Method for determining the drug concentration of sindibulin and carlumab in blood samples based on lc-ms / ms

Abstract

The application discloses a method for determining the drug concentration of sindibuli and carerili beads in a blood sample based on LC-MS / MS. In the pretreatment process of the blood sample, the two kinds of monoclonal antibody drugs in the blood sample are enriched and purified through receptor proteins and magnetic beads, the target objects are better enriched, and the specificity is improved. The detection method removes the protein reduction and alkylation steps in the pretreatment process of the blood sample. Compared with the traditional operation steps, the small-volume protease dosage and the ultrafast enzymolysis are realized, the overall process is simple and fast, and the material and time costs are greatly saved. The detection method for the monoclonal antibody drug concentration of sindibuli and carerili beads has high accuracy, high stability, high sensitivity, high specificity, a simple and fast pretreatment process, and small blood sample requirement.

Description

Technical Field

[0001] This invention relates to drug monitoring technology, and in particular to a method for determining the concentrations of sintilimab and camrelizumab in blood samples based on liquid chromatography-tandem mass spectrometry (LC-MS / MS). Background Technology

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

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

[0004] As therapeutic monoclonal antibody drugs become more widely used, problems are gradually emerging. In the early stages of treatment, response rates can reach 50-90%, and many patients benefit from monoclonal antibody therapy. However, the initial response in most patients disappears over time, leading to disease progression. The lack of or loss of response to monoclonal antibody therapy is caused by a variety of reasons, including a lack of understanding of epigenetic, biomolecular, or pathophysiological mechanisms. Among these, an insufficient understanding of serum monoclonal antibody concentrations is perhaps the most fundamental reason. Therefore, when using sintilimab and camrelizumab, it is necessary to comprehensively consider the patient's specific situation, monitor serum drug concentrations at each stage, and develop a personalized treatment plan.

[0005] Currently, monoclonal antibody drug concentration detection methods include spectroscopic methods, chromatographic methods, immunoassay methods, and liquid chromatography-tandem mass spectrometry (LC-MS / MS). While spectroscopic methods are simple to operate, detection is easily affected by dilution and measurement conditions; chromatographic methods have relatively poor discrimination, and improper experimental conditions can easily affect the results; immunoassay methods have poor specificity, and the detection results are easily interfered with, while the development time for highly specific antibodies is long and the price is 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 Antitumor Biosimilars (2020 Edition)" strongly recommends LC-MS / MS for therapeutic drug monitoring.

[0006] Currently reported LC-MS / MS methods for determining monoclonal antibody concentrations are characterized by complex pretreatment procedures, long enzymatic digestion times, and high detection costs, making them unsuitable for clinical applications. Therefore, a highly efficient, simple, and rapid liquid chromatography-tandem mass spectrometry method is lacking for the simultaneous determination of the concentrations of two monoclonal antibodies, sintilimab and camrelizumab.

[0007] Chinese patent document CN118032966A discloses a highly sensitive LC-MS / MS detection method for monitoring the blood concentration of camrelizumab in humans. This method can only detect one monoclonal antibody drug in plasma, requires large sample and protease volumes, has a long enzyme digestion time, and necessitates a high-sensitivity mass spectrometer (Citrine). TM Triple Quad TM The system is used for testing, so its clinical application is relatively limited. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a method for determining the concentration of sintilimab and camrelizumab 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 rapid pretreatment process, and small blood sample requirements.

[0009] To solve the above-mentioned technical problems, the present invention provides a method for detecting the concentrations of sintilimab and camrelizumab in blood samples, which includes the following steps:

[0010] S1. Take a blood sample, add buffer I and receptor protein enrichment, and utilize the receptor protein's specific binding to the target monoclonal antibody drug;

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

[0012] S3. After magnetic separation, the supernatant of the obtained enriched solution is discarded, and buffer II is added for rinsing. After rinsing, buffer III is added for dilution.

[0013] S4. After denaturing the sample diluted in step S3 at high temperature, add internal standard solution and protease for enzymatic digestion;

[0014] S5. After the enzymatic hydrolysis is completed, add the enzymatic hydrolysis termination solution to stop the enzymatic hydrolysis reaction and obtain the peptide solution to be tested;

[0015] S6. Perform liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis on the peptide solution to be tested. The content of sintilimab and camrelizumab in the blood sample was obtained by detecting the characteristic peptides of sintilimab and camrelizumab respectively.

