A high-sensitivity LC-MS / MS detection method for monitoring the blood drug concentration of camrelizumab in human body

Abstract

The application discloses a high-sensitivity LC-MS / MS detection method for monitoring blood drug concentration of camrelizumab of human bodies, which comprises the following steps: using protein purification magnetic beads to capture and enrich camrelizumab in a sample, using infliximab as an internal standard, denaturing and enzymatically decomposing camrelizumab on the magnetic beads after elution, and detecting by using liquid chromatography tandem mass spectrometry, wherein the liquid chromatography conditions comprise that formic acid aqueous solution is used as mobile phase A, formic acid acetonitrile solution is used as mobile phase B, gradient elution is used, and the volume ratio of mobile phase A to mobile phase B changes in the range of 85:5:15:95 during elution. The monitoring method established by the application has the advantages of good specificity, high sensitivity, small plasma consumption, simple pretreatment process and the like, and can provide a reliable method for clinically monitoring blood drug concentration of patients after injection of camrelizumab.

Description

Technical Field

[0001] This invention belongs to the field of blood testing technology, specifically relating to a detection method for monitoring the blood concentration of camrelizumab in humans. Background Technology

[0002] With the rapid development of biologics, monoclonal antibody drugs have occupied an important position in clinical treatment due to their advantages of low toxicity and high efficacy. They have been widely used in targeted therapy for diseases such as tumors, autoimmune diseases, and infections. However, it is worth noting that the pharmacokinetics and pharmacodynamics of monoclonal antibody drugs in vivo are very complex, and there are large individual differences in clinical efficacy and toxic side effects. Therefore, it is urgent to carry out therapeutic drug monitoring (TDM) of monoclonal antibody drugs to ensure that patients benefit to the greatest extent.

[0003] Camrelizumab (trade name: Ai Ruika) is the first PD-1 immunosuppressant independently developed by Jiangsu Hengrui Medicine to be approved in China for the treatment of advanced liver cancer. In 2023, *The Lancet* published a global multicenter phase III clinical trial of camrelizumab in combination with apatinib, demonstrating significant survival benefits and tolerable safety as first-line treatment for advanced liver cancer, with a median overall survival of 22.1 months. As a domestically developed innovative drug, camrelizumab has shown significant efficacy in various malignant tumors. However, with its increasing clinical application, the incidence of immunotherapy failure and adverse reactions is also gradually increasing. Therefore, developing a rapid, highly sensitive, and accurate detection method to monitor the concentration of camrelizumab in the blood is of great urgent and practical clinical significance.

[0004] Immunoassay is currently a commonly used detection method for monoclonal antibody drugs in clinical practice. However, this method has certain limitations. It has a narrow linear range and cannot accurately quantify samples outside the linear range. It also has poor specificity, depends on the antigen, and is easily affected by interference, such as interference from internal and external factors like the matrix and anti-drug antibodies, which can lead to inaccurate test results.

[0005] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) has gradually become an important method for the quantification of monoclonal antibody drugs in recent years, possessing advantages such as high specificity, high sensitivity, and high throughput. my country's "Expert Consensus on Pharmaceutical Monitoring of Antitumor Biosimilars (2020 Edition)" also recommends using LC-MS / MS to determine the blood concentration of monoclonal antibody drugs. However, currently, there is a lack of LC-MS / MS detection methods that can be used to monitor the blood concentration of camrelizumab in humans. Summary of the Invention

[0006] The purpose of this invention is to provide a highly sensitive LC-MS / MS detection method for monitoring the blood concentration of camrelizumab in humans, based on existing technologies.

[0007] Another object of the present invention is to provide an application of the method of the present invention in detecting the blood concentration of camrelizumab after a patient is injected with it.

[0008] The objective of this invention can be achieved through the following measures:

[0009] A highly sensitive LC-MS / MS method for monitoring the blood concentration of camrelizumab in humans includes the following steps: capturing and enriching camrelizumab in the sample using protein-purified magnetic beads, with infliximab as an internal standard; eluting camrelizumab from the magnetic beads followed by denaturation and enzymatic digestion; and detecting the camrelizumab using liquid chromatography-tandem mass spectrometry. The liquid chromatography conditions include: mobile phase A is an aqueous formic acid solution, mobile phase B is an acetonitrile solution containing formic acid, gradient elution is used, and the volume ratio of mobile phase A to mobile phase B varies within the range of 85–5:15–95 during elution.

[0010] The method of the present invention includes a sample pretreatment step and an LC-MS / MS detection step.

[0011] In the sample pretreatment step of this invention, the protein purification magnetic beads are protein G coupled magnetic beads; the sample is human plasma or serum.

[0012] In the sample pretreatment step, the sample and internal standard working solution were first added to protein G-coupled magnetic beads for incubation. After washing away unbound proteins and interfering substances, the camrelizumab on the magnetic beads was eluted. Then, denaturation and enzymatic digestion were performed, and the resulting enzymatic digest was detected by liquid chromatography-tandem mass spectrometry.

