CN105758953B - A kind of glycosylation modified Method of Mass Spectrographic Quantitative Analysis of monoclonal antibody drug - Google Patents
A kind of glycosylation modified Method of Mass Spectrographic Quantitative Analysis of monoclonal antibody drug Download PDFInfo
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Abstract
A kind of glycosylation modified Method of Mass Spectrographic Quantitative Analysis of monoclonal antibody drug of the invention, belong to the technical field of proteomics.Choose Bevacizumab and its glycosylation modified a certain specific glycopeptide H5N2 is obtained after trypsin hydrolysis, based on Liquid Chromatography-Tandem Mass Spectrometry technology, analysis is scanned to the specific glycopeptide of selection using parallel reaction monitoring pattern, establishes specific sugar stabilized peptide, reliable LC/MS/MS quantitative approach.Hydrophilic enrichment is carried out to the glycopeptide in complex sample simultaneously, quantitative analysis is carried out with Same Way.The present invention can be achieved that the specific sugar-type of monoclonal antibody is analyzed and quantified, and have the characteristics that high flux, high sensitivity, favorable reproducibility;Agents useful for same and medicine are more cheap and easy to get compared with prior art, and compound method is simple, are promoted beneficial to method;Glycosylation modified qualitative and quantitative study to protein medicaments, which has, instructs reference.
Description
Technical field
The invention belongs to the technical field of proteomics, is related to a kind of therapeutic monoclonal antibodies medicine
Bevacizumab glycosylation modified pharmacokinetics Method of Mass Spectrographic Quantitative Analysis.
Background technology
Monoclonal antibody drug is a kind of tool based on γ types immunoglobulin (Immunoglobulin G) structure
There is the pharmaceutical grade protein of special treatment effect.1986, U.S. FDA have approved first monoclonal antibody drug Orthoclone
Listing, has opened the fine new page of biological medicine history.At present, monoclonal antibody drug has been successfully applied to cancer, itself exempted from
The clinical treatment of a variety of diseases such as epidemic disease, virus infection and CNS disorders.With traditional small-molecule drug or Chinese medicine system
Agent is compared, and monoclonal antibody drug has clear and definite pharmacological activity and excellent pharmacokinetics;Monoclonal antibody generation in vivo
It is relatively low to thank to degree, higher concentration can be kept for a long time in body fluid, serum elimination half-life period is averagely up to several weeks to the several months.In addition,
Numerous studies show, monoclonal antibody drug it is glycosylation modified to its stability, solubility, clearance rate, original, antibody is immunized
Dependent cells toxicity and complement dependent cytotoxicity etc. have a certain impact.For example, the glycosylation without core fucose is repaiied
Decorations can improve the compatibility to Fc γ RIII а acceptors, therefore can improve antibody-dependent cellular cytotoxicity;High mannose type glycosylation is repaiied
Decorations can reduce plasma half-life, improve immunogenicity and antibody-dependent cellular cytotoxicity.Food and medicine Surveillance Authority of the U.S. announces
On biological Counterfeit Item instruct draft also explicitly pointed out to monoclonal antibody drug glycosylation research importance.Cause
This, establishes the reliable quantitative analysis method of specification and carries out the glycosylation modified pharmacokinetics of monoclonal antibody drug and grind in time
Study carefully for supporting monoclonal antibody drug research and development tool to be of great significance.
The content of the invention
The technical problem to be solved in the present invention is to establish a kind of monoclonal antibody drug Avastin
(Bevacizumab) glycosylation modified pharmacokinetics Method of Mass Spectrographic Quantitative Analysis.
The present invention adopts the technical scheme that, chooses its glycosylation that Bevacizumab is obtained after trypsin hydrolysis
The a certain specific glycopeptide (H5N2) of modification.Based on liquid chromatography-tandem mass spectrometry (LC/MS/MS) technology, monitored using parallel reaction
(PRM) pattern is scanned analysis to the specific glycopeptide of selection, establishes specific sugar stabilized peptide, reliable LC/MS/MS quantitative squares
Method.Hydrophilic enrichment is carried out to the glycopeptide in complex sample simultaneously, quantitative study is carried out with same LC-MS/MS methods.Take above-mentioned
Method is that the pharmacokinetics mass spectrum quantitative analysis glycosylation modified to Bevacizumab can be achieved.
The concrete technical scheme of the present invention is as described below.