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

[0017] Preferably, in step S1, the buffer solution I is a phosphate buffer or a Tris-HCl buffer with a pH of 6 to 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 5–50 μg / mL; the enrichment conditions are: temperature: 25℃–45℃; time: 10 min–4.0 hr; oscillation speed: 500 rpm–2000 rpm.

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

[0020] Preferably, in step S3, magnetic separation is performed by using a magnetic rack to separate the magnetic beads from the supernatant and then discarding the supernatant.

[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, with a pH of 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 20mM to 200mM and a pH of 6 to 9.

[0023] In step S4, the internal standard solution is a peptide labeled with sintilimab isotope or a peptide labeled with camrelizumab isotope, with a concentration of 0.2–10.0 μg / mL.

[0024] In step S4, the high-temperature denaturation conditions are: temperature: 70℃~100℃; time: 5min~30min;

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

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

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

[0028] Chromatographic column type: One of the following: C4, C8, C12, and C18;

[0029] Mobile phase A: an aqueous solution containing formic acid (0.01%–0.5%); Mobile phase B: an acetonitrile or methanol solution containing formic acid (0.01%–0.5%); Column temperature: 30℃–60℃;

[0030] The mobile phase employs gradient elution, with the following elution program: Mobile phase A + Mobile phase B = 100%; 0–1.0 min, mobile phase B volume remains at 2–20%; 1.0–3.0 min, mobile phase B volume increases to 20–45%; 3.0–3.5 min, mobile phase B volume increases to 70–95%; 3.5–4.5 min, mobile phase B volume remains at 70–95%; 4.5–4.51 min, mobile phase B volume decreases to 2–20%; 4.51–5.0 min, mobile phase B volume remains at 2–20%.

[0031] Flow rate: 0.3–1.0 mL / min;

[0032] Injection volume: 2–50 μL.

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

[0034] Ion source: Electrospray ion source; Ion mode: Positive ion mode;

[0035] Monitoring mode: Multiple response monitoring;

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

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

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

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

[0040] This invention provides a method for determining the concentrations of sintilimab and camrelizumab in blood samples using liquid chromatography-tandem mass spectrometry (LC-MS / MS). During blood sample pretreatment, the two monoclonal antibody drugs are enriched and purified using receptor proteins and magnetic beads, resulting in better target enrichment and improved specificity. This detection method eliminates protein reduction and alkylation steps in the blood sample pretreatment process, achieving smaller protease dosages and ultra-fast enzymatic digestion (within 1 hour) compared to traditional methods. The overall process is simple and rapid, significantly saving material and time costs. This method for detecting the concentrations of sintilimab and camrelizumab monoclonal antibody drugs is highly accurate, stable, sensitive, and specific. The pretreatment process is simple and rapid, requiring only a few microliters of blood sample to achieve a high sensitivity of 1 μg / mL. It can simultaneously perform quantitative detection of both sintilimab and camrelizumab, making it efficient and convenient, and meeting the dual requirements of timeliness and accuracy in clinical testing. Attached Figure Description

[0041] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0042] Figure 1 This is a total chromatogram of two monoclonal antibodies and internal standard from an embodiment of the method for detecting the concentrations of sintilimab and camrelizumab in blood samples according to the present invention;

[0043] Figure 2 This is a chromatogram of sintilimab from an embodiment of the method for detecting the concentrations of sintilimab and camrelizumab in blood samples according to the present invention;

[0044] Figure 3 This is a chromatogram of camrelizumab from an embodiment of the method for detecting the concentrations of sintilimab and camrelizumab in blood samples according to the present invention;

[0045] Figure 4 This is an internal standard chromatogram of an embodiment of the method for detecting the concentrations of sintilimab and camrelizumab in blood samples according to the present invention;

[0046] Figure 5 This is a standard curve of sintilimab from an embodiment of the method for detecting the concentrations of sintilimab and camrelizumab in blood samples according to the present invention;

