CN118032966A

CN118032966A - High-sensitivity LC-MS/MS detection method for monitoring blood concentration of human Carrilizumab

Info

Publication number: CN118032966A
Application number: CN202410057615.1A
Authority: CN
Inventors: 尚红; 戴锦娜; 赵敏; 韩晓旭; 王星琪
Original assignee: First Hospital of China Medical University
Current assignee: First Hospital of China Medical University
Priority date: 2024-01-16
Filing date: 2024-01-16
Publication date: 2024-05-14

Abstract

The invention discloses a high-sensitivity LC-MS/MS detection method for monitoring the blood concentration of human body Carilimumab, which comprises the following steps of capturing and enriching the Carilimumab in a sample by using protein purification magnetic beads, eluting the Carilimumab on the magnetic beads by taking the Infuliximab as an internal standard, then carrying out denaturation and enzymolysis, and adopting a liquid chromatography tandem mass spectrometry to detect, wherein the liquid chromatography conditions comprise: the mobile phase A adopts formic acid aqueous solution, the mobile phase B adopts acetonitrile solution containing formic acid, gradient elution is used, and the mobile phase A and the mobile phase B are between 85 and 5 in the elution process: and the volume ratio of 15 to 95. The monitoring method established by the invention has the advantages of good specificity, high sensitivity, small plasma dosage, simple pretreatment process and the like, and can provide a reliable method for clinically monitoring the blood concentration of patients after the CarRui Li Zhushan antibody is injected.

Description

High-sensitivity LC-MS/MS detection method for monitoring blood concentration of human Carrilizumab

Technical Field

The invention belongs to the technical field of blood detection, and particularly relates to a detection method for monitoring the blood concentration of human Carrilizumab.

Background

With the rapid development of biological agents, monoclonal antibody drugs occupy the important role of clinical treatment with the advantage of low toxicity and high efficiency, and are widely applied to targeted treatment of tumors, autoimmune diseases, infections and other diseases, but the pharmacokinetics and pharmacodynamics processes of the monoclonal antibody drugs in vivo are very complex, and the clinical curative effect and the individual difference of toxic and side effects are large, so that development of therapeutic drug monitoring (therapeutic drug monitoring, TDM) of the monoclonal antibody drugs is needed to ensure the maximum benefit of patients.

Carilizumab (Camrelizumab, trade name Ai Ruika) was the first PD1 immunosuppressant developed independently from Jiangsu Hengrui medicine in China for advanced liver cancer treatment. The global multicenter III clinical study of the Carilizumab and the Apatinib is published in 2023, which shows that the first-line treatment of the advanced liver cancer has obvious survival benefit and tolerable safety, and the total survival period of the middle position reaches 22.1 months. As an innovative drug independently developed in China, the carlizumab shows remarkable curative effects in various malignant tumors, and the conditions of immunotherapy failure and adverse reaction are gradually increased along with the increasing of clinical application, so that the development of a rapid, high-sensitivity and accurate detection method for monitoring the concentration of the carlizumab in blood has urgent and practical clinical application significance.

At present, the immunoassay method is a common detection method of a clinical monoclonal antibody, but the method has certain limitation, has a narrow linear range, and cannot accurately quantify samples in a super linear range; the specificity is poor, the detection result is inaccurate due to the dependence on antigen and the susceptibility to interference, such as the interference of internal and external factors such as matrix, drug-resistant antibody and the like.

In recent years, liquid chromatography tandem mass spectrometry (LC-MS/MS) has become an important means for quantitative determination of monoclonal antibody drugs gradually, and has the advantages of good specificity, high sensitivity, high throughput and the like, and the liquid chromatography-mass spectrometry method is also recommended to measure the blood concentration of the monoclonal antibody drugs in the national "pharmaceutical experts consensus for monitoring anti-tumor biological similar drug therapeutic drugs" (2020 edition). However, at present, an LC-MS/MS detection method for monitoring the blood concentration of human Carilizumab is not available.

Disclosure of Invention

The invention aims to provide a high-sensitivity LC-MS/MS detection method for monitoring the blood concentration of human Carilizumab based on the prior art.

It is another object of the present invention to provide the use of the method of the present invention to detect blood levels in a patient after injection of Carriene Li Zhushan antibody.

The aim of the invention can be achieved by the following measures:

A high-sensitivity LC-MS/MS detection method for monitoring the blood concentration of human body Carilimumab comprises the following steps of capturing and enriching the Carilimumab in a sample by using protein purification magnetic beads, eluting the Carilimumab on the magnetic beads by taking the Infuliximab as an internal standard, then carrying out denaturation and enzymolysis, and adopting a liquid chromatography tandem mass spectrometry to detect, wherein the liquid chromatography conditions comprise: the mobile phase A adopts formic acid aqueous solution, the mobile phase B adopts acetonitrile solution containing formic acid, gradient elution is used, and the mobile phase A and the mobile phase B are between 85 and 5 in the elution process: and the volume ratio of 15 to 95.

