CN110045028A - A kind of Preclinical metabolism and pharmacokinetics study mass spectrometric analysis method of Avastin - Google Patents
A kind of Preclinical metabolism and pharmacokinetics study mass spectrometric analysis method of Avastin Download PDFInfo
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Abstract
A kind of pharmacokinetics mass spectrometric analysis method of monoclonal antibody drug of the invention, belongs to the technical field of proteomics.It adopts the technical scheme that, chooses the specific peptide fragment that obtains through pancreatin hydrolysis Bevacizumab, wherein specificity refers to that peptide fragment is only generated by Bevacizumab through pancreatin hydrolysis, other protein and protein drug can not generate the peptide fragment through pancreatin hydrolysis；Based on liquid chromatography-tandem mass spectrometry technology, analysis is scanned to specific peptide fragment using parallel reaction monitoring pattern, establishes specific peptide fragment stabilization, reliable LC/MS/MS quantitative approach.The present invention has the characteristics that high throughput, high sensitivity, favorable reproducibility；Agents useful for same and drug are more cheap and easy to get, and preparation method is simple, are conducive to method and promote；Sample handling processes and quantitative approach are simple and easy, have to the qualitative and quantitative study of other protein medicaments and polypeptide drugs and instruct reference.
Description
Technical field
The invention belongs to the technical fields of proteomics, are related to a kind of therapeutic monoclonal antibodies drug Avastin
(Bevacizumab) pharmacokinetics mass spectrometric analysis method.
Background technique
Monoclonal antibody drug is a kind of tool based on γ type immunoglobulin (Immunoglobulin G) structure
There is the pharmaceutical grade protein of special treatment effect, it is disorderly to be successfully applied to cancer, autoimmune disease, virus infection and nervous centralis
The clinical treatment of a variety of diseases such as random.Compared with traditional small-molecule drug or Chinese materia medica preparation, monoclonal antibody drug has bright
True pharmacological activity and excellent pharmacokinetics；Extent of metabolism is lower in vivo for monoclonal antibody, can protect for a long time in body fluid
Higher concentration is held, serum elimination half-life period is averagely up to several weeks to the several months.Therefore, the reliable analysis method of specification is established in time simultaneously
Carry out monoclonal antibody drug pharmacokinetic for supporting monoclonal antibody drug research and development to have a very important significance.
Currently, protein medicaments pharmacokinetic mostly uses Enzyme-linked Immunosorbent Assay method (enzyme-linked
Immunosorbent assays, ELISA) and radioimmunoassay labeling method (radioimmunoassay, RIA).Wherein
ELISA is because of its higher sensitivity and is suitble to the advantages that measuring in batches, it has also become the most commonly used protein and peptide drugs biology sample
Product analysis method.However, in monoclonal antibody Pharmacokinetic experiments, ELISA method quantification range it is relatively narrow (usually one or two
The order of magnitude), cannot monoclonal antibody (that is, monoclonal antibody) drug to different shape carry out while analyzing, and it is long there are the development cycle
It is (half a year to several years), at high cost (hundreds of thousands of to million/drug target), cannot be distinguished endogenous protein interference the problems such as, greatly
The quantitative dependable with function of ELISA is affected greatly.For RIA method, the target of some radio-labelled molecules is anti-
Cognition shows the absorption different from proto-drug, distribution and metabolic characteristics；In addition contain radiolabeled monoclonal antibody
Be typically only capable to be stabilized 2-3 weeks, can not adapt in long-term monoclonal antibody pharmacokinetic.Therefore it is reliable to establish specification
Analysis method to monoclonal antibody drug research and development be of great significance.
Summary of the invention
The technical problem to be solved by the present invention is to overcome existing protein medicaments pharmacokinetic analysis method to influence fixed
The shortcomings that dependable with function of amount, establishes the pharmacokinetics mass spectrum point of monoclonal antibody drug Bevacizumab a kind of
Analysis method.
The technical solution adopted by the present invention is that the specific peptide fragment obtained through pancreatin hydrolysis Bevacizumab is chosen, wherein
Specificity refers to that peptide fragment is only generated by Bevacizumab through pancreatin hydrolysis, other protein and protein drug are through pancreatin water
Solution can not generate the peptide fragment.Based on liquid chromatography-tandem mass spectrometry (LC/MS/MS) technology, (PRM) is monitored using parallel reaction
Mode is scanned analysis to specific peptide fragment, establishes specific peptide fragment stabilization, reliable LC/MS/MS quantitative approach.It takes
The method of stating, which can be realized, quantifies Bevacizumab level using normal peptide fragment.Normal peptide fragment is peptide fragment to be measured.