[0047] Figure 6 This is a standard curve of camrelizumab from an embodiment of the method for detecting the concentrations of sintilimab and camrelizumab in blood samples according to the present invention;

[0048] Figure 7 This is a flowchart of an embodiment of the method for detecting the concentrations of sintilimab and camrelizumab in blood samples according to the present invention. Detailed Implementation

[0049] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0050] Example 1

[0051] A method for determining the concentrations of sintilimab and camrelizumab in blood samples based on liquid chromatography-tandem mass spectrometry (LC-MS / MS), such as... Figure 7 As shown, it includes the following steps:

[0052] S1. Take a blood sample, add buffer I and receptor protein enrichment, and utilize the receptor protein's specific binding to the target monoclonal antibody drug;

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

[0054] S3. After magnetic separation, the supernatant of the obtained enriched solution is discarded, and buffer II is added for rinsing. After rinsing, buffer III is added for dilution.

[0055] S4. After denaturing the sample diluted in step S3 at high temperature, add internal standard solution and protease for enzymatic digestion;

[0056] S5. After the enzymatic hydrolysis is completed, add the enzymatic hydrolysis termination solution to stop the enzymatic hydrolysis reaction and obtain the peptide solution to be tested;

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

[0058] Preferably, the blood sample is plasma or serum, with serum being the preferred component.

[0059] Example 1 presents a method for determining the concentrations of sintilimab and camrelizumab in blood samples using liquid chromatography-tandem mass spectrometry (LC-MS / MS). During blood sample pretreatment, the two monoclonal antibody drugs are enriched and purified using receptor proteins and magnetic beads, resulting in better enrichment of the target analytes and improved specificity. This detection method eliminates protein reduction and alkylation steps in the blood sample pretreatment process, achieving smaller protease dosages and ultra-fast enzymatic digestion (within 1 hour) compared to traditional methods. The overall process is simple and rapid, significantly saving material and time costs. This method for detecting the concentrations of sintilimab and camrelizumab monoclonal antibody drugs is highly accurate, stable, sensitive, and specific. The pretreatment process is simple and rapid, requiring only a few microliters of blood sample to achieve a high sensitivity of 1 μg / mL. It can simultaneously quantify both sintilimab and camrelizumab, making it efficient and convenient, and meeting the dual requirements of timeliness and accuracy in clinical testing.

[0060] Example 2

[0061] Based on the method for determining the concentrations of sintilimab and camrelizumab in blood samples using liquid chromatography-tandem mass spectrometry (LC-MS / MS) according to Example 1, in step S1, the buffer I is a phosphate buffer or Tris-HCl buffer with a pH of 6 to 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 5–50 μg / mL; the enrichment conditions are: temperature: 25℃–45℃; time: 10 min–4.0 hr; oscillation speed: 500 rpm–2000 rpm.

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

[0064] Preferably, in step S3, magnetic separation is performed by using a magnetic rack to separate the magnetic beads from the supernatant and then discarding the supernatant.

[0065] Preferably, in step S3, the buffer solution II is a phosphate buffer solution with added surfactant Triton X-100 or a phosphate buffer solution with added Tween 20, 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 20mM to 200mM and a pH of 6 to 9.

[0067] Preferably, in step S4, the internal standard solution is a peptide labeled with sintilimab isotopes or a peptide labeled with camrelizumab isotopes, with a concentration of 0.2–10.0 μg / mL. Using isotopically labeled characteristic peptides of the target analyte as internal standards can better eliminate the influence of sample preparation and instrument response, thereby improving the accuracy of the analytical results.

[0068] Preferably, in step S4, the high-temperature denaturation conditions are: temperature: 70℃~100℃; time: 5min~30min.

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

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

[0071] Example 3

[0072] Based on Example 1, the method for determining the concentrations of sintilimab and camrelizumab in blood samples using liquid chromatography-tandem mass spectrometry, in step S6, the liquid chromatography conditions are as follows:

[0073] Chromatographic column type: One of the following: C4, C8, C12, and C18;

[0074] Mobile phase A: an aqueous solution containing formic acid (0.01%–0.5%); Mobile phase B: an acetonitrile or methanol solution containing formic acid (0.01%–0.5%); Column temperature: 30℃–60℃;

[0075] The mobile phase employs gradient elution, with the following elution program: Mobile phase A + Mobile phase B = 100%; 0–1.0 min, mobile phase B volume remains at 2–20%; 1.0–3.0 min, mobile phase B volume increases to 20–45%; 3.0–3.5 min, mobile phase B volume increases to 70–95%; 3.5–4.5 min, mobile phase B volume remains at 70–95%; 4.5–4.51 min, mobile phase B volume decreases to 2–20%; 4.51–5.0 min, mobile phase B volume remains at 2–20%.