A kind of glycosylation modified Method of Mass Spectrographic Quantitative Analysis of monoclonal antibody drug, described monoclonal antibody drug are
Avastin (Bevacizumab);The quantitative analysis of specific glycopeptide in plasma sample is carried out by following 6 steps:
(1) reduction of plasma sample,
The plasma sample of 2 μ L rats is added in polyethylene pipe,
The pH buffer solutions that 96 μ L contain denaturant are added, vortex mixes,
2 μ L reducing agents are added, vortex mixes,
60 DEG C are incubated 1 hour, obtain reaction solution;
(2) closing of cysteine,
0.75mg solid cysteine sealers are added in reaction solution, vortex mixes;
25 DEG C of lucifuges are incubated 40min;
(3) the pancreatin digestion of plasma sample,
700 μ L digestion buffer solutions are added in reaction solution through cysteine closing, vortex mixing, it is molten to add 3 μ L pancreatin
Liquid, vortex mixing, 37 DEG C are incubated 12~16h and obtain plasma sample enzymolysis liquid, add 5 μ L formic acid terminating reactions;
(4) SPE of blood plasma enzymolysis liquid,
1mL acetonitrile solutions are added in solid-phase extraction column, have been flowed naturally to liquid,
1mL eluent solutions are added in solid-phase extraction column, have been flowed naturally to liquid,
The enzymolysis liquid of plasma sample is taken, has been flowed naturally to liquid,
1mL eluent solutions are added in solid-phase extraction column, have been flowed naturally to liquid, are washed for three times,
1mL elution solution is added in solid-phase extraction column, has been flowed naturally to liquid, has obtained plasma sample eluent, be placed in poly-
In ethylene tube;
(5) concentration, the redissolution of plasma sample,
The plasma sample eluent traditional vacuum obtained in above-mentioned steps is evaporated, adds the formic acid of 100 μ L 0.1%, vortex
Mix, obtain the redissolution liquid of plasma sample.
(6) mass spectral analysis of plasma sample,
Take plasma sample to redissolve the μ L of liquid 2 and carry out liquid chromatography-tandem mass spectrometry analysis, record chromatogram, selected specific sugar
The peak area of peptide represents its relative amount in plasma sample.
A kind of glycosylation modified Method of Mass Spectrographic Quantitative Analysis of monoclonal antibody drug, described monoclonal antibody drug are
Avastin (Bevacizumab);The quantitative analysis of specific glycopeptide after plasma sample enrichment is carried out by following 8 steps:
(1) reduction of plasma sample,
The plasma sample of 2 μ L rats is added in polyethylene pipe,
The pH buffer solutions that 96 μ L contain denaturant are added, vortex mixes,
2 μ L reducing agents are added, vortex mixes,
60 DEG C are incubated 1 hour, obtain reaction solution;
(2) closing of cysteine,
0.75mg solid cysteine sealers are added in reaction solution, vortex mixes;
25 DEG C of lucifuges are incubated 40min;
(3) the pancreatin digestion of plasma sample,
700 μ L digestion buffer solutions are added in reaction solution through cysteine closing, vortex mixing, it is molten to add 3 μ L pancreatin
Liquid, vortex mixing, 37 DEG C are incubated 12~16h and obtain plasma sample enzymolysis liquid, add 5 μ L formic acid terminating reactions;
(4) SPE of blood plasma enzymolysis liquid,
1mL acetonitrile solutions are added in solid-phase extraction column, have been flowed naturally to liquid,
1mL eluent solutions are added in solid-phase extraction column, have been flowed naturally to liquid,
The enzymolysis liquid of plasma sample is taken, has been flowed naturally to liquid,
1mL eluent solutions are added in solid-phase extraction column, have been flowed naturally to liquid, are washed for three times,
1mL elution solution is added in solid-phase extraction column, has been flowed naturally to liquid, has obtained plasma sample eluent, be placed in poly-
In ethylene tube;
(5) concentration, the redissolution of plasma sample,
The plasma sample eluent traditional vacuum obtained in above-mentioned steps is evaporated, adds acetonitrile/1% 3 of 50 μ L 80%
Fluoroacetic acid, vortex mix, and obtain the enrichment solution of plasma sample.