The specific technical solution of the present invention is as described below.
A kind of pharmacokinetics mass spectrometric analysis method of monoclonal antibody drug, the monoclonal antibody drug cut down for shellfish
Pearl monoclonal antibody (Bevacizumab)；The measurement of monoclonal antibody concentration in plasma sample is carried out by following 7 step；
(1) reduction of plasma sample: being added the plasma sample of 2 mL rats in polyethylene pipe, and 96 mL are added and contain denaturant
PH buffer, vortex mix；2 mL reducing agents are added, vortex mixes；60 DEG C are incubated for 1 hour, obtain reaction solution；
(2) closing of cysteine: being added 0.75 mg solid cysteine sealer in reaction solution, and vortex mixes；25 DEG C are kept away
Light is incubated for 40 min；
(3) the pancreatin digestion of plasma sample: being added 700 mL digestion buffer in the closed reaction solution of cysteine, vortex is mixed
It closes, adds 3 mL trypsin solutions, vortex mixing, 37 DEG C of incubation 12-16 h obtain plasma sample enzymolysis liquid, and 5 mL formic acid are added
Terminate reaction；
(4) interior target is introduced into: the internal standard peptide fragment of 10 mL stable isotope labelings being added in enzymolysis liquid, vortex mixes, and obtains blood
The reaction solution of slurry samples；
(5) Solid Phase Extraction of blood plasma reaction solution: 1 mL acetonitrile solution is added in solid-phase extraction column, until liquid has flowed naturally；In
1 mL eluent solution is added in solid-phase extraction column, until liquid has flowed naturally；The reaction solution of plasma sample is taken, until liquid flows naturally
It is complete；1 mL eluent solution is added in solid-phase extraction column, until liquid has flowed naturally, washes for 3 times；1 mL is added in solid-phase extraction column
Elution solution obtains plasma sample eluent, is placed in polyethylene pipe until liquid has flowed naturally；
(6) concentration of plasma sample is redissolved: plasma sample eluent traditional vacuum being evaporated, 100 mL, 0.1% formic acid, whirlpool is added
Stream mixes, and obtains the redissolution liquid of plasma sample；
(7) it the mass spectral analysis of plasma sample: takes plasma sample to redissolve 2 mL of liquid and carries out liquid chromatography-tandem mass spectrometry analysis, record
Selected specific peptide fragment peak area and internal standard peak area ratio are substituted into standard curve y=0.000239987+ by chromatogram
0.0684449x, wherein x is specific peptide fragment peak area and internal standard peak area ratio, and y is that the monoclonal antibody as unit of ng/mL is dense
Degree, acquires peptide fragment concentration, this peptide fragment concentration is Bevacizumab concentration in corresponding plasma sample.
Standard curve y=the 0.000239987+0.0684449x, establishment process have standard serial solution configuration and
The preparation of rat plasma standard curve sample；
The configuration of the monoclonal antibody standard serial solution is to utilize deionized water by monoclonal antibody standard solution stock solution (25 mg/mL)
It is diluted to 5,10,40,100,500,1000,1200 ng/mL respectively；
The preparation of the rat plasma standard curve sample, there is following 7 step,
(1) reduction of rat plasma standard curve sample: the monoclonal antibody mark being separately added into polyethylene pipe in 2 mL above-mentioned steps
Quasi- serial solution；The blank plasma of 2 mL rats is added, vortex mixes；The pH buffer that 94 mL contain denaturant is added, is vortexed
It mixes；2 mL reducing agents are added, vortex mixes；60 DEG C of 1 h of incubation, obtain reaction solution；
(2) closing of cysteine: being added 0.75 mg solid cysteine sealer in reaction solution, and vortex mixes；25 DEG C are kept away
Light is incubated for 40 min；
(3) 700 mL digestion the pancreatin digestion of rat plasma standard curve sample: is added in the closed reaction solution of cysteine
Buffer, vortex mixing add 3 mL trypsin solutions, vortex mixing, and 37 DEG C of incubation 12-16 h obtain monoclonal antibody rat plasma mark
Directrix curve sample enzymolysis liquid, the rear 5 mL formic acid that are added terminate reaction；
(4) interior target is introduced into: 10 mL stable isotope labelings are added in Yu Dankang rat plasma standard curve sample enzymolysis liquid
