CN112129870A - Pharmacokinetic mass spectrometry analysis method of monoclonal antibody drug - Google Patents
Pharmacokinetic mass spectrometry analysis method of monoclonal antibody drug Download PDFInfo
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- G—PHYSICS
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- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8822—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving blood
Abstract
The invention belongs to the technical field of proteomics, and discloses a pharmacokinetic mass spectrometry analysis method of a monoclonal antibody drug, which comprises the following steps: preparing a raw material medicine solution; preparing standard solutions and measuring the concentration of each standard solution; collecting animal blood samples and carrying out plasma sample reduction; performing cysteine blocking treatment; adding a digestion buffer solution and a pancreatin solution in sequence, and incubating to obtain a plasma sample enzymolysis solution; mixing the characteristic peptide segment and the internal standard peptide segment for enzymolysis; and (3) carrying out liquid chromatography-mass spectrometry on the reaction solution after enzymolysis, and substituting the ratio of the peak area of the characteristic peptide segment to the peak area of the internal standard peptide segment into a standard curve to obtain the drug concentration of the monoclonal antibody. The invention analyzes and quantifies the enzymolysis specific peptide segment of the monoclonal antibody medicine, has the characteristics of high flux, high sensitivity, good reproducibility and the like, is not only suitable for the monoclonal antibody medicine, but also has guiding reference significance for qualitative and quantitative research of other protein medicines and polypeptide medicines.
Description
Technical Field
The invention belongs to the technical field of proteomics, and particularly relates to a pharmacokinetic mass spectrometry analysis method of a monoclonal antibody drug.
Background
Currently, monoclonal antibody drugs are protein drugs having a special therapeutic effect based on a gamma-type immunoglobulin (immunoglobulin g) structure, and have been successfully used in clinical treatment of various diseases such as cancer, autoimmune diseases, viral infection, and central nervous system disorders. Compared with the existing micromolecular drugs, the antibody drug has the advantages of strong specificity, high activity, definite biological function, low toxicity, long half-life period, contribution to clinical application and the like.
Pharmacokinetics is the quantitative study of the absorption, distribution, metabolism and excretion rules of drugs in organisms: according to the measured drug concentration in the biological sample, a discipline of the rule that the blood drug concentration changes along with time is explained by applying a mathematical principle and a method. As different patients show different clinical reactions after receiving antibody drug treatment, in order to realize the individuation of the drug administration, ensure the safety of the drug administration, reduce the blindness of the drug administration to the maximum extent, simultaneously better meet the requirements of the regulations, establish a more rapid and accurate blood concentration detection platform, and provide necessary support for the monitoring of the blood concentration in the future.
However, in the monoclonal antibody pharmacokinetic experiment, the quantitative range of the prior art is narrow, monoclonal antibody drugs in different forms cannot be analyzed simultaneously, and the problems of long development period, high cost, incapability of distinguishing interference of endogenous proteins and the like exist, so that the reliability and the practicability of ELISA quantification are greatly influenced. Therefore, establishing a standard and reliable analysis method and developing the pharmacokinetics research of the monoclonal antibody medicine have very important significance for supporting the research and development of the monoclonal antibody medicine.
Through the above analysis, the problems and defects of the prior art are as follows: the prior art has narrow quantitative range, can not simultaneously analyze monoclonal antibody medicines with different forms, has the problems of long development period, high cost, incapability of distinguishing interference of endogenous protein and the like, and greatly influences the reliability and the practicability of ELISA quantification.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a pharmacokinetic mass spectrometry analysis method of a monoclonal antibody drug.