[0013] During the incubation process with the magnetic beads, the sample volume was 10–30 μL, the protein G-coupled magnetic bead suspension volume was 10–30 μL, and the internal standard working solution volume was 10–30 μL. The incubation temperature was 20–30 °C, and the incubation time was 40–80 min.

[0014] In the sample pretreatment step, after incubation, unbound proteins and interfering substances were washed away using bovine serum albumin solution and PBS buffer; the concentration of bovine serum albumin solution was 0.05-0.2%.

[0015] In the sample pretreatment step, formic acid solution was used to elute camrelizumab from the magnetic beads; the concentration of the formic acid solution used for elution was 0.1–0.5%.

[0016] In the sample pretreatment step, protein denaturation was performed using NH4HCO3 solution at high temperature, followed by trypsin digestion, and the enzymatic digestion was stopped using formic acid solution. The concentration of NH4HCO3 solution was 0.5–1.5 mol / L, the protein denaturation temperature was 90–98℃ (preferably 95–96℃), and the denaturation time was 5–15 min. The concentration of trypsin solution was 0.3–0.6 μg / μL, the volume ratio of NH4HCO3 solution to trypsin solution was 0.6–0.8:1, the digestion temperature was 35–39℃, the digestion time was 1.5–2.5 h, and the concentration of formic acid solution used to stop the digestion was 5–15%. After stopping the digestion, the sample was centrifuged, and the supernatant was analyzed by liquid chromatography-tandem mass spectrometry.

[0017] In the LC-MS / MS detection step of the present invention, under the liquid chromatography conditions, mobile phase A is an aqueous solution containing 0.05% to 0.15% formic acid, and mobile phase B is an acetonitrile solution containing 0.05% to 0.15% formic acid; preferably, mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is an acetonitrile solution containing 0.1% formic acid.

[0018] In liquid chromatography conditions, a preferred elution gradient is as follows: 0-1 min, 15% B; 1-4.5 min, 15% B-25% B; 4.5-4.7 min, 25% B-95% B; 4.7-7.4 min, 95% B; 7.4-9 min, 15% B.

[0019] Under liquid chromatography conditions, an Aeris column was used. TM PEPTIDE XB-C18 column, column temperature 35~45℃, injection volume 10μL.

[0020] In mass spectrometry detection, the ion source parameters are as follows: Ion source: electrospray ion source; curtain gas: 30 psi; collision gas: 8 psi; spray capillary voltage: 5000 V; ion source temperature: 450 ℃; ion source nebulizer gas: 45 psi; ion source heating auxiliary gas: 55 psi.

[0021] In mass spectrometry detection, the MRM scanning parameters for the compounds are as follows: Camrelizumab, quantitative characteristic peptide sequence LLIYTATSLADGVPSR, 559.6→630.3, declustering voltage 50V, collision voltage 20V; qualitative characteristic peptide sequence LLIYTATSLADGVPSR, 839.0→359.2, declustering voltage 80V, collision voltage 40V; Infliximab (internal standard), quantitative characteristic peptide sequence SAVYLQMTDLR, 648.8→1039.5, declustering voltage 80V, collision voltage 30V; qualitative characteristic peptide sequence GLEWVAEIR, 536.8→587.3, declustering voltage 80V, collision voltage 40V.

[0022] In mass spectrometry detection, the MRM scan parameters for the compounds are shown in Table 1.

[0023] Table 1. Mass Spectrometry Compound MRM Scan Parameters

[0024]

[0025] This invention discloses a highly sensitive LC-MS / MS detection method for monitoring the blood concentration of camrelizumab in humans, which includes the following steps:

[0026] 1) Prepare a series of calibrator solutions, quality control solutions, and internal standard working solutions;

[0027] 2) Based on the total number of calibrators, quality control materials, blank plasma, and test samples, take Protein G-coupled magnetic bead suspensions and activate and clean them with phosphate buffer solution;

[0028] 3) Add a series of concentrations of calibrator solution, quality control solution, blank plasma and test sample to each of the corresponding magnetic beads in step 2), then add internal standard working solution and PBS buffer, incubate at room temperature to capture and enrich camrelizumab;

[0029] 4) Add a washing solution to wash away non-specific proteins and other impurities in the enrichment solution obtained in step 3);

[0030] 5) Add formic acid solution to elute the camrelizumab on the magnetic beads in the enrichment obtained in step 4), place it on a magnetic rack, and transfer the eluent to a clean EP tube;

[0031] 6) Add NH4HCO3 solution to the eluent obtained in step 5), denature the protein at high temperature, add trypsin solution for enzymatic hydrolysis, and then add formic acid solution to stop the enzymatic hydrolysis reaction. Centrifuge the resulting enzymatic hydrolysate and take the supernatant for later use.