[0076] Flow rate: 0.3–1.0 mL / min;

[0077] Injection volume: 2–50 μL.

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

[0079] Ion source: Electrospray ion source; Ion mode: Positive ion mode;

[0080] Monitoring mode: Multiple response monitoring;

[0081] The ion source temperature is 500℃~750℃, preferably 500℃;

[0082] The spray voltage for positive ions is 4500V to 5500V, preferably 5500V.

[0083] The air curtain gas pressure is 25–40 psi, preferably 30 psi;

[0084] The pressure of the atomizing gas Gas1 and the auxiliary heating gas 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), the chromatographic column is a core component, and its performance 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 suitable for the analysis of different types of compounds. The C18 column, named for its octadecyl carbon chain, possesses high hydrophobicity and a long carbon chain. This structure gives the C18 column excellent separation performance in reversed-phase chromatography, making it particularly suitable for the analysis of long-chain fatty acids and pharmaceutical components. The alkane chain of the C8 column contains eight carbon atoms, exhibiting moderate hydrophobicity. Due to its shorter carbon chain, the C8 column has a relatively short retention time and sharp peak shape, making it suitable for the analysis of small organic compounds. The C4 column has a carbon chain length of four carbon atoms, exhibiting strong hydrophilicity. This characteristic makes the C4 column excellent for separating polar compounds, especially suitable for some difficult-to-separate polar acids and bases. The C18 column has the longest carbon chain and the highest hydrophobicity, making it suitable for the separation of non-polar and weakly polar compounds. C8 columns have moderate hydrophobicity and can be used for a wide range of compound analyses. C4 columns have strong hydrophilicity and are suitable for separating polar compounds. C18 columns have excellent separation performance and can effectively separate long-chain fatty acids, pharmaceutical components, and other compounds. While the separation performance of C8 columns is slightly inferior to that of C18 columns, they perform exceptionally well in the analysis of certain specific compounds. C4 columns have a unique advantage in separating polar compounds, especially suitable for some difficult-to-separate polar acids and bases. Due to the longer carbon chain of C18 columns, their retention time is relatively long, which gives them high resolution when separating complex samples. C8 columns have moderate retention times and are suitable for the analysis of most compounds. C4 columns have shorter retention times and are suitable for rapid analysis. C18, C8, and C4 columns all have good compatibility and can be used in different solvent systems; however, the separation performance of C18 columns may be affected to some extent in strongly polar solvents. C8 and C4 columns 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] Example 4

[0090] The method for determining the concentrations of sintilimab and camrelizumab in blood samples based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) according to Example 1 includes step S1:

[0091] S11. Take 8 μL of serum sample into a 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 hour at room temperature.

[0092] Preferably, step S2 includes:

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

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

[0095] Preferably, step S3 includes:

[0096] S31. Add 1000 μL of buffer II to the first centrifuge tube to rinse and 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 allowed to stand at high temperature for 5 minutes to complete the high-temperature denaturation.

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

[0101] Preferably, step S5 includes:

[0102] S51. Add the enzyme digestion termination solution to the first centrifuge tube in step S42, and shake at 1000 rpm for 2 min; after magnetic separation, collect the supernatant into a 96-well plate to obtain the peptide solution to be tested, ready for analysis.