Internal standard peptide fragment, vortex mix, and obtain monoclonal antibody rat plasma standard curve example reaction liquid；
(5) it is molten that 1 mL acetonitrile the Solid Phase Extraction of monoclonal antibody rat plasma standard curve example reaction liquid: is added in solid-phase extraction column
Liquid, until liquid has flowed naturally；1 mL eluent solution is added in solid-phase extraction column, until liquid has flowed naturally；Step (4) is taken to obtain
Monoclonal antibody rat plasma standard curve example reaction liquid be added in solid-phase extraction column, until liquid has flowed naturally；In Solid Phase Extraction
1 mL eluent solution is added in column, until liquid has flowed naturally, washes for three times；1 mL elution solution is added in solid-phase extraction column, until
Liquid has flowed naturally, obtains monoclonal antibody rat plasma standard curve sample eluent, is placed in polyethylene pipe；
(6) concentration, redissolution of monoclonal antibody rat plasma standard curve sample: monoclonal antibody rat plasma standard curve sample eluent is true
Sky centrifugation is evaporated, and 100 mL, 0.1% formic acid is added, and vortex mixes, and is obtained monoclonal antibody rat plasma standard curve sample and is redissolved liquid；
(7) mass spectral analysis of monoclonal antibody rat plasma standard curve sample:
It takes 2 mL monoclonal antibody rat plasma standard curve samples to redissolve liquid and carries out liquid chromatography-tandem mass spectrometry analysis, record chromatogram,
Using monoclonal antibody concentration in initial monoclonal antibody standard serial solution as abscissa, specific peptide fragment peak area and internal standard peak after selected enzymatic hydrolysis
Area ratio is ordinate, with weighting W=1/x2Least square method carries out regressing calculation, and the linear regression equation acquired is as marked
Directrix curve.
The mass spectrometric analysis method of therapeutic monoclonal antibodies drug of the invention, the pH buffering containing denaturant
Liquid is the 50 mM HEPES buffer solutions (4- hydroxyethyl piperazineethanesulfonic acid buffer solution) containing 8 M urea；The reduction
Agent is the dithiothreitol (DTT) solution of 1 M；The cysteine sealer is solid iodoacetamide reagent；The digestion is slow
Fliud flushing is the HEPES buffer solution (4- hydroxyethyl piperazineethanesulfonic acid buffer solution) of 50 mM；The trypsin solution is 1
The pancreatin aqueous solution of mg/mL；The eluent solution is water/trifluoroacetic acid=100/0.1 solution by volume；Described washes
Precipitation liquid is acetonitrile/water/trifluoroacetic acid=80/20/0.1 solution by volume.
During the preparation of rat plasma standard curve and plasma sample monoclonal antibody concentration mensuration,
Chromatographic condition are as follows: match 3000 series of high efficiency liquid chromatographic system of Mo Feishier company Ultimate in the U.S.；Chromatographic column: from
Peptide C18 capillary chromatographic column processed, 15 cm × 75 μm I.D., 5 μm of partial sizes；Mobile phase: formic acid percentage by volume accounts for
0.1% water and formic acid percentage by volume account for 0.1% acetonitrile, gradient elution；300 nL/min of flow velocity；Sample volume 2mL；
Mass Spectrometry Conditions are as follows: Q-Exactive Orbitrap level four bars-orbit trap liquid chromatography-tandem mass spectrometry instrument are furnished with nanoliter level
2.1 data processing software of electro-spray ionization source and Xcalibur；Ion source: nanoliter level electro-spray ionization source；Cation side
Formula detection；Scanning mode is parallel reaction monitoring；Sweeping resolution ratio entirely is 70000, and targeted scans resolution ratio is 35000, maximum note
The angle of incidence is 200 ms.
In a kind of chromatographic condition of the mass spectrometric analysis method of therapeutic monoclonal antibodies drug of the invention, the gradient
Elution program, process are shown in Table 1,
1 gradient elution program of table
| Time (min) | Flow velocity (mL/min) | %A | %B |
| 0 | 0.3 | 95 | 5 |
| 10 | 0.3 | 95 | 5 |
| 40 | 0.3 | 55 | 45 |
| 42 | 0.3 | 10 | 90 |
| 50 | 0.3 | 10 | 90 |
| 51 | 0.3 | 98 | 2 |
| 60 | 0.3 | 98 | 2 |
Wherein A is the aqueous solution that formic acid percentage by volume accounts for 0.1%, and B is the acetonitrile solution that formic acid percentage by volume accounts for 0.1%.