The invention is realized in such a way that a method for analyzing the pharmacokinetics mass spectrum of a monoclonal antibody drug comprises the following steps:
weighing a monoclonal antibody drug to be analyzed, and preparing a bulk drug solution; the monoclonal antibody drug to be analyzed is a biological targeting drug;
step two, respectively diluting the raw material medicine solution into different concentrations by using a solvent, taking five parts of solutions with different concentrations as series standard solutions, taking the total concentration of the raw material medicine solution as a marked quantity, and measuring the relative concentration of each component in each standard solution;
the concentrations of the series of standard solutions are respectively 10 mu mol/L, 15 mu mol/L, 20 mu mol/L, 25 mu mol/L and 30 mu mol/L;
step three, administering the solution prepared in the step two to healthy tested animals in a gastric perfusion or intravenous injection mode, collecting animal blood samples in a polyethylene tube, and processing the blood samples to obtain plasma samples; preparing corresponding plasma solutions by using the plasma sample and the series of standard solutions;
the healthy test animal is a male Wistar rat with the mass of 200 +/-10 g, and is cultured for 10-15 days before gastric lavage or intravenous injection, wherein the specific culture conditions are as follows: setting the temperature of the culture chamber to be 20-25 ℃, the culture humidity to be 45-60%, and the illumination in the culture chamber to be 12h light and shade alternation;
the method comprises the following steps of collecting an animal blood sample in a polyethylene tube, and processing the blood sample, and specifically comprises the following steps:
collecting blood of healthy test animals at 0.25h, 0.5h, 1h, 2h, 4h, 12h and 24h after administration, and taking 500 mu L venous blood through retrobulbar venous plexus, namely a blood sample;
sucking a blood sample into a centrifuge tube with disodium ethylene diamine tetraacetate anticoagulant, standing for 20min, then centrifuging for 10-12min at the temperature of 4 ℃, wherein the centrifugal speed is 3000rpm, and separating plasma to obtain a plasma sample;
the plasma sample and the series of standard solutions are used for preparing corresponding plasma solutions, and the plasma solutions specifically comprise:
respectively placing 100 mu L of series of standard solutions with different concentrations in a centrifuge tube, and respectively placing in a nitrogen blowing instrument for drying in water bath at 40 ℃;
drying, adding 100 mu L of plasma sample, mixing for 1min in a vortex mode, sequentially adding 10 mu L of internal standard working solution and 300 mu L of methanol, mixing uniformly, and centrifuging for 5min at 12000rpm in a high-speed centrifuge to obtain supernatant;
placing the supernatant in a centrifuge tube, drying in a nitrogen blowing instrument in water bath at 40 deg.C, adding 200 μ L methanol into the dried residue, redissolving, vortexing for 30s, and centrifuging again to obtain supernatant, i.e. plasma solution;
step four, adding 0.5-0.75 mg of solid cysteine sealing agent into the plasma solution obtained in the step three, mixing uniformly by vortex, and sealing cysteine to obtain a reaction solution;
adding 500-700 mu L of digestion buffer solution into the reaction solution sealed by cysteine, mixing in a vortex mode, adding 3 mu L of pancreatin solution, mixing in a vortex mode, and incubating to obtain plasma sample enzymolysis liquid;
adding 10-15 mu L of stable isotope labeled characteristic peptide segment and internal standard peptide segment into the enzymolysis liquid, mixing uniformly by vortex, and mixing and carrying out enzymolysis on the characteristic peptide segment and the internal standard peptide segment;
and seventhly, performing liquid chromatography-mass spectrometry on the reaction liquid after enzymolysis by using a liquid chromatography tandem mass spectrometer, and substituting the ratio of the peak area of the selected characteristic peptide segment to the peak area of the internal standard peptide segment into a standard curve to obtain the drug concentration of the monoclonal antibody.
Further, in the second step, the solvent is chromatographically pure methanol.
Furthermore, in the third step, the experimental animal is a mouse, a rat, a guinea pig, a rabbit, or a dog.
Further, in step four, the conditions for performing the cysteine blocking treatment are as follows: incubating for 40-45 min at 25-27 ℃ in the dark.
Further, in the fifth step, the enzymolysis method is as follows: performing enzyme digestion treatment by using trypsin; BSA is added in the enzymolysis process, and the mass volume percentage of the BSA is 1-1.5%.
