Method for determining concentration of Cindili and Carrayleigh drug in blood sample based on LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry)

Abstract

The invention discloses an LC-MS / MS. In the pretreatment process of a blood sample, two monoclonal antibody drugs in the blood sample are enriched and purified through receptor protein and magnetic beads, a target object is better enriched, and the specificity is improved; according to the detection method, the steps of protein reduction and alkylation are removed in the blood sample pretreatment process, compared with traditional operation steps, small-volume protease dosage and ultra-fast enzymolysis are achieved, the whole process is simple, convenient and fast, and the material and time cost is greatly saved; the method for detecting the drug concentration of the Xindili and Carrayleib monoclonal antibodies has the advantages of high accuracy, high stability, high sensitivity, high specificity, simple and rapid pretreatment process and small demand of blood samples.

Description

Technical Field

[0001] The present invention relates to a drug monitoring technology, and in particular to a method for measuring the drug concentrations of sindili and carrelili beads in a blood sample based on liquid chromatography tandem mass spectrometry (LC-MS / MS). Background Art

[0002] Over the past 30 years, the emergence of therapeutic monoclonal antibodies has changed the way tumors are treated. To date, the FDA has approved more than 100 monoclonal antibodies for the treatment of different diseases, including cancer, autoimmune and chronic inflammatory diseases.

[0003] Both sintilimab and carrelizumab are humanized immunoglobulin G4 (IgG4) monoclonal antibodies that can bind to the PD-1 receptor, block the immunosuppressive response mediated by the PD-1 pathway, and thus inhibit tumor growth.

[0004] As therapeutic monoclonal antibody drugs are used more and more widely in clinical practice, problems are gradually emerging. In the early stages of drug treatment, the response rate can reach 50-90%, and many patients can benefit from monoclonal antibody treatment, but the initial response of most patients will disappear over time, leading to worsening of the disease. The lack or loss of response to monoclonal antibody treatment is caused by a variety of reasons, including a lack of understanding of epigenetic, biomolecular or pathophysiological mechanisms, among which an insufficient understanding of the concentration of monoclonal antibodies in serum may be the most fundamental reason. Therefore, when using sintilimab and carrelizumab for treatment, it is necessary to comprehensively consider the specific situation of the patient, monitor the serum drug concentration at each stage, and formulate a personalized treatment plan.

[0005] At present, the detection of monoclonal antibody drug concentration includes spectroscopy, chromatography, immunoassay and liquid chromatography tandem mass spectrometry (LC-MS / MS). Although the spectroscopy method is simple to operate, the detection is easily affected by dilution and measurement conditions; the chromatographic method has relatively poor discrimination, and improper experimental conditions can easily affect the results; the immunoassay has poor specificity, and the detection results are easily interfered with, and the development time of highly specific antibodies is long and expensive; liquid chromatography tandem mass spectrometry has the advantages of high separation, high specificity and accurate quantification. The "Pharmaceutical Expert Consensus on Therapeutic Drug Monitoring of Anti-tumor Biosimilars (2020 Edition)" strongly recommends the use of LC-MS for therapeutic drug monitoring.

[0006] The LC-MS / MS method for determining the concentration of monoclonal antibodies reported so far has the characteristics of complex pre-treatment process, long enzymatic hydrolysis time, and high detection cost, which is not suitable for clinical application. Therefore, there is a lack of efficient, simple and rapid liquid chromatography tandem mass spectrometry method for simultaneously determining the concentrations of two monoclonal antibodies, Sindili and Carrelizumab.

[0007] Chinese patent document CN118032966A discloses a high-sensitivity LC-MS / MS detection method for monitoring the blood concentration of carrelizumab in human body. This method can only detect one monoclonal antibody drug in plasma, requires large amount of sample and protease, long enzyme cleavage time, and requires a high-sensitivity mass spectrometer (Citrine TM Triple Quad TM System) for detection, so it is relatively limited in clinical application. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a method for determining the drug concentrations of sindili and carrelizumab in blood samples based on liquid chromatography tandem mass spectrometry (LC-MS / MS), which has high accuracy, high stability, high sensitivity, high specificity, simple and fast pretreatment process, and small blood sample requirement.