Present invention has an advantage that
1, the enzymatic hydrolysis specificity peptide fragment of monoclonal antibody is analyzed and is quantified, there are the spies such as high throughput, high sensitivity, favorable reproducibility
Point.2, agents useful for same and drug of the present invention are more cheap and easy to get compared with the prior art, and preparation method is simple, are conducive to method and promote.3,
Sample handling processes and quantitative approach of the invention are simple and easy, are applicable not only to heretofore described monoclonal antibody medicine
Bevacizumab also has the qualitative and quantitative study of other protein medicaments and polypeptide drugs and instructs reference.
Detailed description of the invention
Fig. 1 is the typical chromatogram of specific peptide segment standard product after monoclonal antibody enzymatic hydrolysis.
Fig. 2 is the typical chromatogram of the internal standard peptide fragment of the stable isotope of synthesis.
Fig. 3 is the typical chromatogram of specific peptide fragment in plasma sample that embodiment 1 obtains.
Fig. 4 is the typical chromatogram of internal standard peptide fragment in plasma sample that embodiment 1 obtains.
Fig. 5 is the Drug-time curve of monoclonal antibody medicine Bevacizumab in rat plasma sample that embodiment 1 obtains.
Fig. 6 is the standard curve for the monoclonal antibody rat plasma that embodiment 2 obtains.
Specific embodiment
Embodiment 1: the analysis of monoclonal antibody medicine in rat plasma sample
First part: rat vein administration
A, rat vein administration process
(1) choosing weight is mono- male rat of ± 10 g of 200 g, free diet；
(2) commercially available monoclonal antibody drug Bevacizumab injection is bought, concentration is 25 mg/mL；
(3) according to clinical administration dosage conversions, Rat monoclonal is given by 50 mg/kg dosages using intravenous injection mode and is resisted
Body drug；
B, the preparation of rat plasma sample
Selecting different time points respectively, (0,30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 32 h), from venous collection
Rat whole blood, is centrifugated serum 5min at 13000 g, and -80 DEG C of refrigerators save.
Second part: the assay of monoclonal antibody medicine Bevacizumab in rat plasma sample
A, plasma sample pre-processes
(1) plasma sample of 2 mL rats is added in the reduction of plasma sample in polyethylene pipe, and 96 mL are added and contain denaturant
PH buffer, vortex mix, be added 2 mL reducing agents, vortex mix, 60 DEG C be incubated for 1 hour, obtain reaction solution；
(2) closing of cysteine, is added 0.75 mg solid cysteine sealer in reaction solution, and vortex mixes；25 DEG C incubate
Educate 40 min；
(3) the pancreatin digestion of plasma sample, is added 700 mL digestion buffer in the closed reaction solution of cysteine, vortex is mixed
It closes, adds 3 mL trypsin solutions, vortex mixing, 37 DEG C of incubation 12-16 h obtain plasma sample enzymolysis liquid, and 5 mL formic acid are added
Terminating reaction, (Fig. 3 is the typical chromatogram of specific peptide fragment in plasma sample that the present embodiment obtains, the list provided with Fig. 1
The typical chromatogram of specific peptide segment standard product is identical after resistance to enzymolysis)；
(4) interior target introduces, and the internal standard peptide fragment of 10 mL stable isotope labelings is added in enzymolysis liquid, and vortex mixes, and obtains blood
(Fig. 4 is the typical chromatogram of internal standard peptide fragment in plasma sample that the present embodiment obtains to the reaction solution of slurry samples, is synthesized with Fig. 2
The typical chromatogram of the internal standard peptide fragment of stable isotope is identical)；
(5) 1 mL acetonitrile solution is added in the Solid Phase Extraction of blood plasma reaction solution in solid-phase extraction column, until liquid has flowed naturally, in
1 mL eluent solution is added in solid-phase extraction column, until liquid has flowed naturally, takes the reaction solution of plasma sample, until liquid flows naturally
It is complete, 1 mL eluent solution is added in solid-phase extraction column, until liquid has flowed naturally, washes for three times, 1 is added in solid-phase extraction column
ML elution solution obtains plasma sample eluent, is placed in polyethylene pipe until liquid has flowed naturally；
(6) concentration, redissolution of plasma sample, plasma sample eluent traditional vacuum obtained in above-mentioned steps is evaporated, is added
100 mL, 0.1% formic acid, vortex mix, and obtain the redissolution liquid of plasma sample.