Further, in step five, the incubation method is as follows: incubating at 37 ℃ for 12-16 h to obtain plasma sample enzymolysis liquid, and adding 3-5 mu L of formic acid to terminate the reaction.
Further, in the sixth step, the stable isotope labeling contained in the characteristic peptide fragment and the internal standard peptide fragment is carried out on the N and C elements of proline in the characteristic peptide fragment and the internal standard peptide fragment respectively15N and13and C, marking.
Further, in the seventh step, the liquid chromatography conditions in the liquid chromatography-mass spectrometry are as follows: adopting PeptideC SHTMC18 chromatographic column with specification of 2.1mm × 100mm, 1.8 μm; the column temperature is 40-41 ℃; the mobile phase A is water with the volume percentage of formic acid accounting for 0.15-0.2%; the mobile phase B is acetonitrile with the volume percentage of formic acid accounting for 0.15-0.2%; gradient elution is carried out for 50-60 min, the sample injection amount is 8-10 mu l, and the flow rate is 0.25-0.35 ml/min.
Further, in the seventh step, the conditions of mass spectrometry in the liquid chromatography-mass spectrometry are as follows: the atomization pressure is 55-60 psi, the temperature of the sheath gas is 420-450 ℃, and the capillary tube voltage is 5500-6000V.
Further, in step seven, the standard curve y is 0.000239987+0.0684449x, and the establishment process is carried out by preparing standard series solutions and preparing rat plasma standard curve samples.
By combining all the technical schemes, the invention has the advantages and positive effects that: the pharmacokinetic mass spectrometry analysis method of the monoclonal antibody medicament provided by the invention is used for analyzing and quantifying the enzymolysis specific peptide fragment of the monoclonal antibody medicament, has the characteristics of high flux, high sensitivity, good reproducibility and the like, is simple and easy to implement, is suitable for the monoclonal antibody medicament, and has reference significance for qualitative and quantitative research on other protein medicaments and polypeptide medicaments.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.
FIG. 1 is a flow chart of a method for pharmacokinetic mass spectrometry of a monoclonal antibody drug according to an embodiment of the present invention.
FIG. 2 is a flow chart of the process of collecting a blood sample from an animal in a polyethylene tube and processing the blood sample according to an embodiment of the present invention.
FIG. 3 is a drawing of the plasma solution prepared using a plasma sample and a series of standard solutions as provided in an embodiment of the present invention.
FIG. 4 is a typical chromatogram of a specific peptide fragment standard after enzymatic hydrolysis of a monoclonal antibody provided in an embodiment of the present invention.
Fig. 5 is a typical chromatogram of an internal standard peptide fragment of a synthetic stable isotope provided in an example of the invention.
FIG. 6 is a typical chromatogram of a specific peptide fragment in a plasma sample provided by an embodiment of the present invention.
FIG. 7 is a typical chromatogram of an internal standard peptide fragment in a plasma sample provided by an embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In view of the problems in the prior art, the present invention provides a method for analyzing pharmacokinetic mass spectrum of a monoclonal antibody drug, which is described in detail below with reference to the accompanying drawings.
As shown in fig. 1, the method for analyzing pharmacokinetic mass spectrum of a monoclonal antibody drug provided by the embodiment of the invention includes the following steps:
s101, weighing a monoclonal antibody drug to be analyzed, and preparing a bulk drug solution; the monoclonal antibody drug to be analyzed is a biological target drug.
S102, respectively diluting the raw material medicine solution into different concentrations by using a solvent, taking five parts of solutions with different concentrations as a series of standard solutions, taking the total concentration of the raw material medicine solution as a marked quantity, and measuring the relative concentration of each component in each standard solution.
S103, administering the solution prepared in the step S102 to a healthy test animal by means of intragastric administration or intravenous injection, collecting an animal blood sample in a polyethylene tube, and processing the blood sample to obtain a plasma sample; plasma samples and a series of standard solutions were used to make the corresponding plasma solutions.