[0009] In order to solve the above technical problems, the present invention provides a method for detecting the drug concentration of sindili and carrelili beads in a blood sample, which comprises the following steps:

[0010] S1. Take a blood sample, add buffer I and receptor protein enrichment, and use the receptor protein to specifically bind to the target monoclonal antibody drug;

[0011] S2. Add pre-washed magnetic beads and buffer I to further enrich the target in the blood sample;

[0012] S3. After the obtained enriched solution is separated by magnetic absorption, the supernatant is discarded, and buffer II is added for elution. After elution, buffer III is added for dilution;

[0013] S4. The sample diluted in step S3 is denatured at high temperature, and then an internal standard solution and protease are added for enzymatic hydrolysis;

[0014] S5. 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] S6. The peptide solution to be tested was subjected to liquid chromatography tandem mass spectrometry (LC-MS / MS) analysis, and the contents of sintilimab and carrelizumab in the blood sample were obtained by detecting the characteristic peptides of sintilimab and carrelizumab, respectively.

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

[0017] Preferably, in step S1, the buffer I is a phosphate buffer or a Tris-HCl buffer, with a pH of 6-9.

[0018] Preferably, in step S1, the receptor protein is a PD-1 protein solution or a biotinylated PD-1 protein solution with a concentration of 5 to 50 μg / mL; the enrichment conditions are: temperature: 25°C to 45°C; time: 10 min to 4.0 hr; oscillation speed: 500 rpm to 2000 rpm.

[0019] Preferably, in step S2, the magnetic beads are one of Protein A, Protein G or streptavidin magnetic beads, and the pre-wash adopts one of phosphate buffer or Tris-HCl buffer, pH 6 to 9; the enrichment conditions are: temperature: 25°C to 45°C; time: 10min to 4.0hr; oscillation speed: 500rpm to 2000rpm.

[0020] Preferably, in step S3, the magnetic separation is to separate the magnetic beads from the supernatant using a magnetic frame, and the supernatant is discarded;

[0021] In step S3, the buffer II is a phosphate buffer solution with added surfactant Triton X-100 or a phosphate buffer solution with added Tween 20, pH 6 to 9;

[0022] In step S3, the buffer III is one of bicarbonate buffer, phosphate buffer or Tris-HCl buffer, with a concentration of 20 mM to 200 mM and a pH of 6 to 9;

[0023] In step S4, the internal standard solution is a peptide segment labeled with a sintilimab isotope or a peptide segment labeled with a carrelizumab isotope, and the concentration is 0.2 to 10.0 μg / mL;

[0024] In step S4, the high temperature denaturation conditions are: temperature: 70°C to 100°C; time: 5 min to 30 min;

[0025] In step S4, the protease is trypsin, glycosaminoglycanase or lysine protease (Lys-C), and the enzymatic hydrolysis conditions are: enzymatic hydrolysis ratio: 1:20 to 1:200; enzymatic hydrolysis time: 30 min to 16 hr; enzymatic hydrolysis temperature: 25° C. to 55° C.; oscillation speed: 500 rpm to 2000 rpm;

[0026] In step S5, the enzymatic hydrolysis termination solution is a formic acid aqueous solution or a formic acid acetonitrile solution, and the volume percentage of formic acid is 5% to 30%.

[0027] Preferably, in step S6, the liquid chromatography conditions are:

[0028] Chromatographic column model: one of the following types of columns: C4, C8, C12 and C18;

[0029] 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;

[0030] The mobile phase adopts gradient elution, and the elution procedure is: mobile phase A+mobile phase B=100%; 0-1.0min, the volume of mobile phase B is maintained at 2-20%; 1.0-3.0min, the volume of mobile phase B is increased to 20-45%; 3.0-3.5min, the volume of mobile phase B is gradually increased to 70-95%; 3.5-4.5min, the volume of mobile phase B is maintained at 70-95%; 4.5-4.51min, the volume of mobile phase B is reduced to 2-20%; 4.51-5.0min, the volume of mobile phase B is maintained at 2-20%;

[0031] Flow rate: 0.3~1.0mL / min;

[0032] Injection volume: 2~50μL.