[0032] 7) The supernatant obtained in step 6) was analyzed by liquid chromatography-tandem mass spectrometry to obtain the analytical results.

[0033] As a preferred embodiment of the present invention, the series of concentration calibrator solutions prepared in step 1) are series of concentrations of camrelizumab calibrator solutions prepared using human blank plasma, with concentrations including: 1, 2, 5, 10, 20, 50, 100, and 200 μg / mL, covering the range of blood drug concentrations after injection of camrelizumab.

[0034] In a preferred embodiment of the present invention, in step 1), the quality control solution is a three-level camrelizumab quality control solution prepared using human blank plasma, with concentrations including 2, 75, and 150 μg / mL, covering the C-cell volume after injection of 200 mg camrelizumab. max Concentration range (73.572±11.4634μg / mL).

[0035] As a preferred embodiment of the present invention, in step 1), the internal standard is selected as infliximab with a similar retention time, and the concentration of the solution is 50-150 μg / mL, preferably 100 μg / mL; the solution is PBS buffer.

[0036] As a preferred embodiment of the present invention, in step 2), the amount of Protein G magnetic beads used is 10-30 μL, preferably 20 μL.

[0037] As a preferred embodiment of the present invention, in step 3), the amount of internal standard working solution used is 10-30 μL, preferably 20 μL; the incubation time at room temperature is 0.5h-2h, preferably 1h.

[0038] In a preferred embodiment of the present invention, in step 4), 0.05-0.2% BSA solution and PBS buffer are added sequentially for washing, and the washing is performed twice. Preferably, the concentration of BSA solution is 0.1%.

[0039] As a preferred embodiment of the present invention, in step 5), elution is performed using a 0.1-0.5% formic acid solution, followed by low-speed vortexing at 750 rpm for 10 minutes, preferably with a formic acid concentration of 0.25%.

[0040] As a preferred embodiment of the present invention, in step 6), a trypsin solution of 0.3 to 0.6 μg / μL is added, the amount used is 10 to 30 μL, preferably 25 μL of a trypsin solution of 0.5 μg / μL; and incubated in a water bath at 35 to 39°C for 1.5 to 2.5 h, preferably incubated in a water bath at 37°C for 2 h.

[0041] As a preferred embodiment of the present invention, the liquid chromatography conditions described in step 7) are as follows: Mobile phase: Phase A: aqueous solution containing 0.1% formic acid; Phase B: acetonitrile solution containing 0.1% formic acid, gradient elution. Column temperature: 40℃; Injection volume: 10μL.

[0042] To improve chromatographic selectivity, the polarity of the mobile phase needs to be adjusted. This invention adds formic acid to mobile phase A, which effectively improves the ionization efficiency of the target compound. Combined with the screened characteristic peptides and other conditions, this method achieves higher sensitivity (1 μg / mL) than existing LC-MS / MS methods for detecting other monoclonal antibody drugs in plasma. In a preferred embodiment, mobile phase A is a 0.05%–0.15% aqueous formic acid solution, and mobile phase B is a 0.05%–0.15% formic acid-acetonitrile solution. Preferably, without affecting the effectiveness of this invention, mobile phase A is a 0.1% aqueous formic acid solution, and mobile phase B is a 0.1% formic acid-acetonitrile solution.

[0043] In chromatography, the selection of the chromatographic column is crucial, requiring high column efficiency, good selectivity, and excellent separation. This invention examines two chromatographic columns (ACQUITY UPLC BEH C). 18 100×2.1mm, 1.7μm and Aeris TM PEPTIDE XB-C 18, 2.1 x 100 mm, 2.6 μm), where the liquid chromatography column is an Aeris. TM PEPTIDE XB-C 18 With a peak size of 2.6 μm and a diameter of 2.1 x 100 mm, the chromatographic peak has good shape and can be completely separated from endogenous interfering substances. Under other conditions, the sensitivity is higher, and the accuracy and precision meet the requirements.

[0044] When using the internal standard method, the selection of the internal standard is crucial. An ideal internal standard should have essentially the same or as closely as possible the same physicochemical properties, chromatographic behavior, and response characteristics as the analyte; under chromatographic conditions, the internal standard must be able to fully separate from the components in the sample. This invention uses infliximab as the internal standard. The internal standard and the analyte have similar retention times, chemical properties, and matrix effects. The reproducibility and accuracy of infliximab determination in plasma are good, and it can be used to detect the blood concentration of infliximab after patient injection.

[0045] As a preferred embodiment of the present invention, the ion source parameters of the mass spectrometer in step 7) are as follows: ion source: electrospray ion source; curtain gas: 30 psi; collision gas: 8 psi; spray capillary voltage: 5000 V; ion source temperature: 450 °C; ion source atomizing gas: 45 psi; ion source heating auxiliary gas: 55 psi.