And S104, adding 0.5-0.75 mg of solid cysteine sealing agent into the plasma solution obtained in the step S103, uniformly mixing in a vortex mode, and sealing cysteine to obtain a reaction solution.
And S105, adding 500-700 mu L of digestion buffer solution into the reaction solution sealed by cysteine, carrying out vortex mixing, adding 3 mu L of pancreatin solution, carrying out vortex mixing, and incubating to obtain plasma sample enzymolysis liquid.
S106, adding 10-15 mu L of stable isotope labeled characteristic peptide segment and internal standard peptide segment into the enzymolysis liquid, mixing uniformly by vortex, and mixing and carrying out enzymolysis on the characteristic peptide segment and the internal standard peptide segment.
S107, performing liquid chromatography-mass spectrometry on the reaction liquid after enzymolysis by using a liquid chromatography tandem mass spectrometer, and substituting the ratio of the peak area of the selected characteristic peptide segment to the peak area of the internal standard peptide segment into a standard curve to obtain the drug concentration of the monoclonal antibody.
As shown in fig. 2, in step S103, the collecting a blood sample of an animal in a polyethylene tube and processing the blood sample includes:
s201, collecting blood of healthy test animals at 0.25h, 0.5h, 1h, 2h, 4h, 12h and 24h after administration, and taking 500 mu L venous blood through retrobulbar venous plexus, namely a blood sample.
S202, sucking a blood sample into a centrifuge tube with ethylenediamine tetraacetic acid disodium anticoagulant, and standing for 20 min.
S203, centrifuging for 10-12min at the temperature of 4 ℃, wherein the centrifugal rotating speed is 3000rpm, and separating plasma to obtain a plasma sample.
As shown in fig. 3, in step S103 provided in the embodiment of the present invention, the plasma sample and the series of standard solutions are used to prepare corresponding plasma solutions, which specifically include:
s301, respectively taking 100 mu L of series of standard solutions with different concentrations, placing the solutions in a centrifuge tube, and respectively placing the solutions in a nitrogen blowing instrument for drying in water bath at 40 ℃.
S302, adding 100 mu L of plasma sample after blow-drying, mixing for 1min in a vortex mode, sequentially adding 10 mu L of internal standard working solution and 300 mu L of methanol, mixing uniformly, and placing the mixture in a high-speed centrifuge for centrifugation at 12000rpm for 5min to obtain supernatant.
S303, placing the supernatant into a centrifuge tube, placing the centrifuge tube into a nitrogen blowing instrument for drying in water bath at 40 ℃, adding 200 mu L of methanol into the dried residues for redissolving, carrying out vortex for 30S, and centrifuging again to obtain the supernatant, namely the plasma solution.
In step S102 provided in the embodiment of the present invention, the solvent is chromatographically pure methanol.
In step S103 provided in the embodiment of the present invention, the experimental animal is a mouse, a rat, a guinea pig, a rabbit, or a dog.
In step S104 provided in the embodiment of the present invention, the conditions for performing the cysteine blocking process are as follows: incubating for 40-45 min at 25-27 ℃ in the dark.
In step S105 provided in the embodiment of the present invention, the enzymolysis method includes: performing enzyme digestion treatment by using trypsin; BSA is added in the enzymolysis process, and the mass volume percentage of the BSA is 1-1.5%.
In step S105 provided in the embodiment of the present invention, the incubation method includes: incubating at 37 ℃ for 12-16 h to obtain plasma sample enzymolysis liquid, and adding 3-5 mu L of formic acid to terminate the reaction.
In step S106 provided in the embodiments of the present invention, the characteristic peptide fragment and the internal standard peptide fragment containThe stable isotope labeling is carried out on N and C elements of proline in the characteristic peptide fragment and the internal standard peptide fragment respectively15N and13and C, marking.