[0033] Preferably, in step S6, the mass spectrometry conditions are:

[0034] Ion source: electrospray ion source; Ion mode: positive ion mode;

[0035] Monitoring mode: Multiple reaction monitoring;

[0036] The ion source temperature is 500℃~750℃;

[0037] The spray voltage of positive ions is 4500V~5500V;

[0038] The air curtain gas is 25-40 psi;

[0039] The pressure of the atomizing gas Gas1 and the auxiliary heating gas pressure Gas2 is 30-60 psi.

[0040] The method of the present invention for determining the drug concentration of Sindili and Carrelix beads in a blood sample based on liquid chromatography tandem mass spectrometry (LC-MS / MS) comprises the following steps: in the process of pre-treatment of the blood sample, two monoclonal antibody drugs in the blood sample are enriched and purified by receptor proteins and magnetic beads, so as to better enrich the target and improve the specificity; the detection method removes the protein reduction and alkylation steps in the blood sample pre-treatment process, and compared with the traditional operation steps, a small volume of protease dosage and ultra-fast enzymatic hydrolysis (within 1 hour) are achieved, the overall process is simple and fast, and the material and time costs are greatly saved; the detection method of the drug concentration of Sindili and Carrelix beads monoclonal antibody has high accuracy, high stability, high sensitivity, high specificity, simple and fast pre-treatment process, small blood sample requirement, and only a few microliters are required to achieve a high sensitivity of 1 μg / mL, and can simultaneously perform quantitative detection on two monoclonal antibody drugs of Sindili and Carrelix beads, is efficient and simple, and can simultaneously meet the dual requirements of clinical detection for timeliness and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] 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.

[0042] Figure 1 It is a total chromatogram of two monoclonal antibodies and internal standards in an embodiment of a method for detecting the drug concentrations of sindili and carrelizumab in a blood sample of the present invention;

[0043] Figure 2 is a chromatogram of sintilimab in an embodiment of a method for detecting the drug concentrations of sintilimab and carrelizumab in a blood sample of the present invention;

[0044] Figure 3 is a chromatogram of carrelizumab in accordance with an embodiment of a method for detecting the drug concentrations of sindili and carrelizumab in a blood sample of the present invention;

[0045] Figure 4 It is an internal standard chromatogram of an embodiment of a method for detecting the drug concentration of sindili and carrelili beads in a blood sample of the present invention;

[0046] Figure 5 is a standard curve of sintilimab in an embodiment of a method for detecting the drug concentrations of sintilimab and carrelizumab in a blood sample of the present invention;

[0047] Figure 6 is a carrelizumab standard curve of an embodiment of a method for detecting the drug concentrations of sindili and carrelizumab in a blood sample of the present invention;

[0048] Figure 7 It is a flow chart of an embodiment of a method for detecting the drug concentration of sindili and carrelili beads in a blood sample of the present invention. DETAILED DESCRIPTION

[0049] 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.

[0050] Embodiment 1

[0051] A method for determining the drug concentration of sindili and carrelizumab in blood samples based on liquid chromatography tandem mass spectrometry (LC-MS / MS), such as Figure 7 As shown, the following steps are included:

[0052] S1. Take a blood sample, add buffer I and receptor protein enrichment, and use the receptor protein to specifically bind to the target monoclonal antibody drug;

[0053] S2. Add pre-washed magnetic beads and buffer I to further enrich the target in the blood sample;

[0054] S3. After the obtained enriched solution is separated by magnetic absorption, the supernatant is discarded, and buffer II is added for elution. After elution, buffer III is added for dilution;

[0055] S4. The sample diluted in step S3 is denatured at high temperature, and then an internal standard solution and protease are added for enzymatic hydrolysis;

[0056] S5. 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;

[0057] S6. The peptide solution to be tested was subjected to high performance liquid chromatography-mass spectrometry (LC-MS / MS) analysis, and the contents of sintilimab and carrelizumab in the blood sample were obtained by detecting the characteristic peptides of sintilimab and carrelizumab, respectively.

[0058] Preferably, the blood sample is plasma or serum, preferably serum.