(7) mass spectral analysis of plasma sample takes plasma sample to redissolve 2 mL of liquid and carries out liquid chromatography-tandem mass spectrometry analysis,
Chromatogram is recorded, selected specific peptide fragment peak area and internal standard peak area ratio are substituted into standard curve (referring to embodiment 2),
Peptide fragment concentration is acquired, this peptide fragment concentration is Bevacizumab concentration in corresponding plasma sample.
Solution formula involved in above-mentioned steps is as follows:
PH buffer containing denaturant is the 50 mM HEPES buffer solutions containing 8 M urea；
Reducing agent is the dithiothreitol (DTT) solution of 1 M；
Cysteine sealer is solid iodoacetamide reagent；
Buffer is digested, is the HEPES buffer solution of 50 mM；Trypsin solution is the pancreatin aqueous solution of 1 mg/mL；
Eluent solution is water/trifluoroacetic acid=100/0.1 solution by volume；Elute solution, be by volume acetonitrile/water/
The solution of trifluoroacetic acid=80/20/0.1.
B, monoclonal antibody medicine assay in plasma sample
Different time points after being handled by step A of learning from else's experience respectively redissolve 2 mL of liquid and carry out LC/MS/MS analysis, record chromatogram, blood plasma
Specific peptide fragment typical color spectrogram such as Fig. 3, internal standard peptide fragment typical color spectrogram such as Fig. 4 after plasma sample digests after sample enzymatic hydrolysis, will
The ratio of specific peptide fragment peak area and internal standard peptide fragment peak area substitutes into standard curve, acquires peptide fragment concentration, as plasma sample
Middle monoclonal antibody medicine concentration, Drug-time curve such as Fig. 5 of monoclonal antibody medicine Bevacizumab in obtained rat plasma sample.
Monoclonal antibody Bevacizumab specificity peptide fragment involved in above-mentioned steps and the condition of internal standard peptide fragment measurement are such as
Under:
1, chromatographic condition are as follows: match 3000 series of high efficiency liquid chromatographic system of Mo Feishier company Ultimate in the U.S.；Chromatographic column:
Make peptide C18 capillary chromatographic column, 15 cm × 75 μm I.D., 5 μm of partial sizes by oneself；Mobile phase: formic acid percentage by volume accounts for
0.1% water and formic acid percentage by volume account for 0.1% acetonitrile, gradient elution；300 nL/min of flow velocity；2 μ L of sample volume；
The gradient elution, program can be found in table 1.
2, Mass Spectrometry Conditions are as follows: Q-Exactive Orbitrap level four bars-orbit trap liquid chromatography-tandem mass spectrometry instrument are furnished with
2.1 data processing software of nanoliter level electro-spray ionization source and Xcalibur；Ion source: nanoliter level electro-spray ionization source；Just
Ionic means detection；Scanning mode is parallel reaction monitoring；Sweeping resolution ratio entirely is 70000, and targeted scans resolution ratio is 35000,
Maximum injection length is 200 ms.
3 peptide fragment parallel reaction monitoring and setting value of table
| Peptide section sequence | Parent ion karyoplasmic ratio | Extract ion karyoplasmic ratio | NCE |
| FTFSLDTSK | 523.26 | 797.39 | 28 |
| FTFSLDTSK* | 526.88 | 805.41 | 28 |
A kind of mass spectrometric analysis method linear relationship of monoclonal antibody drug of the present invention is good, accuracy is high, reproducibility
It is good and sensitive, reliable, it can be used for the pharmacokinetic analysis measurement of said monoclonal antibody drug Bevacizumab.
2 rat plasma standard curve sample preparation of embodiment
Monoclonal antibody standard serial solution is configured first: using deionized water that monoclonal antibody standard solution stock solution (25 mg/mL) is dilute respectively
It releases to 5,10,40,100,500,1000,1200 ng/mL.