The method of the present invention comprises a sample pretreatment step and an LC-MS/MS detection step.

In the sample pretreatment step, protein G is adopted for coupling the protein purification magnetic beads; the sample is human plasma or serum.

In the sample pretreatment step, a sample and an internal standard working solution are added into protein G coupling magnetic beads for incubation, after unbound proteins and interferons are washed off, the Carilin bead monoclonal antibodies on the magnetic beads are eluted, denaturation and enzymolysis are carried out, and the obtained enzymolysis solution is detected by a liquid chromatography tandem mass spectrometry.

In the incubation process of the magnetic beads, the dosage of the sample is 10-30 mu L, the dosage of the protein G coupling magnetic bead suspension is 10-30 mu L, and the dosage of the internal standard working solution is 10-30 mu L. In the incubation process of the magnetic beads, the temperature of the sample is 20-30 ℃ and the incubation time is 40-80 min.

In the sample pretreatment step, unbound proteins and interferents are washed away after incubation using a bovine serum albumin solution and PBS buffer; wherein the concentration of the bovine serum albumin solution is 0.05 to 0.2 percent.

In the sample pretreatment step, eluting the karellizumab on the magnetic beads by using a formic acid solution; the concentration of the formic acid solution for elution is 0.1-0.5%.

In the sample pretreatment step, protein denaturation was performed at high temperature using NH ₄HCO₃ solution, enzymatic hydrolysis was performed using trypsin, and enzymatic hydrolysis reaction was stopped using formic acid solution. Wherein the concentration of the NH ₄HCO₃ solution is 0.5-1.5 mol/L, the protein denaturation temperature is 90-98 ℃ (preferably 95-96 ℃), and the denaturation time is 5-15 min; the concentration of the trypsin solution is 0.3-0.6 mug/mu L, and the volume ratio of the NH ₄HCO₃ solution to the trypsin solution is 0.6-0.8: 1, the enzymolysis is carried out at 35-39 ℃ for 1.5-2.5 h, and the concentration of formic acid solution for stopping enzymolysis is 5-15%; and stopping enzymolysis, centrifuging, and taking supernatant to perform liquid chromatography-tandem mass spectrometry detection.

In the LC-MS/MS detection step, in the liquid chromatography condition, the mobile phase A adopts an aqueous solution containing 0.05 to 0.15 percent of formic acid, and the mobile phase B adopts an acetonitrile solution containing 0.05 to 0.15 percent of 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.

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

In the liquid chromatography condition, an Aeris ^TM PEPTIDE XB-C18 column is adopted as a chromatographic column, the column temperature is 35-45 ℃, and the sample injection volume is 10 mu L.

In mass spectrometry detection, ion source parameters of a mass spectrum are as follows: ion source: an electrospray ion source; air curtain gas: 30psi; collision gas: 8psi; spray capillary voltage: 5000V; ion source temperature: 450 ℃; ion source atomizing gas: 45psi; the ion source heats the auxiliary gas: 55psi.

In mass spectrometry detection, the compound MRM scan parameters of the mass spectrum are as follows: the carlizumab has quantitative characteristic peptide segment sequences LLIYTATSLADGVPSR, 559.6-630.3, declustering voltage of 50V and collision voltage of 20V; qualitative characteristic peptide fragment sequences LLIYTATSLADGVPSR, 839.0-359.2, declustering voltage 80V and collision voltage 40V; infliximab (internal standard), quantitative characteristic peptide fragment sequence SAVYLQMTDLR, 648.8-1039.5, declustering voltage 80V and collision voltage 30V; qualitative feature peptide fragment sequence GLEWVAEIR, 536.8- & gt 587.3, declustering voltage 80V, collision voltage 40V.

In mass spectrometry detection, the compound MRM scan parameters of the mass spectrum are shown in table 1.

Table 1, mass spectrometry compound MRM scan parameters

The invention discloses a specific high-sensitivity LC-MS/MS detection method for monitoring the blood concentration of human Carrilizumab, which comprises the following steps:

1) Preparing a calibrator solution, a quality control solution and an internal standard working solution with serial concentrations;

2) According to the total number of the calibrator, the quality control product, the blank plasma and the sample to be tested, respectively taking Protein G coupling magnetic bead suspension, and using phosphate buffer salt solution for activation and cleaning;

3) Respectively adding a series of concentration calibrator solution, quality control solution, blank plasma and a sample to be tested into the corresponding magnetic beads in the step 2), then adding an internal standard working solution and a PBS buffer solution, incubating at room temperature, and capturing and enriching the Carilizumab;