[0059] In the method for determining the drug concentration of Sindili and Carrelix beads in a blood sample based on liquid chromatography tandem mass spectrometry (LC-MS / MS) in Example 1, during the pretreatment of the blood sample, two monoclonal antibody drugs in the blood sample are enriched and purified by receptor proteins and magnetic beads, so as to better enrich the target and improve the specificity; the detection method removes the protein reduction and alkylation steps in the blood sample pretreatment process, and compared with the traditional operation steps, a small volume of protease is used and ultra-fast enzymatic hydrolysis (within 1 hour) is achieved. The overall process is simple and fast, which greatly saves material and time costs; the detection method of the drug concentration of Sindili and Carrelix beads monoclonal antibody has high accuracy, high stability, high sensitivity, high specificity, simple and fast pretreatment process, small blood sample requirement, and only a few microliters are required to achieve a high sensitivity of 1 μg / mL. It can simultaneously perform quantitative detection on two monoclonal antibody drugs, Sindili and Carrelix beads, which is efficient and simple, and can simultaneously meet the dual requirements of clinical testing for timeliness and accuracy.

[0060] Embodiment 2

[0061] Based on the method for determining the drug concentration of sintili and carrelili beads in a blood sample based on liquid chromatography tandem mass spectrometry (LC-MS / MS) in Example 1, in step S1, the buffer I adopts phosphate buffer or Tris-HCl buffer, pH 6-9.

[0062] Preferably, in step S1, the receptor protein is a PD-1 protein solution or a biotinylated PD-1 protein solution with a concentration of 5 to 50 μg / mL; the enrichment conditions are: temperature: 25°C to 45°C; time: 10 min to 4.0 hr; oscillation speed: 500 rpm to 2000 rpm.

[0063] Preferably, in step S2, the magnetic beads are one of Protein A, Protein G or streptavidin magnetic beads, and the pre-wash adopts one of phosphate buffer or Tris-HCl buffer, pH 6 to 9; the enrichment conditions are: temperature: 25°C to 45°C; time: 10min to 4.0hr; oscillation speed: 500rpm to 2000rpm.

[0064] Preferably, in step S3, the magnetic separation is to separate the magnetic beads from the supernatant using a magnetic frame, and the supernatant is discarded.

[0065] Preferably, in step S3, the buffer II is a phosphate buffer solution to which the surfactant Triton X-100 is added or a phosphate buffer solution to which Tween 20 is added, with a pH of 6 to 9.

[0066] Preferably, in step S3, the buffer III is one of bicarbonate buffer, phosphate buffer or Tris-HCl buffer, with a concentration of 20 mM to 200 mM and a pH of 6 to 9.

[0067] Preferably, in step S4, the internal standard solution is a peptide segment labeled with a sintilimab isotope or a peptide segment labeled with a carrelizumab isotope, and the concentration is 0.2 to 10.0 μg / mL. Using isotope-labeled characteristic peptide segments of the target as internal standards can better eliminate the effects of sample preparation and instrument response, and improve the accuracy of the analysis results.

[0068] Preferably, in step S4, the high temperature denaturation conditions are: temperature: 70°C to 100°C; time: 5 min to 30 min.

[0069] Preferably, in step S4, the protease is trypsin, glycosaminoglycanase or lysine protease (Lys-C), and the enzymatic hydrolysis conditions are: enzymatic hydrolysis ratio: 1:20 to 1:200; enzymatic hydrolysis time: 30 min to 16 hr; enzymatic hydrolysis temperature: 25°C to 55°C; oscillation speed: 500rpm to 2000rpm.

[0070] Preferably, in step S5, the enzymatic hydrolysis termination solution is a formic acid aqueous solution or a formic acid acetonitrile solution, and the volume percentage of formic acid is 5% to 30%.

[0071] Embodiment 3

[0072] Based on the method for determining the drug concentration of Sindili and Carrelix beads in a blood sample based on liquid chromatography tandem mass spectrometry in Example 1, in step S6, the liquid chromatography conditions are:

[0073] Chromatographic column model: one of the following types of columns: C4, C8, C12 and C18;

[0074] 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;

[0075] The mobile phase adopts gradient elution, and the elution procedure is: mobile phase A+mobile phase B=100%; 0-1.0min, the volume of mobile phase B is maintained at 2-20%; 1.0-3.0min, the volume of mobile phase B is increased to 20-45%; 3.0-3.5min, the volume of mobile phase B is gradually increased to 70-95%; 3.5-4.5min, the volume of mobile phase B is maintained at 70-95%; 4.5-4.51min, the volume of mobile phase B is reduced to 2-20%; 4.51-5.0min, the volume of mobile phase B is maintained at 2-20%;

[0076] Flow rate: 0.3~1.0mL / min;

[0077] Injection volume: 2~50μL.