[0046] The MRM scanning parameters for the compounds in the mass spectrometer are shown in Table 1. Segmented scanning analysis was performed using an ESI ion source and positive ion mode.

[0047] The method of this invention can be applied to the detection of camrelizumab blood concentration in patients.

[0048] The beneficial effects of this invention are:

[0049] Compared with traditional immunoassay methods and existing LC-MS / MS methods for detecting other monoclonal antibody drugs in plasma, this invention only requires 20 μL of human plasma to achieve a high sensitivity of 1 μg / mL, which can meet the detection requirements of human camrelizumab blood drug concentration. The sample pretreatment operation of this invention is simple, without the need for reduction and alkylation, solid phase extraction and other steps, and the experimental cycle is short, which meets the needs of therapeutic drug monitoring. Based on the liquid chromatography-tandem mass spectrometry platform, combined with the selected characteristic peptides for quantitative and qualitative analysis, and the optimized sample pretreatment and chromatographic / mass spectrometry conditions, the monitoring method established by this invention has many advantages such as good specificity, high sensitivity, small plasma volume and simple pretreatment process, which can provide a reliable method for clinical monitoring of the blood drug concentration of camrelizumab after injection in patients. Attached Figure Description

[0050] Figure 1 This is a chromatogram of the lower limit of quantification (1 μg / mL) of camrelizumab in Example 1.

[0051] Figure 2 This is a chromatogram of infliximab (internal standard) in Example 1.

[0052] Figure 3 This is the chromatogram of blank plasma (without medication) from Example 1.

[0053] Figure 4 This is a chromatogram of camrelizumab in the plasma sample of the patient after medication in Example 1.

[0054] Figure 5 This is the standard curve of camrelizumab in Example 1.

[0055] Figure 6 This is a chromatogram of camrelizumab in the plasma sample of patient 1 at the trough concentration time point in Example 1.

[0056] Figure 7 This is a chromatogram of camrelizumab in plasma samples from patient 2 at the trough concentration time point in Example 1.

[0057] Figure 8 This is the chromatogram of blank serum (without medication) in Example 2.

[0058] Figure 9 This is a chromatogram of camrelizumab in the serum sample of the patient after medication in Example 2.

[0059] Figure 10 This is the chromatogram of camrelizumab in the quality control sample after pretreatment using "Sample Pretreatment 1" in Comparative Example 1.

[0060] Figure 11 This is the chromatogram of camrelizumab in the quality control sample after pretreatment using "sample pretreatment 2" in Comparative Example 1. Detailed Implementation

[0061] The technical solution of the present invention will be described below with reference to specific embodiments and accompanying drawings. The following embodiments will enable those skilled in the art to better understand and implement the present invention.

[0062] Example 1

[0063] A highly sensitive LC-MS / MS method for monitoring the blood concentration of camrelizumab in humans is presented in this embodiment for monitoring therapeutic drugs in cancer patients after camrelizumab injection. The samples were obtained from plasma samples collected from inpatients of the Department of Oncology at the First Affiliated Hospital of China Medical University after routine testing following camrelizumab injection.

[0064] (I) Sample Pretreatment

[0065] 1. Preparation of standard curve, quality control, and internal standard: A standard curve for camrelizumab was prepared using blank plasma, with eight concentrations: 1, 2, 5, 10, 20, 50, 100, and 200 μg / mL. Quality control samples for camrelizumab were prepared using blank plasma, with three concentration levels: 2, 75, and 150 μg / mL. Infliximab was prepared with PBS buffer to a concentration of 100 μg / mL as an internal standard.

[0066] 2. Capture and enrichment of camrelizumab: Resuspend protein G-conjugated magnetic beads (Cytiva) under inverted conditions. Take 20 μL of the magnetic bead suspension into a 2.0 mL EP tube, add 400 μL of PBS buffer, and vortex at 750 rpm for 1 min. Activate and wash twice, place on a magnetic rack, and discard the supernatant. Add 20 μL of sample (standard curve, quality control, or plasma sample to be tested), 10 μL of 100 μg / mL internal standard, and 250 μL of PBS buffer sequentially, and incubate at room temperature for 60 min.

[0067] 3. Wash away unbound proteins and other interfering substances: Place the obtained camrelly bead enrichment solution on a magnetic rack and discard the supernatant; add 500 μL of 0.1% BSA solution and 500 μL of PBS buffer in sequence to wash away unbound proteins and other interfering substances, place on a magnetic rack and discard the supernatant, and repeat 2 times.