In step S107 provided in the embodiment of the present invention, the liquid chromatography conditions in the liquid chromatography-mass spectrometry are as follows: adopting PeptideC SHTMC18 chromatographic column with specification of 2.1mm × 100mm, 1.8 μm; the column temperature is 40-41 ℃; the mobile phase A is water with the volume percentage of formic acid accounting for 0.15-0.2%; the mobile phase B is acetonitrile with the volume percentage of formic acid accounting for 0.15-0.2%; gradient elution is carried out for 50-60 min, the sample injection amount is 8-10 mu l, and the flow rate is 0.25-0.35 ml/min.
In step S107 provided in the embodiment of the present invention, the conditions of mass spectrometry in the liquid chromatography-mass spectrometry are as follows: the atomization pressure is 55-60 psi, the temperature of the sheath gas is 420-450 ℃, and the capillary tube voltage is 5500-6000V.
In step S107 provided in the embodiment of the present invention, the standard curve positions are: y-0.000239987 +0.0684449x, the set-up procedure was from the preparation of a standard series of solutions and the preparation of rat plasma standard curve samples.
The present invention will be further described with reference to the following examples.
Examples
1. Intravenous administration procedure in rats
(1) Selecting a male rat with the weight of 200g +/-10 g, and freely eating;
(2) and (3) purchasing a commercially available monoclonal antibody medicament, weighing the monoclonal antibody medicament to be analyzed, and preparing a raw material medicament solution.
(3) The raw material medicine solution is respectively diluted into different concentrations by the same solvent to be used as a series of standard solutions, and the relative concentration of each component in each standard solution is measured by taking the total concentration of the raw material medicine solution as a marked quantity.
(4) According to the clinical dosage conversion, the rat monoclonal antibody drug is administered by intravenous injection according to the dosage of 50 mg/kg.
2. Preparation of rat plasma samples
And (3) administering the solution to healthy test animals by intragastric administration or intravenous injection, collecting animal blood samples in a polyethylene tube, and reducing plasma samples to prepare corresponding plasma solutions.
3. Single-resistant drug content determination in rat plasma sample
And adding 0.5-0.75 mg of solid cysteine blocking agent into the obtained plasma solution, uniformly mixing by vortex, and carrying out cysteine blocking treatment to obtain reaction liquid.
Adding 500-700 mu L of digestion buffer solution into the reaction solution sealed by cysteine, mixing in a vortex mode, adding 3 mu L of pancreatin solution, mixing in a vortex mode, and incubating to obtain plasma sample enzymatic hydrolysate (FIG. 6 is a typical chromatogram of a specific peptide fragment in a plasma sample obtained in the embodiment of the invention, and is the same as the typical chromatogram of a specific peptide fragment standard product after monoclonal antibody enzymatic hydrolysis given in FIG. 4);
adding 10-15 mu L of stable isotope labeled characteristic peptide segment and internal standard peptide segment into the enzymolysis solution, mixing uniformly by vortex, and carrying out mixed enzymolysis on the characteristic peptide segment and the internal standard peptide segment (FIG. 7 is a typical chromatogram of the internal standard peptide segment in the plasma sample obtained by the embodiment of the invention, and is the same as the typical chromatogram of the internal standard peptide segment of the stable isotope synthesized in FIG. 5);
and (3) carrying out liquid chromatography-mass spectrometry on the reaction solution after enzymolysis, and substituting the ratio of the peak area of the selected characteristic peptide segment to the peak area of the internal standard peptide segment into a standard curve to obtain the drug concentration of the monoclonal antibody.