Rat plasma standard curve sample is prepared again, and point following 7 step carries out:
(1) reduction of rat plasma standard curve sample is separately added into 2 mL monoclonal antibody standard serial solutions in polyethylene pipe, adds
Enter the blank plasma of 2 mL rats, vortex mixes, and the pH buffer that 94 mL contain denaturant is added, and vortex mixes, and 2 mL are added
Reducing agent, vortex mix, and 60 DEG C of 1 h of incubation obtain reaction solution；
(2) closing of cysteine, is added 0.75 mg solid cysteine sealer in reaction solution, and vortex mixes；25 DEG C are kept away
Light is incubated for 40 min；
(3) the pancreatin digestion of rat plasma standard curve sample, is added 700 mL digestion in the closed reaction solution of cysteine
Buffer, vortex mixing add 3 mL trypsin solutions, vortex mixing, and 37 DEG C of incubation 12-16 h obtain monoclonal antibody rat plasma mark
The enzymolysis liquid of directrix curve sample, the rear 5 mL formic acid that are added terminate reaction；
(4) interior target introduces, and the internal standard peptide fragment of 10 mL stable isotope labelings is added in enzymolysis liquid, and vortex mixes, and obtains list
The reaction solution of anti-rat plasma standard curve sample；
(5) it is molten that 1 mL acetonitrile is added in the Solid Phase Extraction of monoclonal antibody rat plasma standard curve example reaction liquid in solid-phase extraction column
1 mL eluent solution is added until liquid has flowed naturally in liquid in solid-phase extraction column, until liquid has flowed naturally, takes monoclonal antibody rat serum
1 mL eluent solution is added until liquid has flowed naturally in the reaction solution of slurry standard curve sample in solid-phase extraction column, until liquid is certainly
It has so flowed, has washed for three times, 1 mL elution solution is added in solid-phase extraction column until liquid has flowed naturally and obtains monoclonal antibody rat plasma mark
Directrix curve sample eluent, is placed in polyethylene pipe；
(6) concentration, redissolution of monoclonal antibody rat plasma standard curve sample, by monoclonal antibody rat plasma standard obtained in above-mentioned steps
Curve sample eluent traditional vacuum is evaporated, and 100 mL, 0.1% formic acid is added, and vortex mixes, and obtains monoclonal antibody rat plasma standard
The redissolution liquid of curve sample.
(7) mass spectral analysis of monoclonal antibody rat plasma standard curve sample takes 2 mL to carry out liquid chromatography-tandem mass spectrometry point
Analysis records chromatogram, using monoclonal antibody concentration in initial monoclonal antibody standard serial solution as abscissa, specific peptide fragment after selected enzymatic hydrolysis
Peak area and internal standard peak area ratio are ordinate, with weighting W=1/x2Least square method carries out regressing calculation, the straight line acquired
Regression equation, as standard curve.Liquid chromatography-tandem mass spectrometry condition is the same as embodiment 1.In Fig. 6 regression equation, x is specificity
Peptide fragment peak area and internal standard peak area ratio, y are monoclonal antibody concentration.
Claims (5)
1. a kind of pharmacokinetics mass spectrometric analysis method of monoclonal antibody drug, the monoclonal antibody drug is that shellfish cuts down pearl
Monoclonal antibody；The measurement of monoclonal antibody concentration in plasma sample is carried out by following 7 step；
(1) reduction of plasma sample: being added the plasma sample of 2 mL rats in polyethylene pipe, and 96 mL are added and contain denaturant
PH buffer, vortex mix；2 mL reducing agents are added, vortex mixes；60 DEG C are incubated for 1 hour, obtain reaction solution；
(2) closing of cysteine: being added 0.75 mg solid cysteine sealer in reaction solution, and vortex mixes；25 DEG C are kept away
Light is incubated for 40 min；
(3) the pancreatin digestion of plasma sample: being added 700 mL digestion buffer in the closed reaction solution of cysteine, vortex is mixed
It closes, adds 3 mL trypsin solutions, vortex mixing, 37 DEG C of 12 ~ 16 h of incubation obtain plasma sample enzymolysis liquid, and 5 mL formic acid are added
Terminate reaction；
(4) interior target is introduced into: the internal standard peptide fragment of 10 mL stable isotope labelings being added in enzymolysis liquid, vortex mixes, and obtains blood
The reaction solution of slurry samples；
(5) Solid Phase Extraction of blood plasma reaction solution: 1 mL acetonitrile solution is added in solid-phase extraction column, until liquid has flowed naturally；In
1 mL eluent solution is added in solid-phase extraction column, until liquid has flowed naturally；The reaction solution of plasma sample is taken, until liquid flows naturally
It is complete；1 mL eluent solution is added in solid-phase extraction column, until liquid has flowed naturally, washes for 3 times；1 mL is added in solid-phase extraction column
Elution solution obtains plasma sample eluent, is placed in polyethylene pipe until liquid has flowed naturally；
(6) concentration of plasma sample is redissolved: plasma sample eluent traditional vacuum being evaporated, 100 mL, 0.1% formic acid, whirlpool is added
Stream mixes, and obtains the redissolution liquid of plasma sample；
(7) it the mass spectral analysis of plasma sample: takes plasma sample to redissolve 2 mL of liquid and carries out liquid chromatography-tandem mass spectrometry analysis, record
Selected specific peptide fragment peak area and internal standard peak area ratio are substituted into standard curve y=0.000239987+ by chromatogram
0.0684449x, wherein x is specific peptide fragment peak area and internal standard peak area ratio, and y is that the monoclonal antibody as unit of ng/mL is dense
Degree, acquires peptide fragment concentration, this peptide fragment concentration is Avastin concentration in corresponding plasma sample.
