 5% of the peak area corresponding to the internal standard working concentration.

[0132] Table 7 Pembrolizumab carryover contamination

[0133]

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A LC-MS / MS method for detecting pembrolizumab monoclonal antibody drugs in blood samples, characterized in that: The following steps are involved: S1. Take a blood sample, add enrichment reagent I for enrichment, and use the pre-washed magnetic beads to capture the target monoclonal antibody drug; S2. After the enriched blood sample is separated by magnetic absorption, the supernatant is discarded, and buffer I is added for elution. After elution, buffer II is added for dilution; S3. The diluted blood sample of step S2 is denatured at high temperature, and then an internal standard solution and a protease are added to the sample; S4. After the enzymatic hydrolysis is completed, an enzymatic hydrolysis stop solution is added to terminate the enzymatic hydrolysis reaction to obtain a peptide solution to be tested; S5. The peptide solution to be tested is analyzed by liquid chromatography-tandem mass spectrometry, and the content of pembrolizumab monoclonal antibody drug in the blood sample is obtained by respectively detecting the characteristic peptides of pembrolizumab.

2. The LC-MS / MS detection method of pembrolizumab monoclonal antibody drug in blood samples according to claim 1, characterized in that: The blood sample is plasma or serum.

3. The LC-MS / MS detection method of pembrolizumab monoclonal antibody drug in blood samples according to claim 1, characterized in that: In step S1, the enrichment reagent is PD-1 protein with a concentration of 5 to 30 μg / ml.

4. The LC-MS / MS detection method of pembrolizumab monoclonal antibody drug in blood samples according to claim 1, characterized in that: In step S1, the magnetic beads used are one of Protein A, Protein G or streptavidin, and the pre-washing uses one of phosphate buffer solution or Tris buffer solution, pH 6 to 9; the enrichment conditions are: temperature 8°C to 40°C, time 10min to 1.5hr, and oscillation speed 500rpm to 2000rpm.

5. The LC-MS / MS detection method of pembrolizumab monoclonal antibody drug in blood samples according to claim 1, characterized in that: In step S2, the buffer I is a phosphate buffer solution or a Tris buffer solution, with a pH of 6-9.

6. The LC-MS / MS detection method of pembrolizumab monoclonal antibody drug in blood samples according to claim 1, characterized in that: In step S2, magnetic separation is to separate the magnetic beads from the supernatant using a magnetic frame, and the supernatant is discarded; In step S2, the buffer II is one of ammonium bicarbonate solution or Tris buffer solution, with a concentration of 20 mM to 200 mM and a pH of 6 to 8; In step S3, the high temperature denaturation conditions are: temperature 60°C to 100°C; time 5min to 20min; shaking speed 500rpm to 2000rpm; In step S3, the internal standard solution used contains the isotopic peptide fragment of Pablolide beads at a concentration of 0.1 to 20 μg / mL; In step S3, the protease is one of trypsin or lysine protease (Lys-C), and the enzymatic hydrolysis conditions are: enzymatic hydrolysis ratio: 1:10-1:200; enzymatic hydrolysis time: 20min-16hr; enzymatic hydrolysis temperature: 15°C-60°C; oscillation speed: 500rpm-2000rpm; In step S4, the enzymatic hydrolysis termination solution is one of a formic acid aqueous solution or a formic acid acetonitrile solution, and the volume percentage of formic acid is 5% to 25%.

7. The LC-MS / MS detection method of pembrolizumab monoclonal antibody drug in blood samples according to claim 1, characterized in that: In step S5, the liquid chromatography conditions are: Chromatographic column model: one of C4, C8, C12 and C18 column types; Mobile phase A: aqueous solution containing formic acid (0.01% to 0.5%); Mobile phase B: acetonitrile or methanol solution containing formic acid (0.01% to 0.5%); Column temperature: 30°C to 60°C; The mobile phase adopts gradient elution, and the elution program is: mobile phase A+mobile phase B=100%; 0-0.5min, the volume of mobile phase B is maintained at 2-20%; 3.0-3.5min, the volume of mobile phase B increases to 20-95%; 3.5-4.5min, the volume of mobile phase B increases to 70-95%; 4.5-5.0min, the volume of mobile phase B decreases to 2-20%; Flow rate: 0.3~1.0mL / min; Injection volume: 2~50μL.