(6) enrichment of plasma sample
10 μ L liquid-transfering gun pipette tips tips are blocked with appropriate absorbent cotton, the hydrophilic enrichment glycopeptide materials of 5mg are dissolved in 50 μ L
The trifluoroacetic acid solution of 80% acetonitrile/1%, inserts pipette tips, and 700g is centrifuged to liquid from dry, and tip posts are made,
In the enrichment solution that plasma sample is added in tip posts, 700g is centrifuged off liquid,
In adding the trifluoroacetic acid solution of acetonitriles of 50 μ L 80%/1% in tip posts, 700g is centrifuged off liquid, washes for three times,
In adding the acetonitrile solutions of 50 μ L 10% in tip posts, 700g is centrifuged to liquid from dry, is washed for two times, collects plasma sample
Enrichment eluent;
(7) concentration, the redissolution of blood plasma enriched sample
The enrichment eluent traditional vacuum of the plasma sample obtained in above-mentioned steps is evaporated, adds the first of 20 μ L 0.1%
Acid, vortex mix, and obtain the redissolution liquid of blood plasma enriched sample.
(8) mass spectral analysis of blood plasma enriched sample,
Take blood plasma enriched sample to redissolve the μ L of liquid 4 and carry out liquid chromatography-tandem mass spectrometry analysis, record chromatogram, selected spy
The peak area for determining glycopeptide represents its relative amount in plasma sample.
The quantitative analysis of specific glycopeptide in plasma sample, after plasma sample enrichment the quantitative analysis of specific glycopeptide determined
Cheng Zhong,
Chromatographic condition is:The series of high efficiency liquid chromatographic systems of U.S. match Mo Feishier company Ultimate 3000;Chromatogram
Post:Make peptide C18 capillary chromatographic columns, 15cm × 75 μm I.D., 5 μm of particle diameters by oneself;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 300nL/min;
Mass Spectrometry Conditions are:Q-Exactive Orbitrap level Four bar-orbit trap liquid chromatography-tandem mass spectrometry instrument, equipped with receiving
Upgrade electro-spray ionization source and the data processing softwares of Xcalibur 2.1;Ion gun:Nanoliter level electro-spray ionization source;Just from
Submode detects;Scan mode monitors for parallel reaction;Full resolution ratio of sweeping is 70000, and targeted scans resolution ratio is 35000, most
Big injection length is 200ms.
The glycosylated mass spectrometric analysis method of therapeutic monoclonal antibodies medicine of the present invention, the pH containing denaturant
Buffer solution, it is 50mM 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) cushioning liquid containing 8M urea;Described reducing agent, it is 1M
Dithiothreitol (DTT) solution;Described cysteine sealer, it is solid iodoacetamide reagent;Described digestion buffer solution, it is
50mM 4- hydroxyethyl piperazineethanesulfonic acids (HEPES) cushioning liquid;Described trypsin solution, it is 1mg/mL trypsin solution;Institute
The eluent solution stated, it is the solution of water/trifluoroacetic acid=100/0.1 by volume;Described elution solution, it is second by volume
The solution of nitrile/water/trifluoroacetic acid=80/20/0.1.
In a kind of chromatographic condition of the glycosylation modified mass spectrometric analysis method of therapeutic monoclonal antibodies medicine of the invention,
Described gradient elution program, process are shown in Table 1.
The gradient elution program of table 1
In table 1, A is the aqueous solution that formic acid percentage by volume accounts for 0.1%, and B is the acetonitrile that formic acid percentage by volume accounts for 0.1%
Solution.
Advantage of the invention is that:
1st, the specific sugar-type of monoclonal antibody is analyzed and quantified, there is high flux, high sensitivity, favorable reproducibility.
2nd, agents useful for same and medicine of the present invention are more cheap and easy to get compared with prior art, and compound method is simple, is pushed away beneficial to method
Extensively.
3rd, sample handling processes and quantitative approach of the invention are simple and easy, are applicable not only to heretofore described monoclonal antibody
The medicine Bevacizumab sugar-type, the glycosylation modified qualitative and quantitative study to other protein medicaments are also borrowed with guidance
Mirror meaning.
Brief description of the drawings
Fig. 1 is the typical chromatogram of H5N2 glycopeptide standard items after monoclonal antibody enzymolysis.
Fig. 2 is the MS/MS spectrograms of H5N2 glycopeptide standard items after monoclonal antibody enzymolysis.
Fig. 3 is the typical chromatogram of H5N2 glycopeptides in the plasma sample that embodiment 1 obtains.
Fig. 4 is the typical chromatogram of H5N2 glycopeptides after the plasma sample enrichment that embodiment 1 obtains.
It is bent when Fig. 5 is the medicine of the H5N2 glycopeptides of monoclonal antibody medicine Bevacizumab in the rat plasma sample that embodiment 2 obtains
Line.