4) Adding a cleaning solution to wash off non-specific proteins and other impurities in the enrichment solution obtained in the step 3);

5) Adding formic acid solution, eluting the Carelin bead monoclonal antibody on the magnetic beads in the enrichment liquid obtained in the step 4), placing the magnetic beads on a magnetic rack, and transferring the eluent to a clean EP tube;

6) Adding NH ₄HCO₃ solution into the eluent obtained in the step 5), carrying out enzymolysis by adding trypsin solution after protein denaturation at high temperature, then adding formic acid solution to stop enzymolysis reaction, centrifuging the obtained enzymolysis solution, and taking the supernatant for later use;

7) And (3) detecting the supernatant obtained in the step (6) by using a liquid chromatography-tandem mass spectrometry method to obtain an analysis result.

As a preferred embodiment of the present invention, the series of concentration calibrator solutions prepared in step 1) are series of concentration calicheamicin calibrator solutions prepared using human blank plasma, the concentrations comprising: 1.2, 5, 10, 20, 50, 100, 200 μg/mL, covering the blood concentration range after injection of kari Li Zhushan antibody.

As a preferred embodiment of the present invention, in step 1), the quality control solution is a three-level karilizumab quality control solution prepared from human blank plasma, and the concentration includes: 2.75, 150 μg/mL, covering the C _max concentration range (73.572 + -11.4634 μg/mL) after 200mg dose of Carir Li Zhushan antibody.

As a preferred scheme of the invention, in the step 1), infliximab with similar retention time is selected as an internal standard, and the concentration of the solution is 50-150 mug/mL, preferably 100 mug/mL; the solution was PBS buffer.

As a preferred embodiment of the present invention, in step 2), the amount of Protein G magnetic beads used is 10 to 30. Mu.L, preferably 20. Mu.L.

In a preferred embodiment of the present invention, in step 3), the internal standard working fluid is used in an amount of 10 to 30. Mu.L, preferably 20. Mu.L; the incubation time at room temperature is 0.5h to 2h, preferably 1h.

As a preferable embodiment of the present invention, in the step 4), the washing is performed by sequentially adding 0.05 to 0.2% BSA solution and PBS buffer, and washing is performed twice, and the concentration of the BSA solution is preferably 0.1%.

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

As a preferred embodiment of the present invention, in step 6), 0.3 to 0.6. Mu.g/. Mu.L of trypsin solution is added, and the amount of trypsin solution used is 10 to 30. Mu.L, preferably 25. Mu.L of trypsin solution of 0.5. Mu.g/. Mu.L; incubation in a water bath at 35-39℃for 1.5-2.5 h, preferably in a water bath at 37℃for 2h.

As a preferred embodiment of the present invention, the liquid chromatography conditions described in step 7) are as follows: mobile phase: phase A: an aqueous solution containing 0.1% formic acid; and B phase: acetonitrile solution containing 0.1% formic acid, gradient elution. Column temperature: 40 ℃; sample injection amount: 10 mu L.

In order to improve the chromatographic selectivity, it is necessary to consider the adjustment of the polarity of the mobile phase. According to the invention, formic acid is added into the mobile phase A, so that the ionization efficiency of a target compound can be effectively improved, and the sensitivity of detecting other monoclonal antibodies in plasma by adopting an LC-MS/MS method in the prior art is higher and reaches 1 mug/mL in combination with the screened characteristic peptide fragment and other conditions. In a preferred embodiment, mobile phase A is 0.05% to 0.15% formic acid in water and mobile phase B is 0.05% to 0.15% formic acid in acetonitrile, and the preferred mobile phase A is 0.1% formic acid in water and mobile phase B is 0.1% formic acid in acetonitrile without affecting the effect of the invention.

In chromatography, the choice of the chromatographic column is important, and the requirements for the chromatographic column are: high column efficiency, good selectivity, good separation effect, etc. The invention examines two chromatographic columns (ACQUITY UPLC BEH C ₁₈, 100 multiplied by 2.1mm,1.7 mu m and Aeris ^TMPEPTIDE XB-C_18,, 2.1mm, 2.6 mu m), wherein the types of the liquid chromatographic columns are Aeris ^TM PEPTIDE XB-C₁₈, 2.6 mu m,2.1 mm and 100mm, the chromatographic peaks have good peak shapes, can be completely separated from endogenous interfering substances, and have higher sensitivity and accuracy and precision which meet the requirements under the matching of other conditions.

The selection of the internal standard is a very important task when using the internal standard method. The ideal internal standard should have substantially the same or as consistent as possible physicochemical properties, chromatographic behavior and response characteristics as the sample being analyzed; the internal standard must be sufficiently separated from the components of the sample under chromatographic conditions. The invention adopts infliximab as an internal standard, the internal standard and an object to be detected have similar retention time, chemical property and matrix effect, the reproducibility and accuracy are good when the carlizumab in blood plasma is measured, and the invention can be used for detecting the blood concentration of patients after the carlizumab is injected into the patients.