[0078] In step S6, the mass spectrometry conditions are:

[0079] Ion source: electrospray ion source; Ion mode: positive ion mode;

[0080] Monitoring mode: Multiple reaction monitoring;

[0081] The ion source temperature is 500° C. to 750° C., preferably, the ion source temperature is 500° C.;

[0082] The spray voltage of positive ions is 4500V to 5500V, preferably, the spray voltage of positive ions is 5500V;

[0083] The air curtain gas is 25-40 psi, preferably, the air curtain gas is 30 psi;

[0084] The pressure of the atomizing gas Gas1 and the auxiliary heating gas pressure Gas2 is 30-60 psi, preferably 55 psi.

[0085] In step S6, the characteristic peptide information of the target monoclonal antibody drug is shown in Table 1.

[0086] Table 1

[0087]

[0088] 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.

[0089] Embodiment 4

[0090] Based on the method for determining the drug concentration of sindili and carrelizumab in a blood sample based on liquid chromatography tandem mass spectrometry (LC-MS / MS) in Example 1, step S1 comprises:

[0091] S11. Take 8 μL of serum sample into the first 1.5 mL centrifuge tube, add 200 μL of buffer I and 20 μL of receptor protein solution for enrichment, and shake at 800 rpm for 1 hr at room temperature.

[0092] Preferably, step S2 includes:

[0093] S21. Take a certain volume of magnetic beads (calculated as 10.0 μL / sample) to a 1.5 mL second centrifuge tube, add 1000 μL buffer I; separate by magnetic attraction, and discard the supernatant; repeat the washing twice to complete the pre-washing of the magnetic beads; take a certain volume of buffer I (calculated as 50.0 μL / sample) and add it to the second centrifuge tube;

[0094] S22. Take 50 μL of magnetic beads and 150 μL of buffer I after pre-washing in step S21, add them to the first centrifuge tube, and shake at 1000 rpm for 30 min at room temperature; separate by magnetic attraction, and discard the supernatant.

[0095] Preferably, step S3 includes:

[0096] S31. Add 1000 μL of buffer II to the first centrifuge tube to wash away unbound proteins and other interfering substances, separate by magnetic attraction, and discard the supernatant;

[0097] S32. Add 125.0 μL of buffer III to the first centrifuge tube for dilution.

[0098] Preferably, step S4 includes:

[0099] S41. The sample diluted in step S3 is placed under high temperature conditions for 5 minutes to complete high temperature denaturation;

[0100] S42. After the first centrifuge tube returns to room temperature, add 10 μL of internal standard solution and 1.5 μg of protease, and shake at 1100 rpm for 1 hr at 37°C.

[0101] Preferably, step S5 includes:

[0102] S51. Add the enzymatic hydrolysis stop solution to the first centrifuge tube in step S42, and shake at 1000 rpm for 2 min; collect the supernatant into a 96-well plate through magnetic separation to obtain a peptide solution to be tested, and wait for analysis on the machine.

[0103] Embodiment 5

[0104] Based on the method for determining the drug concentration of sindili and carrelili beads in a blood sample based on liquid chromatography tandem mass spectrometry (LC-MS / MS) in Example 1, in step S6, the peptide solution to be tested is subjected to liquid chromatography tandem mass spectrometry (LC-MS / MS) analysis:

[0105] Liquid chromatography conditions: 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 program is: mobile phase A+mobile phase B=100%; 0-1.0min, the volume of mobile phase B is maintained at 15%; 1.0-3.0min, the volume of mobile phase B is increased from 10% to 38%; 3.0-3.5min, the volume of mobile phase B is increased from 38% 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 15%; 4.51-5.0min, the volume of mobile phase B is maintained at 15%; flow rate: 0.6mL / min; injection volume: 10μL;

[0106] Mass spectrometry parameters: SCIEX 4500MD was used; ion source: electrospray ion source; ion mode: positive ion mode; monitoring mode: multiple reaction monitoring (parameters are shown in Table 2, multiple reaction monitoring mass spectrometry parameters of two monoclonal antibody drugs); ion source temperature was 500°C; the spray voltage of the positive ion was 5500V; the curtain gas was 30psi; the nebulizer gas Gas1 pressure was 55psi; the auxiliary heating gas pressure Gas2 pressure was 55psi.