[0103] Example 5

[0104] Based on the method for determining the concentrations of sintilimab and camrelizumab in blood samples using liquid chromatography-mass spectrometry (LC-MS / MS) according to Example 1, in step S6, the peptide solution to be tested is analyzed by liquid chromatography-mass spectrometry (LC-MS / MS):

[0105] Liquid chromatography conditions: SCIEX 4500MD; column type: C18; column temperature: 45℃; mobile phase A: aqueous solution containing formic acid (0.1%); mobile phase B: acetonitrile solution containing formic acid (0.1%); gradient elution was used, with the following elution program: mobile phase A + mobile phase B = 100%; 0–1.0 min, mobile phase B volume maintained at 15%; 1.0–3.0 min, mobile phase B volume increased from 10% to 38%; 3.0–3.5 min, mobile phase B volume increased from 38% to 95%; 3.5–4.5 min, mobile phase B volume maintained at 95%; 4.50–4.51 min, mobile phase B volume decreased from 95% to 15%; 4.51–5.0 min, mobile phase B volume maintained at 15%; flow rate: 0.6 mL / min; injection volume: 10 μL;

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

[0107] Table 2

[0108]

[0109] Example 5 describes a method for determining the concentrations of sintilimab and camrelizumab in blood samples using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The total chromatograms of the two monoclonal antibodies and the internal standard are shown below. Figure 1 As shown; the chromatogram of sintilimab is as follows. Figure 2 As shown; the chromatogram of camrelizumab is as follows. Figure 3 As shown; the internal standard chromatogram is as follows. Figure 4As shown, the chromatograms of the two monoclonal antibody drugs and the internal standard obtained by the method for detecting the concentrations of sintilimab and camrelizumab in blood samples of the present invention all have good peak shapes, stable detection baselines, and no obvious interference. By using the indicated concentration (X) of each calibrator and the peak area ratio (Y) of the characteristic peptide of each monoclonal antibody drug in the series of calibrators to the characteristic peptide of the internal standard, calibration curves were plotted and fitted to obtain the linear regression equation and correlation coefficient, as shown below. Figure 5 , Figure 6 As shown in Table 3, the linear regression equations and correlation coefficients of the calibration curves for the two monoclonal antibody drugs are presented. In this detection method, both monoclonal antibody drugs exhibited good linearity in the range of 1–50 μg / mL, with a linear correlation coefficient r0. 2 All values ​​are greater than 0.99.

[0110] Table 3

[0111] 1 sintilimab 1~50μg / mL y = 0.9724x + 0.2964 0.9995 2 Camrelizumab 1~50μg / 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 to reflect the common biochemical properties of human serum and exclude the influence of individual differences; the specific quantity can be determined according to experimental needs) was selected as the routine sample. An equal volume of solvent without the analyte was added to the routine sample (mixed human serum) to prepare the baseline sample. Different amounts of the analyte standard were added to the routine sample (mixed human serum) (the volume of the added standard solution did not exceed 5% of the total sample volume), preparing three recovery samples with different concentrations (low, medium, and high concentrations). The concentrations of sintilimab and camrelizumab in blood samples were measured using the method of this invention. The sample processing method was the same as in Example 4, and the chromatographic conditions and mass spectrometry parameters were the same as in Example 5. Each concentration level sample was analyzed three times in the same analytical batch, and the average value was used for calculation. The recovery rates of sintilimab and camrelizumab using the method of this invention are shown in Table 4 and Table 5, respectively. The recovery rates of both monoclonal antibody drugs are within the range of 85% to 115%, which meets the technical requirements, indicating that the detection results of the method of this 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] Quality control samples of certain concentrations were prepared by adding the analyte standard to a mixed human serum sample (the same as the mixed human serum sample in Example 6): low, medium, and high concentrations. Six replicates were performed daily at each concentration level for three consecutive days, resulting in three batches. The sample processing method was the same as in Example 4, and the chromatographic conditions and mass spectrometry parameters were the same as in Example 5. The coefficient of variation (CV%) for each concentration level was calculated, and this 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% = (Standard deviation of 3 batches / Mean of 3 batches) × 100%). The intra-batch and inter-batch precision detection results and calculation results for sintilimab are shown in Table 6, and those for camrelizumab are shown in Table 7. The intra-batch and inter-batch precision of both monoclonal antibody drugs were within 15.0%, indicating that the detection method of this invention has good precision.