Respectively taking 2 mu L of complex solution at different time points to perform LC/MS/MS analysis, recording chromatograms, wherein a typical chromatogram of the specific peptide fragment after the plasma sample is subjected to enzymolysis is shown in figure 6, and a typical chromatogram of the internal standard peptide fragment after the plasma sample is subjected to enzymolysis is shown in figure 7.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A method for analyzing a pharmacokinetic mass spectrum of a monoclonal antibody drug, the method comprising the steps of:
weighing a monoclonal antibody drug to be analyzed, and preparing a bulk drug solution; the monoclonal antibody drug to be analyzed is a biological targeting drug;
step two, respectively diluting the raw material medicine solution into different concentrations by using a solvent, taking five parts of solutions with different concentrations as series standard solutions, taking the total concentration of the raw material medicine solution as a marked quantity, and measuring the relative concentration of each component in each standard solution;
the concentrations of the series of standard solutions are respectively 10 mu mol/L, 15 mu mol/L, 20 mu mol/L, 25 mu mol/L and 30 mu mol/L;
step three, administering the solution prepared in the step two to healthy tested animals in a gastric perfusion or intravenous injection mode, collecting animal blood samples in a polyethylene tube, and processing the blood samples to obtain plasma samples; preparing corresponding plasma solutions by using the plasma sample and the series of standard solutions;
the healthy test animal is a male Wistar rat with the mass of 200 +/-10 g, and is cultured for 10-15 days before gastric lavage or intravenous injection, wherein the specific culture conditions are as follows: setting the temperature of the culture chamber to be 20-25 ℃, the culture humidity to be 45-60%, and the illumination in the culture chamber to be 12h light and shade alternation;
the method comprises the following steps of collecting an animal blood sample in a polyethylene tube, and processing the blood sample, and specifically comprises the following steps:
collecting blood of healthy test animals at 0.25h, 0.5h, 1h, 2h, 4h, 12h and 24h after administration, and taking 500 mu L venous blood through retrobulbar venous plexus, namely a blood sample;
sucking a blood sample into a centrifuge tube with disodium ethylene diamine tetraacetate anticoagulant, standing for 20min, then centrifuging for 10-12min at the temperature of 4 ℃, wherein the centrifugal speed is 3000rpm, and separating plasma to obtain a plasma sample;
the plasma sample and the series of standard solutions are used for preparing corresponding plasma solutions, and the plasma solutions specifically comprise:
respectively placing 100 mu L of series of standard solutions with different concentrations in a centrifuge tube, and respectively placing in a nitrogen blowing instrument for drying in water bath at 40 ℃;
drying, adding 100 mu L of plasma sample, mixing for 1min in a vortex mode, sequentially adding 10 mu L of internal standard working solution and 300 mu L of methanol, mixing uniformly, and centrifuging for 5min at 12000rpm in a high-speed centrifuge to obtain supernatant;
placing the supernatant in a centrifuge tube, drying in a nitrogen blowing instrument in water bath at 40 deg.C, adding 200 μ L methanol into the dried residue, redissolving, vortexing for 30s, and centrifuging again to obtain supernatant, i.e. plasma solution;
step four, adding 0.5-0.75 mg of solid cysteine sealing agent into the plasma solution obtained in the step three, mixing uniformly by vortex, and sealing cysteine to obtain a reaction solution;
adding 500-700 mu L of digestion buffer solution into the reaction solution sealed by cysteine, mixing in a vortex mode, adding 3 mu L of pancreatin solution, mixing in a vortex mode, and incubating to obtain plasma sample enzymolysis liquid;
adding 10-15 mu L of stable isotope labeled characteristic peptide segment and internal standard peptide segment into the enzymolysis liquid, mixing uniformly by vortex, and mixing and carrying out enzymolysis on the characteristic peptide segment and the internal standard peptide segment;
and seventhly, performing liquid chromatography-mass spectrometry on the reaction liquid after enzymolysis by using a liquid chromatography tandem mass spectrometer, and substituting the ratio of the peak area of the selected characteristic peptide segment to the peak area of the internal standard peptide segment into a standard curve to obtain the drug concentration of the monoclonal antibody.
2. The method for pharmacokinetic mass spectrometry of a monoclonal antibody drug according to claim 1, wherein in step two, the solvent is chromatographically pure methanol.