As a preferred embodiment of the present invention, the ion source parameters of the mass spectrum in the step 7) are as follows: ion source: an electrospray ion source; air curtain gas: 30psi; collision gas: 8psi; spray capillary voltage: 5000V; ion source temperature: 450 ℃; ion source atomizing gas: 45psi; the ion source heats the auxiliary gas: 55psi.

The MRM scan parameters for the compounds of the mass spectrum are shown in table 1. And adopting ESI ion source and positive ion mode to perform sectional scanning analysis.

The method of the invention can be applied to the aspect of detecting the blood concentration of the carlizumab in a patient.

The invention has the beneficial effects that:

Compared with the traditional immunization method and the existing LC-MS/MS method for detecting other monoclonal antibodies in blood plasma, the invention can reach high sensitivity of 1 mug/mL by only taking 20 mug of human blood plasma, and can meet the detection requirement of human Carrilizumab blood concentration; the pretreatment operation of the sample is simple and convenient, the steps of reduction, alkylation, solid phase extraction and the like are not needed, the experimental period is short, and the requirements of therapeutic drug monitoring are met; the method relies on a liquid chromatography tandem mass spectrometry platform, combines the optimized characteristic peptide segments for quantification and qualitative, and optimized sample pretreatment and chromatography/mass spectrometry conditions, has the advantages of good specificity, high sensitivity, less plasma dosage, simple pretreatment process and the like, and can provide a reliable method for clinically monitoring the plasma concentration of patients after the Carry Li Zhushan antibody is injected.

Drawings

FIG. 1 is a chromatogram of the lower limit of quantitation (1. Mu.g/mL) of Carlizumab in example 1.

FIG. 2 is a chromatogram of infliximab (internal standard) in example 1.

FIG. 3 is a chromatogram of hollow white blood plasma (not administered) of example 1.

Fig. 4 is a chromatogram of carlizumab in a plasma sample of a patient following administration in example 1.

FIG. 5 is a standard curve of the Caririnotecan in example 1.

Fig. 6 is a chromatogram of carlizumab in plasma samples at patient 1 trough concentration time point in example 1.

Fig. 7 is a chromatogram of carlizumab in plasma samples at patient 2 trough concentration time points in example 1.

FIG. 8 is a chromatogram of hollow white blood (not taken) of example 2.

Fig. 9 is a chromatogram of carlizumab in serum samples of patients following administration in example 2.

Fig. 10 is a chromatogram of carlizumab in the quality control after pretreatment with "sample pretreatment 1" in comparative example 1.

FIG. 11 is a chromatogram of Caririnotecan in the control after pretreatment with "sample pretreatment 2" in comparative example 1.

Detailed Description

The technical scheme of the invention is described below with reference to specific embodiments and drawings. The present invention may be better understood and implemented by those skilled in the art in light of the following examples.

Example 1

A high-sensitivity LC-MS/MS detection method for monitoring the blood concentration of human Carilizumab is applied to the treatment drug monitoring after the injection of Carilin Li Zhushan antibody to tumor patients in the embodiment. The sample is derived from a plasma sample which is reserved by routine detection after the injection of Carry Li Zhushan antibody by a patient in the medical department of oncology in a first hospital affiliated to China university.

Sample pretreatment

1. Standard curve, quality control and internal standard configuration: the calichealizumab standard curve was configured with blank plasma, set up: 1. 8 concentrations of 2, 5, 10, 20, 50, 100, 200 μg/mL; the karilizumab quality control was configured with blank plasma, setting three concentration levels: 2. 75, 150 μg/mL; infliximab was formulated at 100 μg/mL with PBS buffer as an internal standard;

2. Capturing and enriching the karilizumab: re-suspending protein G coupled magnetic beads (Cytiva) under the reciprocal of the jolt, taking 20 mu L of magnetic bead suspension into a 2.0mL EP tube, adding 400 mu L of PBS buffer solution, oscillating at 750rpm for 1min at low speed, activating, cleaning for 2 times, placing in a magnetic frame, and discarding supernatant; sequentially adding 20 mu L of sample (standard curve, quality control product or plasma sample to be tested), 10 mu L of 100 mu g/mL internal standard and 250 mu L of PBS buffer solution, and incubating for 60min at room temperature;

3. Washing away unbound proteins and other interferents: placing the obtained Carril bead enrichment solution in a magnetic frame, and discarding the supernatant; sequentially adding 500 mu L of 0.1% BSA solution and 500 mu L of PBS buffer solution to wash unbound proteins and other interferents, placing the mixture on a magnetic rack, discarding the supernatant, and repeating for 2 times;