2. the pharmacokinetics mass spectrometric analysis method of monoclonal antibody drug according to claim 1, characterized in that described
Standard curve y=0.000239987+0.0684449x, establishment process has the configuration and rat plasma standard of standard serial solution
The preparation of curve sample；
The configuration of the monoclonal antibody standard serial solution is to utilize deionized water by monoclonal antibody standard solution stock solution (25 mg/mL)
It is diluted to 5,10,40,100,500,1000,1200 ng/mL respectively；
The preparation of the rat plasma standard curve sample, there is following 7 step,
(1) reduction of rat plasma standard curve sample: the monoclonal antibody mark being separately added into polyethylene pipe in 2 mL above-mentioned steps
Quasi- serial solution；The blank plasma of 2 mL rats is added, vortex mixes；The pH buffer that 94 mL contain denaturant is added, is vortexed
It mixes；2 mL reducing agents are added, vortex mixes；60 DEG C of 1 h of incubation, obtain reaction solution；
(2) closing of cysteine: being added 0.75 mg solid cysteine sealer in reaction solution, and vortex mixes；25 DEG C incubate
It educates and is protected from light 40 min；
(3) 700 mL digestion the pancreatin digestion of rat plasma standard curve sample: is added in the closed reaction solution of cysteine
Buffer, vortex mixing add 3 mL trypsin solutions, vortex mixing, and 37 DEG C of 12 ~ 16 h of incubation obtain monoclonal antibody rat plasma mark
Directrix curve sample enzymolysis liquid, the rear 5 mL formic acid that are added terminate reaction；
(4) interior target is introduced into: 10 mL stable isotope labelings are added in Yu Dankang rat plasma standard curve sample enzymolysis liquid
Internal standard peptide fragment, vortex mix, and obtain monoclonal antibody rat plasma standard curve example reaction liquid；
(5) it is molten that 1 mL acetonitrile the Solid Phase Extraction of monoclonal antibody rat plasma standard curve example reaction liquid: is added in solid-phase extraction column
Liquid, until liquid has flowed naturally；1 mL eluent solution is added in solid-phase extraction column, until liquid has flowed naturally；Step (4) is taken to obtain
Monoclonal antibody rat plasma standard curve example reaction liquid be added in solid-phase extraction column, until liquid has flowed naturally；In Solid Phase Extraction
1 mL eluent solution is added in column, until liquid has flowed naturally, washes for three times；1 mL elution solution is added in solid-phase extraction column, until
Liquid has flowed naturally, obtains monoclonal antibody rat plasma standard curve sample eluent, is placed in polyethylene pipe；
(6) concentration, redissolution of monoclonal antibody rat plasma standard curve sample: monoclonal antibody rat plasma standard curve sample eluent is true
Sky centrifugation is evaporated, and 100 mL, 0.1% formic acid is added, and vortex mixes, and is obtained monoclonal antibody rat plasma standard curve sample and is redissolved liquid；
(7) mass spectral analysis of monoclonal antibody rat plasma standard curve sample:
It takes 2 mL monoclonal antibody rat plasma standard curve samples to redissolve liquid and carries out liquid chromatography-tandem mass spectrometry analysis, record chromatogram,
Using monoclonal antibody concentration in initial monoclonal antibody standard serial solution as abscissa, specific peptide fragment peak area and internal standard peak after selected enzymatic hydrolysis
Area ratio is ordinate, with weighting W=1/x2Least square method carries out regressing calculation, and the linear regression equation acquired is as marked
Directrix curve.