[0107] Table 2

[0108]

[0109] Example 5 is a method for determining the drug concentration of Sindili and Carrelizumab in a blood sample based on liquid chromatography tandem mass spectrometry (LC-MS / MS). The total chromatograms of the two monoclonal antibodies and the internal standard are as follows: Figure 1 As shown; the chromatogram of sintilimab is as shown Figure 2 As shown; the chromatogram of carrelizumab is as shown Figure 3 The internal standard chromatogram is shown in Figure 4As shown, it can be seen that the chromatograms of the two monoclonal antibody drugs and the internal standard obtained by the detection method of the drug concentration of sindili and carrelizumab in the blood sample of the present invention have good peak shapes, the detection baseline is stable, and there is no obvious interference. Through the marked 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 in Figure 5 , Figure 6 Table 3 shows the linear regression equations and correlation coefficients of the calibration curves of the two monoclonal antibody drugs. In this detection method, the two monoclonal antibody drugs have a good linear relationship in the range of 1 to 50 μg / mL, and the linear correlation coefficient r 2 The values ​​were all greater than >0.99.

[0110] Table 3

[0111] Serial number Monoclonal antibody drug name Linear range Linear regression equation <![CDATA[Coefficient of correlation r 2 > 1 Sintilimab 1–50 μg / mL y=0.9724x+0.2964 0.9995 2 Camrelizumab 1–50 μg / mL y=0.904x+0.9094 0.9969

[0112] Example 6, Accuracy of the Detection Method

[0113] Mixed human serum (obtained by mixing a certain number of human serum samples, used to extract the common biochemical properties of human serum and exclude the influence of individual differences, and the specific amount can be determined according to experimental requirements) is selected as a routine sample. A solvent without the substance to be tested is added to the routine sample (mixed human serum) 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) (the volume of the added standard solution does not exceed 5% of the total volume of the sample), and three recovered samples with different added concentrations (low, medium, and high concentrations) are prepared. The recovered samples and the basic samples are measured by the detection method of the drug concentration of Sindili and Carrelili beads in the blood sample of the present invention. 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. Each concentration level sample is analyzed three times, which is completed in the same analysis batch, and the mean is taken for calculation. The detection results of the spiked recovery rate of sintilimab by the method of the present invention are shown in Table 4, and the detection results of the spiked recovery rate of carrelizumab are shown in Table 5. The recoveries of the two monoclonal antibody drugs are both within the range of 85% to 115%, which meets the technical requirements, indicating that the detection results of the method of the present invention are accurate and reliable.

[0114] Table 4

[0115]

[0116] Table 5

[0117]

[0118]

[0119] Example 7, intra-batch and inter-batch precision of the detection method

[0120] The standard substance to be tested was added to the mixed human serum sample (the same as the mixed human serum sample of Example 6) to prepare a certain concentration of quality control samples: low, medium and high concentrations. Each concentration level was measured 6 times a day, and the measurement was continued for 3 days, i.e., 3 batches. The sample processing method was the same as that of Example 4, and the chromatographic conditions and mass spectrometry parameters were the same as those of Example 5. The coefficient of variation (CV%) of each concentration level was calculated, and the CV% was used to represent the corresponding intra-batch and inter-batch precision (intra-batch precision formula: CV% = (intra-batch standard deviation / intra-batch mean) × 100%. Inter-batch precision CV% = 3 batches of data standard deviation / 3 batches of data mean) × 100%). The intra-batch and inter-batch precision test results and calculation results of sindilimab are shown in Table 6, and the intra-batch and inter-batch precision test results and calculation results of carrelizumab are shown in Table 7. The intra-batch and inter-batch precision of the two monoclonal antibody drugs were within 15.0%, indicating that the detection method of the present invention has good precision.