[0068] 4. Elution: Add 100 μL of 0.25% formic acid solution to elute, vortex at 750 rpm for 10 min to elute the camrelizumab on the magnetic beads, place on a magnetic rack, and transfer the eluent to a clean EP tube;

[0069] 5. Denaturation and Enzymatic Hydrolysis: Add 17 μL of 1 mol / L NH4HCO3 solution to the eluent, incubate at 95°C for 10 min, cool to room temperature, add 25 μL of 0.5 μg / μL trypsin solution, and hydrolyze at 37°C for 2 h. Then, add 10% formic acid solution to stop the enzymatic hydrolysis reaction. Centrifuge the resulting hydrolysate, collect 10 μL of the supernatant, and analyze it using liquid chromatography-tandem mass spectrometry (LC-MS / MS) with Citrine. TM Triple Quad TM The analysis was performed using a System mass spectrometer.

[0070] (II) Liquid Chromatography Conditions

[0071] 1. Chromatographic column: Aeris TM PEPTIDE XB-C18 2.6μm, 2.1×100mm, column temperature 40℃, injection volume 10μL.

[0072] 2. Mobile phase: Mobile phase A is 0.1% formic acid water, and mobile phase B is 0.1% formic acid-acetonitrile.

[0073] 3. Elution gradient: 0-1 min, 15% B; 1-4.5 min, 15% B-25% B; 4.5-4.7 min, 25% B-95% B; 4.7-7.4 min, 95% B; 7.4-9 min, 15% B.

[0074] (III) Mass Spectrometry Conditions

[0075] Ion source: electrospray ion source; Detection mode: positive ion mode; Air curtain gas: 30psi; Collision gas: 8psi; Spray capillary voltage: 5000V; Ion source temperature: 450℃; Ion source atomizing gas: 45psi; Ion source heating auxiliary gas: 55psi.

[0076] The MRM scan parameters for the compounds obtained by mass spectrometry are shown in Table 1. The chromatogram for the limit of quantitation (1 μg / mL) of camrelizumab is shown in [reference needed]. Figure 1 The chromatogram of the internal standard infliximab is shown below. Figure 2 .

[0077] (iv) Methodological Examination

[0078] A complete methodological investigation was conducted on the high-sensitivity LC-MS / MS detection method for monitoring human camrelizumab blood concentration provided by this invention. The investigation included: specificity, standard curve and linear range, limit of quantitation, carryover, precision, accuracy, and stability of the injection tray after 24 hours.

[0079] Specificity: Six blank plasma samples (untreated) were randomly selected and processed according to the method described in "(I) Sample Pretreatment". No significant interfering peaks were observed at the retention times of the camrelizumab and internal standard peaks, indicating good specificity of the method. The chromatogram of the blank plasma (untreated) is shown below. Figure 3 The chromatogram of camrelizumab in the patient's plasma sample after medication is shown in the figure below. Figure 4 .

[0080] Standard Curve and Linear Range: Data were collected using Analyst software, and chromatographic peaks were integrated and calculated. A weighted curve regression was performed with the peak area ratio of camrelizumab and internal standard (infliximab) in the calibrator as the ordinate (y) and concentration as the coordinate (x). The regression equation was y = ax + b. The peak area ratio of camrelizumab and internal standard (infliximab) in the test sample was substituted into the standard curve equation to calculate the concentration of camrelizumab in the test sample. Results showed that camrelizumab exhibited good linearity in the range of 1–200 μg / mL. 2 All values ​​are greater than 0.99; a representative standard curve can be found here. Figure 5 .

[0081] Table 2. Lower Limit of Quantitative Quantification (LOQ) Data

[0082]

[0083] Limit of Quantitation (LOQ): The LOQ of the method was evaluated by detecting 12 samples (1 μg / mL). The repeatability of LOQ was expressed as the CV value, which was required to be less than 20%. The results of the LOQ detection are shown in Table 2. The results showed that the average LOQ was 0.9530, and the CV was 9.11%, which met the detection requirements.

[0084] Table 3. Data on contamination testing of camrelizumab.

[0085]

[0086] Table 4. Test Data on Internal Standard Carryover Pollution

[0087]

[0088] Carryover contamination: After the linear peak of the injection, a blank sample was injected to examine the carryover contamination of the method. This was repeated 6 times, and the peak area of ​​camrelizumab in the blank sample and the LOQ sample were compared. The result was expressed as residual amount (%), and the residual amount was required to be less than 20%. The carryover contamination of camrelizumab and internal standard is shown in Tables 3 and 4. The results showed that the carryover contamination of camrelizumab was ≤13.81%, and the carryover contamination of the internal standard was ≤0.08%, which met the detection requirements.

[0089] Table 5. Method precision data

[0090]

[0091]

[0092] Table 6. Method accuracy data

[0093]

[0094] Precision and accuracy: Camrelizumab standard solution was added to blank plasma to prepare samples at three concentration levels (2, 75, and 150 μg / mL). Each concentration was tested three times daily for three consecutive days. Intra-assay and inter-assay precision were expressed as CV values, with a requirement of CV values ​​less than 15%. The recovery rate of camrelizumab was determined by comparing the measured values ​​with the labeled concentrations. The recovery rate was used to express the method accuracy, with a requirement of 85%–115%. The precision data are shown in Table 5, and the accuracy data are shown in Table 6. The results showed that the intra-assay precision CV value was ≤4.42%, the inter-assay precision CV value was ≤9.66%, and the recovery rate was 86.53%–113.20%, meeting the detection requirements.