3. the pharmacokinetics mass spectrometric analysis method of monoclonal antibody drug according to claim 1 or 2, characterized in that
The pH buffer containing denaturant is the 50 mM 4- hydroxyethyl piperazineethanesulfonic acid buffer solutions containing 8 M urea；Institute
The reducing agent stated is the dithiothreitol (DTT) solution of 1M；The cysteine sealer is solid iodoacetamide reagent；It is described
Digestion buffer, be the 4- hydroxyethyl piperazineethanesulfonic acid buffer solution of 50 mM；The trypsin solution is the pancreas of 1mg/mL
Enzyme aqueous solution；The eluent solution is water/trifluoroacetic acid=100/0.1 solution by volume；The elution solution is
Acetonitrile/water/trifluoroacetic acid=80/20/0.1 solution by volume.
4. the pharmacokinetics mass spectrometric analysis method of monoclonal antibody drug according to claim 1 or 2, characterized in that
The liquid chromatography-tandem mass spectrometry analysis, chromatographic condition are as follows: it is high that the U.S. matches 3000 series of Mo Feishier company Ultimate
Effect liquid phase chromatogram system；Chromatographic column: self-control peptide C18 capillary chromatographic column, 15 cm × 75 μm I.D., 5 μm of partial sizes；
Mobile phase: formic acid percentage by volume account for 0.1% water and formic acid percentage by volume account for 0.1% acetonitrile, gradient elution；Flow velocity 300
nL/min；Sample volume 2mL；Mass Spectrometry Conditions are as follows: Q-Exactive Orbitrap level four bars-orbit trap liquid chromatography-tandem mass spectrometry
Instrument is furnished with 2.1 data processing software of nanoliter level electro-spray ionization source and Xcalibur；Ion source: nanoliter level electron spray ion
Change source；Positive ion mode detection；Scanning mode is parallel reaction monitoring；Sweeping resolution ratio entirely is 70000, and targeted scans resolution ratio is
35000, maximum injection length is 200 ms.
5. the pharmacokinetics mass spectrometric analysis method of monoclonal antibody drug according to claim 4, characterized in that described
Gradient elution, process is shown in Table 1,
1 gradient elution program of table
Wherein A is the aqueous solution that formic acid percentage by volume accounts for 0.1%, and B is the acetonitrile solution that formic acid percentage by volume accounts for 0.1%.
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| CN113219117A (en) * | 2021-05-27 | 2021-08-06 | 杭州广科安德生物科技有限公司 | Mass spectrometry method of TIMP1 protein standard substance |
| CN113804804A (en) * | 2021-09-08 | 2021-12-17 | 杭州佰辰医学检验所有限公司 | Monitoring method for rapidly determining monoclonal antibody drug in plasma by ultra-high performance liquid chromatography tandem mass spectrometry |
| CN113899894A (en) * | 2021-09-14 | 2022-01-07 | 上海中科新生命生物科技有限公司 | Method for simultaneously detecting drug concentrations of bevacizumab and trastuzumab |
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| CN111366655A (en) * | 2020-04-01 | 2020-07-03 | 上海中科新生命生物科技有限公司 | Mass spectrum detection method for bevacizumab bioanalysis based on immunoaffinity |
| CN113219117A (en) * | 2021-05-27 | 2021-08-06 | 杭州广科安德生物科技有限公司 | Mass spectrometry method of TIMP1 protein standard substance |
| CN113804804A (en) * | 2021-09-08 | 2021-12-17 | 杭州佰辰医学检验所有限公司 | Monitoring method for rapidly determining monoclonal antibody drug in plasma by ultra-high performance liquid chromatography tandem mass spectrometry |
| CN113899894A (en) * | 2021-09-14 | 2022-01-07 | 上海中科新生命生物科技有限公司 | Method for simultaneously detecting drug concentrations of bevacizumab and trastuzumab |
| CN113899894B (en) * | 2021-09-14 | 2023-11-14 | 上海中科新生命生物科技有限公司 | Method for simultaneously detecting drug concentration of bevacizumab and trastuzumab |
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