4. eluting: adding 100 mu L of 0.25% formic acid solution for eluting, vortexing at 750rpm for 10min, eluting the Carilizumab on the magnetic beads, placing on a magnetic rack, and transferring the eluent to a clean EP tube;

5. Denaturation and enzymolysis: 17 mu L of NH ₄HCO₃ solution with the concentration of 1mol/L is added into the eluent, water bath is carried out for 10min at the temperature of 95 ℃, after cooling to room temperature, 25 mu L of 0.5 mu g/mu L trypsin solution is added, enzymolysis is carried out for 2h at the temperature of 37 ℃, then 10% formic acid solution is added to stop the enzymolysis reaction, the obtained enzymolysis solution is centrifuged, 10 mu L of supernatant is taken, detection is carried out by adopting a liquid chromatography tandem mass spectrometry, and analysis is carried out by using a Citrine ^TM Triple Quad^TM System mass spectrometer.

(II) liquid chromatography conditions

1. Chromatographic column: aeris ^TM PEPTIDE XB-C18.6 μm, 2.1X100 mm, column temperature 40℃and sample volume 10. Mu.L.

2. Mobile phase: mobile phase a was 0.1% formic acid water and mobile phase B was 0.1% formic acid-acetonitrile.

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

(III) Mass Spectrometry Condition

Ion source: an electrospray ion source; detection mode: a positive ion mode; air curtain gas: 30psi; collision gas: 8psi; spray capillary voltage: 5000V; ion source temperature: 450 ℃; ion source atomizing gas: 45psi; the ion source heats the auxiliary gas: 55psi.

The MRM scan parameters for the compounds of the mass spectrum are shown in table 1. The lower limit of quantitation (1. Mu.g/mL) chromatogram of Carelimumab is shown in FIG. 1. The chromatogram of the internal standard infliximab is shown in figure 2.

(IV) methodology investigation

The invention provides a high-sensitivity LC-MS/MS detection method for monitoring human Carlizumab plasma concentration, which is subjected to complete methodology investigation, and comprises the following steps: specificity, standard curve and linear range, lower limit of quantification, carrying pollution, precision, accuracy and stability of sample tray placed for 24 h.

Specificity: 6 human blank plasma (not taken) was randomly selected, and the operation was performed according to the "(first) sample pretreatment" method, and no significant interference peak was seen at the retention time position of the peak of the karirinotecan, indicating that the method was well specific. The chromatogram of blank plasma (without drug administration) is shown in fig. 3, and the chromatogram of karilizumab in the plasma sample of the patient after drug administration is shown in fig. 4.

Standard curve and linear range: adopting analysis software to collect data, and integrating and calculating chromatographic peaks; and (3) taking the area ratio of the peak areas of the kari Li Zhushan antibody and the internal standard (infliximab) in the calibrator as an ordinate (y), taking the concentration as a coordinate (x), and carrying out curve regression operation by weighting, wherein a regression equation is as follows: y=ax+b. Substituting the peak area ratio of the karil Li Zhushan antibody and the internal standard (infliximab) in the sample to be detected into a standard curve equation, and calculating the concentration of the karil bead monoclonal antibody in the sample to be detected. The result shows that the carlizumab has good linear relation in the range of 1-200 mu g/mL, r ² is larger than 0.99, and a representative standard curve is shown in figure 5.

TABLE 2 Low quantitative Limit (LOQ) data sheet

Lower limit of quantification: the lower limit of quantification of the method was evaluated by detecting 12 samples of the limit of quantification (1. Mu.g/mL), the reproducibility of LOQ was expressed as CV value, the CV value was required to be less than 20%, and the lower limit of quantification detection results are shown in Table 2. The results show that: the average value of the lower limit of the quantification is 0.9530, the CV is 9.11 percent, and the detection requirement is met.

Table 3 karellizumab carryover contamination test data sheet

Table 4 internal standard carried pollution test data table

Carrying pollution: and after the linear highest point is sampled, a blank sample is sampled again to examine the carrying pollution condition of the method, the method is repeated for 6 times, the peak areas of the blank sample and the LOQ sample Carilizumab are compared, the peak areas are expressed by residual quantity (%), the required residual quantity is less than 20%, and the carrying pollution conditions of the Carilizumab and the internal standard are shown in tables 3 and 4. The results show that: the Carilib monoclonal antibody has the carrying pollution less than or equal to 13.81 percent and the carrying pollution of an internal standard less than or equal to 0.08 percent, and meets the detection requirement.