[0095] Table 7. Stability test of sample tray after 24 hours of placement.

[0096]

[0097]

[0098] Stability: The stability of the samples was investigated in the injection tray (10℃) for 24 hours. Camrelizumab standard solution was added to blank plasma to prepare samples at three concentration levels (2, 75, and 150 μg / mL), which were then placed in the injection tray (10℃). The detection values ​​at 0 h and 24 h were compared. Each concentration was repeated three times. Stability was expressed as RE (%), and the RE value was required to be less than ±15%. The results are shown in Table 7. The results showed that after 24 h, the average RE values ​​of the three concentration levels of camrelizumab were -3.95%, -1.31%, and -0.90%, respectively, indicating that the test samples were stable after 24 h in the injection tray (10℃).

[0099] (V) Monitoring of therapeutic drugs after injection of camrelizumab in cancer patients

[0100] Plasma samples were collected from patients who had received two or more cycles of camrelizumab treatment in the oncology ward (blood collection time was 30-60 minutes before the next injection cycle). After sample pretreatment as described in "(I) Sample Pretreatment", 10 μL of the supernatant was collected and processed using Citrine. TM Triple Quad TM The analysis was performed using a System mass spectrometer.

[0101] The chromatogram of camrelizumab in the plasma sample at the trough concentration time point of patient 1 is shown in the figure. Figure 6 .

[0102] Chromatograms of camrelizumab in plasma samples at the trough concentration time point of patient 2 are shown in the figure. Figure 7 .

[0103] (vi) Discussion

[0104] This invention establishes a highly sensitive LC-MS / MS detection method for monitoring the blood concentration of camrelizumab in humans. It achieves a high sensitivity of 1 μg / mL with a small plasma volume (only 20 μL), and the pretreatment is simple, eliminating the need for reduction, alkylation, solid-phase extraction, and other steps. The experimental cycle is reduced by 4–12 hours, meeting the needs of therapeutic drug monitoring and providing a reliable method for clinical monitoring of the blood concentration of camrelizumab after injection in patients.

[0105] Using infliximab as an internal standard for quantification can not only greatly eliminate matrix interference, but also ensure accurate quantification as the results are not affected by pretreatment processes, instrument response fluctuations, or other conditions.

[0106] The method showed good specificity, with no significant interfering peaks observed at the retention times of the analyte camrelizumab and the internal standard. Camrelizumab exhibited good linearity within the range of 1–200 μg / mL. 2 All values ​​are greater than 0.99; the sensitivity is high, with a limit of quantitation of 1 μg / mL; the method has good repeatability and accuracy, with intra-batch precision CV value ≤4.42%, inter-batch precision CV value ≤9.66%, and recovery rate ranging from 86.53% to 113.20%, all of which meet the detection requirements.

[0107] In summary, the method of this invention combines quantitative and qualitative characteristic peptides, a simple pretreatment method, and optimized sample pretreatment and chromatographic / mass spectrometry conditions. It has many advantages such as high specificity, high sensitivity, low plasma consumption, and simple pretreatment process, and can provide a reliable method for monitoring monoclonal antibody therapeutic drugs in clinical applications.

[0108] Example 2

[0109] A highly sensitive LC-MS / MS method for monitoring the blood concentration of camrelizumab in humans was applied to serum samples in Example 2. The samples were serum samples collected from inpatients of the Department of Oncology at the First Affiliated Hospital of China Medical University after routine testing following camrelizumab injection.

[0110] (I) Sample Pretreatment

[0111] 1. Preparation of standard curve, quality control, and internal standard: A standard curve for camrelizumab was prepared using blank serum, with eight concentrations: 1, 2, 5, 10, 20, 50, 100, and 200 μg / mL. Quality control samples for camrelizumab were prepared using blank serum, with three concentration levels: 2, 75, and 150 μg / mL. Infliximab was prepared with PBS to a concentration of 100 μg / mL as an internal standard.

[0112] 2. Capture and enrichment of camrelizumab: Resuspend protein G-conjugated magnetic beads (Cytiva) under inverted conditions. Take 20 μL of the magnetic bead suspension into a 2.0 mL EP tube, add 400 μL of PBS buffer, and vortex at 750 rpm for 1 min. Activate and wash twice, place on a magnetic rack, and discard the supernatant. Add 20 μL of sample (standard curve, quality control, or serum sample to be tested), 10 μL of 100 μg / mL internal standard, and 250 μL of PBS buffer in sequence, and incubate at room temperature for 50 min.