3. The method for pharmacokinetic mass spectrometry of a monoclonal antibody drug according to claim 1, wherein in step three, the experimental animal is a mouse, a rat, a guinea pig, a rabbit, or a dog.
4. The method for mass spectrometric pharmacokinetic analysis of a monoclonal antibody drug of claim 1, wherein in step four, the cysteine blocking treatment is performed under the following conditions: incubating for 40-45 min at 25-27 ℃ in the dark.
5. The method for pharmacokinetic mass spectrometry of a monoclonal antibody drug according to claim 1, wherein in step five, the enzymatic hydrolysis method comprises: performing enzyme digestion treatment by using trypsin; BSA is added in the enzymolysis process, and the mass volume percentage of the BSA is 1-1.5%.
6. The method for pharmacokinetic mass spectrometry of a monoclonal antibody drug according to claim 1, wherein in step five, the incubation method is: incubating at 37 ℃ for 12-16 h to obtain plasma sample enzymolysis liquid, and adding 3-5 mu L of formic acid to terminate the reaction.
7. The method for pharmacokinetic mass spectrometry of a monoclonal antibody drug according to claim 1, wherein in the sixth step, the stable isotope labeling included in the characteristic peptide fragment and the internal standard peptide fragment is performed on the N and C elements of proline in the characteristic peptide fragment and the internal standard peptide fragment respectively15N and13and C, marking.
8. The method for pharmacokinetic mass spectrometry of a monoclonal antibody drug according to claim 1, wherein in step seven, the liquid chromatography conditions in the liquid chromatography-mass spectrometry are as follows: adopting a Peptide CSHTM C18 chromatographic column, wherein the specification of the chromatographic column is 2.1mm multiplied by 100mm and 1.8 mu m; the column temperature is 40-41 ℃; the mobile phase A is water with the volume percentage of formic acid accounting for 0.15-0.2%; the mobile phase B is acetonitrile with the volume percentage of formic acid accounting for 0.15-0.2%; gradient elution is carried out for 50-60 min, the sample injection amount is 8-10 mu l, and the flow rate is 0.25-0.35 ml/min.
9. The method for pharmacokinetic mass spectrometry of a monoclonal antibody drug according to claim 1, wherein in step seven, the conditions for mass spectrometry in the liquid chromatography-mass spectrometry are: the atomization pressure is 55-60 psi, the temperature of the sheath gas is 420-450 ℃, and the capillary tube voltage is 5500-6000V.
10. The method for mass spectrometric pharmacokinetic analysis of a monoclonal antibody drug of claim 1, wherein in step seven, the standard curve y is 0.000239987+0.0684449x, and the establishment process is performed by preparing standard series solutions and rat plasma standard curve samples.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113884561A (en) * | 2021-05-14 | 2022-01-04 | 谱天（天津）生物科技有限公司 | Quality control method for evaluating proteome reduction alkylation efficiency |
| CN114019065A (en) * | 2021-10-20 | 2022-02-08 | 澳门科技大学 | Pharmacokinetic analysis method for covalent drug and metabolite thereof |
| CN114236010A (en) * | 2021-12-18 | 2022-03-25 | 苏州莱奥生物技术有限公司 | Pharmacokinetic analysis method of bioactive drug |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113884561A (en) * | 2021-05-14 | 2022-01-04 | 谱天（天津）生物科技有限公司 | Quality control method for evaluating proteome reduction alkylation efficiency |
| CN113884561B (en) * | 2021-05-14 | 2022-02-22 | 谱天（天津）生物科技有限公司 | Quality control method for evaluating proteome reduction alkylation efficiency |
| CN114019065A (en) * | 2021-10-20 | 2022-02-08 | 澳门科技大学 | Pharmacokinetic analysis method for covalent drug and metabolite thereof |
| CN114236010A (en) * | 2021-12-18 | 2022-03-25 | 苏州莱奥生物技术有限公司 | Pharmacokinetic analysis method of bioactive drug |
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