Table 5 method precision data

Table 6 method accuracy data

Precision and accuracy: adding a calicheamicin monoclonal antibody standard solution into blank plasma to prepare low, medium and high concentration level (2, 75, 150 mug/mL) samples, repeatedly detecting each concentration 3 times a day, continuously detecting for 3 days, wherein the precision in and among batches is expressed by CV values, and the CV value is required to be less than 15%; the recovery rate of the karirinotecan is obtained by comparing the measured value with the marked concentration, the recovery rate is used for representing the accuracy of the method, the requirement is between 85 and 115 percent, the obtained precision data are shown in table 5, and the accuracy data are shown in table 6. The results show that: the precision CV value in the batch is less than or equal to 4.42 percent, the precision CV value between batches is less than or equal to 9.66 percent, the recovery rate is 86.53-113.20 percent, and the detection requirement is met.

Table 7 sample tray placement 24h stability test

Stability: the stability of the sample in a sample injection disc (10 ℃) is examined for 24 hours, a standard substance solution of the Carilib monoclonal antibody is added into blank plasma, low, medium and high concentration level (2, 75, 150 mug/mL) samples are prepared, the samples are placed in the sample injection disc (10 ℃) and respectively compared with detection values of 0h and 24h, each concentration is repeated for 3 times, the stability is expressed by RE (%), the RE value is required to be smaller than +/-15%, and the detection results are shown in Table 7. The results show that: after 24 hours, the average RE values of the low, medium and high 3 concentration levels of the karellizumab are respectively-3.95%, -1.31%, -0.90%, which indicates that the sample to be tested is stable when placed in a sample injection disc (10 ℃) for 24 hours.

(V) monitoring of therapeutic drug after injection of Carry Li Zhushan antibody in tumor patients

Plasma samples of patients with more than two cycles of treatment by injecting the Carilizumab in the oncology ward (blood sampling time is 30-60 min before medication in the next injection cycle), 10 mu L of supernatant is taken after pretreatment of the sample, and analysis is carried out by using a Citrine ^TM Triple Quad^TM System Mass spectrometer.

The chromatogram of the carlizumab in the patient 1 trough plasma samples at time point is shown in fig. 6.

The chromatogram of carlizumab in patient 2 trough plasma samples at time point is shown in fig. 7.

Discussion of (sixth)

The invention establishes a high-sensitivity LC-MS/MS detection method for monitoring the blood concentration of human Caririnotecan monoclonal antibody, the dosage of plasma is small (only 20 mu L) and can reach 1 mu g/mL high sensitivity, the pretreatment is simple, the steps of reduction, alkylation, solid phase extraction and the like are not needed, the experimental period is saved by 4-12 hours, the requirement of therapeutic drug monitoring is met, and a reliable method can be provided for clinically monitoring the blood concentration of patients after the Caririn Li Zhushan antibody is injected.

The infliximab is used as an internal standard for quantification, so that not only can the matrix interference be greatly eliminated, but also the result is not influenced by the conditions of pretreatment process, instrument response fluctuation and the like, and the accurate quantification can be achieved.

The method has good specificity, and no obvious interference peak is seen at the retention time position of the internal standard peak of the to-be-detected substance karirizumab; the carlizumab has good linear relation within the range of 1-200 mug/mL, and r ² is larger than 0.99; the sensitivity is high, and the lower limit of the quantification is 1 mug/mL; the method has good repeatability and accuracy, the precision CV value in the batch is less than or equal to 4.42 percent, the precision CV value between batches is less than or equal to 9.66 percent, and the recovery rate is 86.53-113.20 percent, thereby being capable of meeting the detection requirement.

In a word, the method combines quantitative and qualitative characteristic peptide fragments, a simple pretreatment method, optimized sample pretreatment and chromatographic/mass spectrum conditions, has the advantages of good specificity, high sensitivity, small plasma dosage, simple pretreatment process and the like, and can provide a reliable monoclonal antibody therapeutic drug monitoring method for clinical application.

Example 2

A high sensitivity LC-MS/MS detection method for monitoring human carlizumab plasma concentration was applied to the detection of serum samples in example 2. The sample is derived from a serum sample which is conventionally detected and reserved after the Karui Li Zhushan antibody is injected into a patient in the department of oncology in a first hospital affiliated to China university of medical science.

Sample pretreatment

1. Standard curve, quality control and internal standard configuration: the standard curve of the karellizumab is prepared by using blank serum, and the following steps are set: 1. 8 concentrations of 2, 5, 10, 20, 50, 100, 200 μg/mL; the karellizumab quality control was configured with blank serum, setting three concentration levels: 2. 75, 150 μg/mL; infliximab was formulated with PBS at 100 μg/mL as an internal standard;

2. Capturing and enriching the karilizumab: re-suspending protein G coupled magnetic beads (Cytiva) under the reciprocal of the jolt, taking 20 mu L of magnetic bead suspension into a 2.0mL EP tube, adding 400 mu L of PBS buffer solution, oscillating at 750rpm for 1min at low speed, activating, cleaning for 2 times, placing in a magnetic frame, and discarding supernatant; sequentially adding 20 mu L of a sample (a standard curve, a quality control product or a serum sample to be detected), 10 mu L of an internal standard of 100 mu g/mL and 250 mu L of PBS buffer solution, and incubating for 50min at room temperature;