8. The LC-MS / MS detection method of pembrolizumab monoclonal antibody drug in blood samples according to claim 1, characterized in that: In step S5, the mass spectrometry conditions are: Ion source: electrospray ion source; Ion mode: positive ion mode; Monitoring mode: Multiple reaction monitoring; The ion source temperature is 500℃~750℃; The spray voltage of positive ions is 4500V~5500V; The air curtain gas is 25-40 psi; The pressure of the atomizing gas Gas1 and the auxiliary heating gas pressure Gas2 is 30-60 psi.

9. The LC-MS / MS detection method of pembrolizumab monoclonal antibody drug in a blood sample according to claim 1, characterized in that: Step S1 includes: S11. Accurately pipette 20.0 μL of enrichment reagent I, 200.0 μL of buffer I working solution, and 5.0 μL of calibration, quality control, or blood sample to be tested into a 1.5 mL low-absorption centrifuge tube and incubate at room temperature for 1 hour; S12. Accurately pipette 50.0 μL of magnetic beads and 100.0 μL of buffer I working solution into all low-adsorption centrifuge tubes in step S11, incubate at 30°C for 30 min, and then place under magnetic suction. When the solution becomes clear, discard the supernatant to complete the capture of the target monoclonal antibody drug. Step S2 includes: S21. Accurately transfer 1000.0 μL of buffer I working solution to all low-adsorption centrifuge tubes in step S12, incubate at 30°C for 2 min, place under magnetic suction, and discard the supernatant after the solution becomes clear; S22. Accurately transfer 125.0 μL of buffer II solution to all low-adsorption centrifuge tubes in step S21, incubate at 100°C for 5 min, return to room temperature, and centrifuge at 1000 rpm for 30 sec at room temperature; Step S3 includes: S31. Accurately transfer 10.0 μL of internal standard and 3.0 μL of protease solution to all low-adsorption centrifuge tubes in step S22, incubate at 37°C, 1100 rpm for 1 hr, and then centrifuge at 1000.0 rpm for 30 sec at room temperature for enzymatic hydrolysis; Step S4 includes: S41. Accurately transfer 7.0 μL of the enzymatic hydrolysis stop solution to all low-adsorption centrifuge tubes in step S31, and shake at 1000 rpm for 5 min at room temperature to terminate the enzymatic hydrolysis reaction; S42. After terminating the enzyme digestion reaction, all centrifuge tubes were magnetically aspirated and allowed to stand. After the solution became clear, 120.0 μL of the supernatant was transferred to a new 96-well plate. After shaking at 1300.0 rpm for 2 min at room temperature, the plate was centrifuged at 4000.0 rpm for 5 min at 4°C to obtain a peptide solution to be tested. Step S5 includes: S51. Place the 96-well plate in the automatic sampler of the liquid chromatography-tandem mass spectrometer for sample injection analysis with an injection volume of 5.0 μL. The content of the pembrolizumab monoclonal antibody drug in the blood sample is obtained by detecting the characteristic peptide segments of pembrolizumab respectively.

10. The LC-MS / MS detection method of pembrolizumab monoclonal antibody drug in a blood sample according to claim 1, characterized in that: In step S5, the peptide solution to be tested is analyzed by liquid chromatography tandem mass spectrometry: Liquid chromatography conditions: AB SCIEX 4500MD; Chromatographic column model: C18; Column temperature: 45°C; Mobile phase A: aqueous solution containing formic acid (0.1%); Mobile phase B: acetonitrile solution containing formic acid (0.1%); The mobile phase adopts gradient elution, and the elution procedure is: mobile phase A+mobile phase B=100%; 0-0.5min, the volume of mobile phase B is maintained at 5%; 0.5-3.0min, the volume of mobile phase B is increased from 5% to 35%; 3.0-3.5min, the volume of mobile phase B is increased from 35% to 95%; 3.5-4.5min, the volume of mobile phase B is maintained at 95%; 4.50-4.51min, the volume of mobile phase B is reduced from 95% to 5%; 4.51-5.0min, the volume of mobile phase B is maintained at 5%; Flow rate: 0.5 mL / min; Injection volume: 5 μL. Mass spectrometry parameters: AB SCIEX 4500MD was used; Ion source: electrospray ion source; Ion mode: positive ion mode; Monitoring mode: multiple reaction monitoring; The ion source temperature was 600°C; the spray voltage for positive ions was 5500 V; The pressure of the curtain gas is 35psi; the pressure of the atomizing gas Gas1 is 55psi; the pressure of the auxiliary heating gas Gas2 is 55psi.

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