[0113] 3. Wash away unbound proteins and other interfering substances: Place the obtained camrelly bead enrichment solution on a magnetic rack and discard the supernatant; add 450 μL of 0.1% BSA solution and 450 μL of PBS buffer in sequence to wash away unbound proteins and other interfering substances, place on a magnetic rack and discard the supernatant, and repeat 2 times.

[0114] 4. Elution: Add 100 μL of 0.3% formic acid solution to elute, vortex at 750 rpm for 10 min to elute the camrelizumab on the magnetic beads, place on a magnetic rack, and transfer the eluent to a clean EP tube;

[0115] 5. Denaturation and Enzymatic Hydrolysis: Add 15 μL of 1 mol / L NH4HCO3 solution to the eluent, incubate at 95°C for 10 min, cool to room temperature, add 22 μL of 0.5 μg / μL trypsin solution, hydrolyze at 37°C for 100 min, then add 10% formic acid solution to stop the enzymatic hydrolysis reaction. Centrifuge the obtained hydrolysate, take 10 μL of the supernatant, and analyze it using liquid chromatography-tandem mass spectrometry (LC-MS / MS) with a Citrine mass spectrometer. TM Triple Quad TM The analysis was performed using a System mass spectrometer.

[0116] The liquid chromatography and mass spectrometry conditions used in Example 1 were adopted to conduct methodological investigations, including specificity, standard curve and linear range, limit of quantitation, carryover, precision, accuracy, and stability of the injection tray after 24 hours. All of these conditions met the detection requirements.

[0117] Serum samples were selected from patients who had received two or more cycles of camrelizumab treatment in the oncology ward (blood collection time was 30-60 minutes before the next injection cycle). After sample pretreatment according to "(I) Sample Pretreatment" in Example 2, 10 μL of the supernatant was collected and processed using Citrine. TM Triple Quad TM Analysis was performed using a System mass spectrometer. The chromatogram of blank serum (untreated) is shown below. Figure 8 The chromatogram of camrelizumab in the patient's serum sample after medication is shown below. Figure 9 .

[0118] In summary, the method of the present invention is also applicable to the accurate detection of camrelizumab blood concentration in human serum samples, and can provide multiple sample types for clinical applications.

[0119] Comparative Example 1

[0120] The method for detecting the blood concentration of camrelizumab in humans provided in this comparative example includes the following steps:

[0121] (I) Sample pretreatment 1. The sample pretreatment steps of Example 1 are adopted.

[0122] (II) Sample Pretreatment 2

[0123] 1. The configuration methods for the standard curve, quality control materials, and internal standard remain unchanged;

[0124] 2. Capture and enrichment of camrelizumab: Using another brand of magnetic beads (Gentic), resuspend the magnetic beads by inverting several times, take 15 μL of the magnetic bead suspension into a 2.0 mL EP tube, add 400 μL of PBS buffer, shake at 750 rpm for 1 min, activate and wash twice, place on a magnetic rack and discard the supernatant; add 20 μL of sample (standard curve, quality control or plasma sample to be tested) and 10 μL of 100 μg / mL internal standard and 250 μL of PBS in sequence, and incubate at room temperature for 120 min;

[0125] 3. Wash away unbound proteins and other interfering substances: Place the obtained camrelizumab enrichment solution on a magnetic rack and discard the supernatant; add 600 μL of 0.1% BSA solution and 600 μL of PBS in sequence to wash away unbound proteins and other interfering substances, place on a magnetic rack and discard the supernatant, and repeat 2 times.

[0126] 4. Elution: Add 100 μL of 0.2% formic acid solution to elute, vortex at 750 rpm for 20 min to elute the camrelizumab on the magnetic beads, place on a magnetic rack, and transfer the eluent to a clean EP tube;

[0127] 5. Denaturation and enzymatic hydrolysis: Add 20 μL of 1M NH4HCO3 solution to the eluent, incubate in a 95℃ water bath for 20 min, cool to room temperature, add 60 μL of 0.4 μg / μL trypsin solution, and incubate at 37℃ for 8 h. Then add 10% formic acid solution to stop the enzymatic hydrolysis reaction. Centrifuge the obtained enzymatic hydrolysate and take 10 μL of the supernatant for later use.

[0128] Using the liquid chromatography and mass spectrometry conditions of Example 1, the standard and quality control samples were pretreated using two methods, "Sample Pretreatment 1" and "Sample Pretreatment 2," respectively, to investigate the differences in response intensity of camrelizumab under different types of magnetic beads and under different conditions (differences in the amount, concentration, and incubation time of purification, elution, enzymatic digestion, denaturation-related solvents). The chromatograms of camrelizumab in the quality control samples after pretreatment using "Sample Pretreatment 1" and "Sample Pretreatment 2" are shown below. Figure 10 and Figure 11 .Depend on Figure 10 and Figure 11 It can be seen that, in terms of response intensity, the processing efficiency of "sample pretreatment 1" is about 80 times higher than that of "sample pretreatment 2", and the overall time is reduced by 7.3 hours. In other words, the "sample pretreatment 2" method has low sensitivity and is time-consuming, which cannot meet the needs of clinical applications.