3. washing away unbound proteins and other interferents: placing the obtained Carril bead enrichment solution in a magnetic frame, and discarding the supernatant; sequentially adding 450 mu L of 0.1% BSA solution and 450 mu L of PBS buffer solution to wash unbound proteins and other interferents, placing the mixture on a magnetic rack, discarding the supernatant, and repeating for 2 times;

4. eluting: adding 100 mu L of 0.3% formic acid solution for eluting, vortexing at 750rpm for 10min, eluting the Carilizumab on the magnetic beads, placing on a magnetic rack, and transferring the eluent to a clean EP tube;

5. Denaturation and enzymolysis: adding 15 mu L of 1mol/L NH ₄HCO₃ solution into the eluent, carrying out water bath at 95 ℃ for 10min, cooling to room temperature, adding 22 mu L of 0.5 mu g/mu L trypsin solution, carrying out enzymolysis for 100min at 37 ℃, adding 10% formic acid solution to stop enzymolysis reaction, centrifuging the obtained enzymolysis solution, taking 10 mu L of supernatant, detecting by adopting a liquid chromatography tandem mass spectrometry, and analyzing by using a Citrine ^TM Triple Quad^TM System mass spectrometer.

The liquid chromatography conditions and the mass spectrometry conditions in the embodiment 1 are adopted for methodology investigation, and the method comprises specificity, standard curve and linear range, quantitative lower limit, carrying pollution, precision, accuracy and sample tray placement stability for 24 hours all meet detection requirements.

Serum samples of patients with more than two cycles of treatment with Carilizumab in the oncology ward (blood sampling time is 30-60 min before administration in the next injection cycle) were selected, 10 μl of supernatant was taken after "(one sample pretreatment" according to example 2), and analyzed by Citrine ^TM Triple Quad^TM System mass spectrometer. The chromatogram of the blank serum (not taken) is shown in fig. 8. The chromatogram of the carlizumab in the serum sample after patient administration is shown in fig. 9.

In conclusion, the method is also suitable for accurately detecting the blood concentration of the karelimumab in the human serum sample, and can provide various sample type selections for clinical application.

Comparative example 1

The method for detecting the blood concentration of human Carelimumab provided by the comparative example comprises the following steps:

Sample pretreatment 1 (one) the sample pretreatment procedure of example 1 was used.

(II) sample pretreatment 2

1. The standard curve, the quality control product and the internal standard configuration method are unchanged;

2. Capturing and enriching the karilizumab: using another brand of magnetic beads (Gentic), re-suspending the magnetic beads under reversed numbers, taking 15 mu L of magnetic bead suspension into a 2.0mL EP tube, adding 400 mu L of PBS buffer solution, oscillating at 750rpm for 1min at low speed, activating, cleaning for 2 times, placing in a magnetic rack, and discarding supernatant; sequentially adding 20 mu L of sample (standard curve, quality control product or plasma sample to be tested), 10 mu L of 100 mu g/mL internal standard and 250 mu L of PBS, and incubating for 120min at room temperature;

3. Washing away unbound proteins and other interferents: placing the obtained Carril bead enrichment solution in a magnetic frame, and discarding the supernatant; sequentially adding 600 mu L of 0.1% BSA solution and 600 mu L of PBS to wash off unbound proteins and other interferents, placing the mixture on a magnetic rack, discarding the supernatant, and repeating the steps for 2 times;

4. Eluting: adding 100 mu L of 0.2% formic acid solution for eluting, vortexing at 750rpm for 20min, eluting the Carilizumab on the magnetic beads, placing on a magnetic rack, and transferring the eluent to a clean EP tube;

5. denaturation and enzymolysis: adding 20 mu L of 1M NH ₄HCO₃ solution into the eluent, carrying out water bath at 95 ℃ for 20min, cooling to room temperature, adding 60 mu L of 0.4 mu g/mu L trypsin solution, carrying out enzymolysis for 8h at 37 ℃, adding 10% formic acid solution to stop the enzymolysis reaction, centrifuging the obtained enzymolysis solution, and taking 10 mu L of supernatant for later use.

The liquid chromatography condition and the mass spectrometry condition of the example 1 are adopted, and the standard substance and the quality control substance are respectively pretreated by adopting two treatment modes of 'sample pretreatment 1' and 'sample pretreatment 2', so as to examine the difference of the Carilizumab in response intensity under different types of magnetic beads and different conditions (different conditions of the use amount, concentration and incubation time of the relevant solvents of purification, elution, enzymolysis and denaturation). After pretreatment with "sample pretreatment 1" and "sample pretreatment 2", the chromatograms of the carlizumab in the quality control are shown in fig. 10 and 11. As can be seen from fig. 10 and 11, the treatment efficiency of the "sample pretreatment 1" is about 80 times higher than that of the "sample pretreatment 2" in terms of the response intensity, and the overall time is saved by 7.3 hours. That is, the method using "sample pretreatment 2" is low in sensitivity and takes a long time, and cannot meet the clinical application requirements.