[0121] Table 6

[0122]

[0123]

[0124] Table 7

[0125]

[0126] Example 8: Carryover of Detection Methods

[0127] A high-concentration sample was prepared by adding a standard substance to a mixed human serum sample (the same as the mixed human serum sample in Example 6), and the sample was evaluated by injecting 5 injections of a blank sample continuously after the high-concentration sample was injected; the measurement was continued for 3 days, the sample processing method was the same as in Example 3, the chromatographic conditions and mass spectrometry parameters were the same as in Example 4, the results of the contamination detection of sintilimab carryover are shown in Table 8, the results of the contamination detection of carrelizumab carryover are shown in Table 9, and the results of the contamination detection of the internal standard carryover are shown in Table 10. The peak area residues of the blank sample compounds of the two monoclonal antibody drugs were both less than 20% of the peak area of ​​the quantitative lower limit compound, which met the technical requirements (the peak area of ​​the blank solution <20% 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.

[0128] Table 8

[0129]

[0130] Table 9

[0131]

[0132] Table 10

[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 method for determining the drug concentration of sindili and carrelizumab in blood samples based on LC-MS / MS, characterized in that: The following steps are involved: S1. Take a blood sample, add buffer I and receptor protein, and use the receptor protein to specifically bind to the target monoclonal antibody drug; S2. Add pre-washed magnetic beads and buffer I to further enrich the target in the blood sample; S3. After the obtained enriched solution is separated by magnetic absorption, the supernatant is discarded, and buffer II is added for elution. After elution, buffer III is added for dilution; S4. The sample diluted in step S3 is denatured at high temperature, and then an internal standard solution and protease are added for enzymatic hydrolysis; S5. 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; S6. Perform LC-MS / MS analysis on the peptide solution to be tested, and obtain the contents of sintilimab and carrelizumab in the blood sample by detecting the characteristic peptides of sintilimab and carrelizumab, respectively.

2. The method for determining the drug concentration of sindili and carrelizumab in blood samples based on LC-MS / MS according to claim 1, characterized in that: The blood sample is plasma or serum.

3. The method for determining the drug concentration of sindili and carrelizumab in a blood sample based on LC-MS / MS according to claim 1, characterized in that: In step S1, the buffer I is a phosphate buffer or a Tris-HCl buffer with a pH of 6-9.

4. The method for determining the drug concentration of sindili and carrelizumab in a blood sample based on LC-MS / MS according to claim 1, characterized in that: In step S1, the receptor protein is a PD-1 protein solution or a biotinylated PD-1 protein solution with a concentration of 5 to 50 μg / mL; the enrichment conditions are: Temperature: 25°C ~ 45°C; Time: 10min ~ 4.0hr; Oscillation speed: 500rpm ~ 2000rpm.

5. The method for determining the drug concentration of sindili and carrelizumab in a blood sample based on LC-MS / MS according to claim 1, characterized in that: In step S2, the magnetic beads are one of Protein A, Protein G or streptavidin magnetic beads, and the pre-washing is carried out using one of phosphate buffer or Tris-HCl buffer, pH 6 to 9; the enrichment conditions are: Temperature: 25°C ~ 45°C; Time: 10min ~ 4.0hr; Oscillation speed: 500rpm ~ 2000rpm.

6. The method for determining the drug concentration of sindili and carrelizumab in a blood sample based on LC-MS / MS according to claim 1, characterized in that: In step S3, magnetic separation is to separate the magnetic beads from the supernatant using a magnetic frame, and the supernatant is discarded; In step S3, the buffer II is a phosphate buffer solution with added surfactant Triton X-100 or a phosphate buffer solution with added Tween 20, pH 6 to 9; In step S3, the buffer III is one of bicarbonate buffer, phosphate buffer or Tris-HCl buffer, with a concentration of 20 mM to 200 mM and a pH of 6 to 9; In step S4, the internal standard solution is a peptide segment labeled with a sintilimab isotope or a peptide segment labeled with a carrelizumab isotope, and the concentration is 0.2 to 10.0 μg / mL; In step S4, the high temperature denaturation conditions are: temperature: 70°C to 100°C; time: 5 min to 30 min; In step S4, the protease is trypsin, glycosaminoglycanase or lysine protease (Lys-C), and the enzymatic hydrolysis conditions are: enzymatic hydrolysis ratio: 1:20 to 1:200; enzymatic hydrolysis time: 30 min to 16 hr; enzymatic hydrolysis temperature: 25° C. to 55° C.; oscillation speed: 500 rpm to 2000 rpm; In step S5, the enzymatic hydrolysis termination solution is a formic acid aqueous solution or a formic acid acetonitrile solution, and the volume percentage of formic acid is 5% to 30%.