[0129] The above embodiments are only used to illustrate and not limit the technical solutions of the present invention. Although the above embodiments have described the present invention in detail, those skilled in the art should understand that modifications or equivalent substitutions can be made to the present invention, but any modifications and partial substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A highly sensitive LC-MS / MS detection method for monitoring the blood concentration of camrelizumab in humans, characterized in that... The process includes the following steps: using protein purification magnetic beads to capture and enrich camrelizumab in the sample, using infliximab as an internal standard, eluting camrelizumab from the magnetic beads, followed by denaturation and enzymatic digestion, and then detecting it using liquid chromatography-tandem mass spectrometry. The liquid chromatography conditions included: mobile phase A was an aqueous formic acid solution, and mobile phase B was an acetonitrile solution containing formic acid; gradient elution was used, with the volume ratio of mobile phase A to mobile phase B varying within the range of 85–5:15–95 during elution; the chromatographic column used was an Aeris column. TM PEPTIDE XB-C18 column, column temperature 35–45℃, injection volume 10 μL; The ion source parameters for mass spectrometry are as follows: Ion source: electrospray ionization source; curtain gas: 30 psi; collision gas: 8 psi; spray capillary voltage: 5000 V; ion source temperature: 450 °C; ion source nebulizer gas: 45 psi; ion source heating auxiliary gas: 55 psi. The MRM scanning parameters for the compounds in the mass spectrometer were as follows: Camrelizumab: quantitative characteristic peptide sequence LLIYTATSLADGVPSR, 559.6→630.3, declustering voltage 50V, collision voltage 20V; qualitative characteristic peptide sequence LLIYTATSLADGVPSR, 839.0→359.2, declustering voltage 80V, collision voltage 40V; infliximab (internal standard): quantitative characteristic peptide sequence SAVYLQMTDLR, 648.8→1039.5, declustering voltage 80V, collision voltage 30V; qualitative characteristic peptide sequence GLEWVAEIR, 536.8→587.3, declustering voltage 80V, collision voltage 40V.

2. The method according to claim 1, characterized in that... The protein purification magnetic beads are protein G-coupled magnetic beads; the sample is human plasma or serum.

3. The method according to claim 2, characterized in that... First, the sample and internal standard working solution were added to protein G-coupled magnetic beads for incubation. After washing away unbound proteins and interfering substances, the camrelizumab on the magnetic beads was eluted. Then, denaturation and enzymatic digestion were performed. The resulting enzymatic digest was detected by liquid chromatography-tandem mass spectrometry.

4. The method according to claim 3, characterized in that... The concentration of infliximab in the internal standard working solution is 50–150 μg / mL, and the solution is PBS buffer. Unbound proteins and interfering substances were washed away using bovine serum albumin solution and PBS buffer; camrelizumab on the magnetic beads was eluted with formic acid solution. Protein denaturation was performed using NH4HCO3 solution at high temperature, followed by enzymatic hydrolysis using trypsin, and the enzymatic hydrolysis reaction was stopped using formic acid solution.

5. The method according to claim 4, characterized in that... The sample volume is 10-30 μL, the protein G-coupled magnetic bead suspension volume is 10-30 μL, and the internal standard working solution volume is 10-30 μL; the concentration of the bovine serum albumin solution is 0.05-0.2%; the concentration of the formic acid solution for elution is 0.1-0.5%; the concentration of the NH4HCO3 solution is 0.5-1.5 mol / L; the protein denaturation temperature is 90-98℃, and the denaturation time is 5-15 min; the enzymatic hydrolysis temperature is 35-39℃, the hydrolysis time is 1.5-2.5 h, and the concentration of the formic acid solution used to stop the hydrolysis is 5-15%; after stopping the hydrolysis, the sample is centrifuged, and the supernatant is collected for detection by liquid chromatography-tandem mass spectrometry.

6. The method according to claim 1, characterized in that... In the liquid chromatography conditions, mobile phase A is an aqueous solution containing 0.05% to 0.15% formic acid, and mobile phase B is an acetonitrile solution containing 0.05% to 0.15% formic acid.

7. The method according to claim 6, characterized in that... In the liquid chromatography conditions, mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is an acetonitrile solution containing 0.1% formic acid.

8. The method according to claim 1, characterized in that... The elution gradients in the liquid chromatography conditions were as follows: 0-1 min, 15% B; 1-4.5 min, 15% B-25% B; 4.5-4.7 min, 25% B-95% B; 4.7-7.4 min, 95% B; 7.4-9 min, 15% B.

9. The use of the method of claim 1 in detecting the blood concentration of camrelizumab in a patient.

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