The above embodiments are only for illustrating the technical solution of the present invention, and although the above embodiments are described in detail, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention, and any modifications and equivalents are intended to be included within the scope of the claims.

Claims

1. The high-sensitivity LC-MS/MS detection method for monitoring the blood concentration of human body Carilimumab is characterized by comprising the following steps of capturing and enriching the Carilimumab in a sample by using protein purification magnetic beads, eluting the Carilimumab on the magnetic beads by taking the Infuliximab as an internal standard, then carrying out denaturation and enzymolysis, and adopting a liquid chromatography tandem mass spectrometry to detect, wherein the liquid chromatography conditions comprise: the mobile phase A adopts formic acid aqueous solution, the mobile phase B adopts acetonitrile solution containing formic acid, gradient elution is used, and the mobile phase A and the mobile phase B are between 85 and 5 in the elution process: and the volume ratio of 15 to 95.

2. The method of claim 1, wherein the protein-purifying magnetic beads are protein G-coupled magnetic beads; the sample is human plasma or serum.

3. The method of claim 2, wherein the sample and the internal standard working solution are added into protein G-coupled magnetic beads for incubation, and after unbound proteins and interferons are washed away, the karellizumab on the magnetic beads is eluted, and then denatured and enzymatically hydrolyzed, and the obtained enzymatic hydrolysate is detected by liquid chromatography-tandem mass spectrometry.

4. The method according to claim 3, wherein the concentration of infliximab in the internal standard working solution is 50-150 μg/mL, and the solution is PBS buffer; washing unbound protein and interferons using bovine serum albumin solution and PBS buffer; eluting the karilizumab on the magnetic beads by using a formic acid solution; protein denaturation was performed at high temperature using NH ₄HCO₃ solution, enzymatic hydrolysis was performed using trypsin, and enzymatic hydrolysis reaction was stopped using formic acid solution.

5. The method according to claim 4, wherein the sample is used in an amount of 10 to 30. Mu.L, the protein G-coupled magnetic bead suspension is used in an amount of 10 to 30. Mu.L, and the internal standard working solution is used in an amount of 10 to 30. Mu.L; 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 NH ₄HCO₃ solution is 0.5-1.5 mol/L, the protein denaturation temperature is 90-98 ℃, and the denaturation time is 5-15 min; the enzymolysis is carried out at the temperature of 35-39 ℃ for 1.5-2.5 hours, and the concentration of formic acid solution for stopping enzymolysis is 5-15%; and stopping enzymolysis, centrifuging, and taking supernatant to perform liquid chromatography-tandem mass spectrometry detection.

6. The method according to claim 1, wherein 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; preferably, mobile phase A adopts an aqueous solution containing 0.1% formic acid, and mobile phase B adopts an acetonitrile solution containing 0.1% formic acid; the chromatographic column adopts an Aeris ^TM PEPTIDEXB-C18 column with the column temperature of 35-45 ℃ and the sample injection volume of 10 mu L.

7. The method according to claim 1, characterized in that in the liquid chromatography conditions, the elution gradient is: 0-1min,15% B;

1-4.5min，15％B-25％B；4.5-4.7min，25％B-95％B；4.7-7.4min，95％B；7.4-9min，15％B。

8. The method of claim 1, wherein the ion source parameters of the mass spectrum are as follows: ion source: an electrospray ion source; air curtain gas: 30psi; collision gas: 8psi; spray capillary voltage: 5000V; ion source temperature: 450 ℃; ion source atomizing gas: 45psi; the ion source heats the auxiliary gas: 55psi.

9. The method according to claim 1, characterized in that the compound MRM scan parameters of the mass spectrum are as follows: the carlizumab has quantitative characteristic peptide segment sequences LLIYTATSLADGVPSR, 559.6-630.3, declustering voltage of 50V and collision voltage of 20V; qualitative characteristic peptide fragment sequences LLIYTATSLADGVPSR, 839.0-359.2, declustering voltage 80V and collision voltage 40V; infliximab (internal standard), quantitative characteristic peptide fragment sequence SAVYLQMTDLR, 648.8-1039.5, declustering voltage 80V and collision voltage 30V; qualitative feature peptide fragment sequence GLEWVAEIR, 536.8- & gt 587.3, declustering voltage 80V, collision voltage 40V.

10. Use of the method of claim 1 for detecting the plasma concentration of carlizumab in a patient.