[0121] Table 6

[0122]

[0123]

[0124] Table 7

[0125]

[0126] Example 8: Carryover of contaminants in detection methods

[0127] A high-concentration sample was prepared by adding the analyte standard to a mixed human serum sample (the same as the mixed human serum sample in Example 6). The sample was evaluated by injecting five consecutive blank samples after injecting the high-concentration sample. The samples were measured for three consecutive days. The sample processing method was the same as in Example 3, and the chromatographic conditions and mass spectrometry parameters were the same as in Example 4. The contamination detection results of sintilimab are shown in Table 8, the contamination detection results of camrelizumab are shown in Table 9, and the contamination detection results of internal standard are shown in Table 10. The residual peak area of ​​the compound in the blank samples of both monoclonal antibody drugs was less than 20% of the peak area of ​​the compound at the lower limit of quantitation, which met the technical requirements (peak area of ​​blank solution < 20% C1 peak area). The residual peak area of ​​the internal standard in the blank sample was less than 5% of the peak area corresponding to the working concentration of the internal standard.

[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 within the scope of protection of the present invention.

Claims

1. A method for determining the concentrations of sintilimab and camrelizumab in blood samples based on LC-MS / MS, characterized by comprising the following steps: S1. Take a blood sample, add buffer I and receptor protein, and utilize the receptor protein to specifically bind to the target monoclonal antibody drug; the receptor protein is a PD-1 protein solution or a biotinylated PD-1 protein solution, with a concentration of 5-50 μg / mL; the enrichment conditions are: temperature: 25℃-45℃; time: 10 min-4.0 hr; shaking speed: 500 rpm-2000 rpm; S2. Add pre-washed magnetic beads and buffer I to further enrich the target analytes in the blood sample; the magnetic beads are one of Protein A, Protein G, or streptavidin magnetic beads; the pre-washing is performed using one of phosphate buffer or Tris-HCl buffer, pH 6-9; the enrichment conditions are: temperature: 25℃-45℃; time: 10min-4.0hr; shaking speed: 500rpm-2000rpm; S3. After magnetic separation, the supernatant of the enriched solution is discarded, and buffer II is added for rinsing. After rinsing, buffer III is added for dilution. S4. After denaturing the sample diluted in step S3 at high temperature, add internal standard solution and protease for enzymatic digestion; S5. After the enzymatic hydrolysis is completed, add the enzymatic hydrolysis termination solution to stop the enzymatic hydrolysis reaction and obtain the peptide solution to be tested; S6. Perform LC-MS / MS analysis on the peptide solution to be tested, and obtain the content of sintilimab and camrelizumab in blood samples by detecting the characteristic peptides of sintilimab and camrelizumab respectively; The liquid chromatography conditions are as follows: Column type: C18; Mobile phase A: an aqueous solution containing 0.01%–0.5% formic acid; Mobile phase B: an acetonitrile or methanol solution containing 0.01%–0.5% formic acid; Column temperature: 30℃–60℃; The mobile phase is subjected to gradient elution, and the elution program is as follows: mobile phase A + mobile phase B = 100%; 0-1.0 min, the volume of mobile phase B is maintained at 2-20%; From 1.0 to 3.0 min, the volume of mobile phase B increases to 20% to 45%; from 3.0 to 3.5 min, the volume of mobile phase B increases to 70% to 95%; from 3.5 to 4.5 min, the volume of mobile phase B remains at 70% to 95%; from 4.5 to 4.51 min, the volume of mobile phase B decreases to 2% to 20%; from 4.51 to 5.0 min, the volume of mobile phase B remains at 2% to 20%. Flow rate: 0.3–1.0 mL / min; Injection volume: 2–50 μL; Mass spectrometry conditions are: Ion source: Electrospray ion source; Ion mode: Positive ion mode; Monitoring mode: Multiple response monitoring; The ion source temperature is 500℃~750℃; The spray voltage for positive ions is 4500V~5500V; The air curtain gas pressure is 25–40 psi; The pressure of the atomizing gas Gas1 and the auxiliary heating gas Gas2 is 30-60 psi.

2. The method for determining the concentrations of sintilimab and camrelizumab in blood samples based on LC-MS / MS according to claim 1, characterized in that, The blood sample is either plasma or serum.

3. The method for determining the concentrations of sintilimab and camrelizumab in blood samples based on LC-MS / MS according to claim 1, characterized in that, In step S1, the buffer solution I is either phosphate buffer or Tris-HCl buffer, with a pH of 6 to 9.