7. The method for determining the drug concentration of sindili and carrelizumab in a blood sample based on LC-MS / MS according to claim 1, characterized in that: In step S6, the liquid chromatography conditions are: Chromatographic column model: one of the following types of columns: C4, C8, C12 and C18; 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 procedure is: mobile phase A+mobile phase B=100%; 0-1.0min, the volume of mobile phase B is maintained at 2-20%; 1.0-3.0min, the volume of mobile phase B increases to 20-45%; 3.0-3.5min, the volume of mobile phase B increases to 70-95%; 3.5-4.5min, the volume of mobile phase B is maintained at 70-95%; 4.5-4.51min, the volume of mobile phase B decreases to 2-20%; 4.51-5.0min, the volume of mobile phase B is maintained at 2-20%; Flow rate: 0.3~1.0mL / min; Injection volume: 2~50μL.

8. The method for determining the drug concentration of sindili and carrelizumab in a blood sample based on LC-MS / MS according to claim 1, characterized in that: In step S6, 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 method for determining the drug concentration of sindili and carrelizumab in a blood sample based on LC-MS / MS according to claim 1, characterized in that: Step S1 includes: S11. Take 8 μL of serum sample into a 1.5 mL first centrifuge tube, add 200 μL of buffer I and 20 μL of receptor protein solution for enrichment, and shake at 800 rpm for 1 hr at room temperature; Step S2 includes: S21. Take a certain volume of magnetic beads (calculated as 10.0 μL / sample) to a 1.5 mL second centrifuge tube, add 1000 μL buffer I; separate by magnetic attraction, and discard the supernatant; repeat the washing twice to complete the pre-washing of the magnetic beads; take a certain volume of buffer I (calculated as 50.0 μL / sample) and add it to the second centrifuge tube; S22. Take 50 μL of magnetic beads and 150 μL of buffer I after pre-washing in step S21, add them to the first centrifuge tube, shake at 1000 rpm for 30 min at room temperature; separate by magnetic absorption, and discard the supernatant; Step S3 includes: S31. Add 1000 μL of buffer II to the first centrifuge tube to wash away unbound proteins and other interfering substances, separate by magnetic attraction, and discard the supernatant; S32. Add 125.0 μL of buffer III to dilute the first centrifuge tube; Step S4 includes: S41. The sample diluted in step S3 is placed under high temperature conditions for 5 minutes to complete high temperature denaturation; S42. After the first centrifuge tube returns to room temperature, add 10 μL of internal standard solution and 1.5 μg of protease, and shake at 1100 rpm for 1 hr at 37°C; Step S5 includes: S51. Add the enzymatic hydrolysis stop solution to the first centrifuge tube in step S42, and shake at 1000 rpm for 2 min; collect the supernatant into a 96-well plate through magnetic separation to obtain a peptide solution to be tested.

10. The method for determining the drug concentration of sintili and carrelizumab in a blood sample based on LC-MS / MS according to claim 1, characterized in that: In step S6, the peptide solution to be tested is subjected to LC-MS / MS analysis: Liquid chromatography conditions: 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 program is: mobile phase A+mobile phase B=100%; 0-1.0min, the volume of mobile phase B is maintained at 15%; 1.0-3.0min, the volume of mobile phase B is increased from 10% to 38%; 3.0-3.5min, the volume of mobile phase B increased from 38% to 95%; 3.5-4.5min, the volume of mobile phase B remained at 95%; 4.50-4.51min, the volume of mobile phase B decreased from 95% to 15%; 4.51-5.0min, the volume of mobile phase B remained at 15%; flow rate: 0.6mL / min; injection volume: 10μL; Mass spectrometry parameters: SCIEX 4500MD; ion source: electrospray ion source; ion mode: positive ion mode; Monitoring mode: multiple reaction monitoring; ion source temperature: 500°C; positive ion spray voltage: 5500V; curtain gas: 30psi; The pressure of atomizing gas Gas1 is 55psi; the pressure of auxiliary heating gas Gas2 is 55psi.

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