4. The method for determining the concentrations of sintilimab and camrelizumab in blood samples based on LC-MS / MS according to claim 1, characterized in that, In step S3, magnetic separation is achieved by using a magnetic rack to separate the magnetic beads from the supernatant and then discarding the supernatant. 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, with a pH of 6 to 9; In step S3, the buffer III is one of bicarbonate buffer, phosphate buffer, or Tris-HCl buffer, with a concentration of 20mM to 200mM and a pH of 6 to 9. In step S4, the internal standard solution is a peptide labeled with sintilimab isotope or a peptide labeled with camrelizumab isotope, with a concentration of 0.2–10.0 μg / mL. In step S4, the high-temperature denaturation conditions are: temperature: 70℃~100℃; time: 5min~30min; In step S4, the protease is trypsin or lysine protease, and the enzymatic hydrolysis conditions are as follows: hydrolysis ratio: 1:20 to 1:200; hydrolysis time: 30 min to 16 hr; hydrolysis temperature: 25℃ to 55℃; shaking speed: 500 rpm to 2000 rpm. In step S5, the enzymatic hydrolysis termination solution is an aqueous solution of formic acid or a formic acid-acetonitrile solution, with a volume percentage of formic acid of 5% to 30%.

5. The method for determining the concentrations of sintilimab and camrelizumab in blood samples 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 centrifuge tube, add 200 μL of buffer I and 20 μL of receptor protein solution for enrichment, and shake at 800 rpm for 1 hour at room temperature; Step S2 includes: S21. Based on a sample volume of 10.0 μL, transfer a certain volume of magnetic beads to a 1.5 mL second centrifuge tube and add 1000 μL of buffer I; separate by magnetic attraction and discard the supernatant; repeat the washing process twice to complete the pre-washing of the magnetic beads; based on a sample volume of 50.0 μL, transfer a certain volume of buffer I to a second centrifuge tube. S22. Take 50 μL of pre-washed magnetic beads and 150 μL of buffer I from 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; 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 the first centrifuge tube for dilution; Step S4 includes: S41. The sample diluted in step S3 is allowed to stand at high temperature for 5 minutes to complete the high-temperature denaturation. S42. After the first centrifuge tube has returned to room temperature, add 10 μL of internal standard solution and 1.5 μg of protease, and shake at 1100 rpm for 1 hour at 37°C. Step S5 includes: S51. Add the enzymatic hydrolysis termination solution to the first centrifuge tube in step S42, and shake at 1000 rpm for 2 min; after magnetic separation, collect the supernatant into a 96-well plate to obtain the peptide solution to be tested.

6. The method for determining the concentrations of sintilimab and camrelizumab in blood samples based on LC-MS / MS according to claim 1, characterized in that, In step S6, the solution of the peptide to be tested is analyzed by LC-MS / MS: Liquid chromatography conditions: SCIEX 4500 MD; column type: C18; column temperature: 45℃; mobile phase A: aqueous solution containing 0.1% formic acid; mobile phase B: acetonitrile solution containing 0.1% formic acid; gradient elution was used, with the following elution program: mobile phase A + mobile phase B = 100%; 0–1.0 min, mobile phase B volume maintained at 15%; 1.0–3.0 min, mobile phase B volume increased from 10% to 38%; From 3.0 to 3.5 min, the volume of mobile phase B increased from 38% to 95%; from 3.5 to 4.5 min, the volume of mobile phase B remained at 95%; from 4.50 to 4.51 min, the volume of mobile phase B decreased from 95% to 15%; from 4.51 to 5.0 min, the volume of mobile phase B remained at 15%; flow rate: 0.6 mL / min; injection volume: 10 μL. Mass spectrometry parameters: SCIEX 4500 MD; Ion source: electrospray ionization source; Ion mode: positive ion mode; Monitoring mode: multiple reaction monitoring; Ion source temperature: 500℃; Positive ion spray voltage: 5500V; Curtain gas pressure: 30psi; Nebulizer gas pressure: Gas1: 55psi; Auxiliary heating gas pressure: Gas2: 55psi.

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