Fig. 6 is the medicine of monoclonal antibody medicine Bevacizumab H5N2 glycopeptides after the rat plasma sample that embodiment 2 obtains is enriched with
When curve.
Embodiment
Embodiment 1:The quantitative analysis of H5N2 glycopeptides in rat plasma sample
Part I:Rat vein is administered
A, rat vein administration process
(1) it is mono- male rat of 200g ± 10g to choose body weight, free diet;
(2) commercially available Bevacizumab parenteral solutions, concentration 25mg/mL are bought;
(3) according to clinical administration dosage conversions, Rat monoclonal is given by 50mg/kg dosages using intravenous injection mode
Antibody drug;
B, the preparation of rat plasma sample
Different time point (0,30min, 1h, 2h, 4h, 8h, 12h, 24h, 32h) is selected respectively, from venous collection rat
Whole blood, 13000g, 5min centrifuge serum, and -80 DEG C of refrigerators preserve.
Part II:The assay of monoclonal antibody medicine Bevacizumab H5N2 glycopeptides in rat plasma sample
A, the pretreatment of plasma sample
(1) reduction of plasma sample,
The plasma sample of 2 μ L rats is added in polyethylene pipe,
The pH buffer solutions that 96 μ L contain denaturant are added, vortex mixes,
2 μ L reducing agents are added, vortex mixes,
60 DEG C are incubated 1 hour, obtain reaction solution;
(2) closing of cysteine,
0.75mg solid cysteine sealers are added in reaction solution, vortex mixes;
25 DEG C of lucifuges are incubated 40min;
(3) the pancreatin digestion of plasma sample,
700 μ L digestion buffer solutions are added in reaction solution through cysteine closing, vortex mixing, it is molten to add 3 μ L pancreatin
Liquid, vortex mixing, 37 DEG C are incubated 12~16h and obtain plasma sample enzymolysis liquid, add 5 μ L formic acid terminating reactions;
(4) SPE of blood plasma enzymolysis liquid,
1mL acetonitrile solutions are added in solid-phase extraction column, have been flowed naturally to liquid,
1mL eluent solutions are added in solid-phase extraction column, have been flowed naturally to liquid,
The enzymolysis liquid of plasma sample is taken, has been flowed naturally to liquid,
1mL eluent solutions are added in solid-phase extraction column, have been flowed naturally to liquid, are washed for three times,
1mL elution solution is added in solid-phase extraction column, has been flowed naturally to liquid, has obtained plasma sample eluent, be placed in poly-
In ethylene tube;
(5) concentration, the redissolution of plasma sample,
The plasma sample eluent traditional vacuum obtained in above-mentioned steps is evaporated, adds the formic acid of 100 μ L 0.1%, vortex
Mix, obtain the redissolution liquid of plasma sample.
(6) mass spectral analysis of plasma sample,
Take plasma sample to redissolve the μ L of liquid 2 and carry out liquid chromatography-tandem mass spectrometry analysis, record chromatogram, selected specific sugar
The peak area of peptide represents its relative amount in plasma sample.
It is as follows to be related to solution formula in above-mentioned steps:
PH buffer solutions containing denaturant, it is the 50mM HEPES cushioning liquid containing 8M urea;
Reducing agent, it is 1M dithiothreitol (DTT) solution;
Cysteine sealer, it is solid iodoacetamide reagent;
Buffer solution is digested, is 50mM HEPES cushioning liquid;
Trypsin solution, it is 1mg/mL trypsin solution;
Eluent solution, it is the solution of water/trifluoroacetic acid=100/0.1 by volume;
Solution is eluted, is the solution of acetonitrile/water/trifluoroacetic acid=80/20/0.1 by volume.
B, monoclonal antibody medicine H5N2 glycopeptide assays in plasma sample
Different time points after being handled by step A of learning from else's experience respectively redissolve the μ L of liquid 2 and carry out LC/MS/MS analyses, record chromatogram,
H5N2 glycopeptide typical case chromatograms such as Fig. 3 after plasma sample enzymolysis, the peak area of selected specific glycopeptide (H5N2) represent its
Relative amount in plasma sample, in obtained rat plasma sample during the medicine of monoclonal antibody medicine Bevacizumab H5N2 glycopeptides
Curve such as Fig. 5.
The condition for being related to monoclonal antibody Bevacizumab H5N2 glycopeptides measure in above-mentioned steps is as follows:
1st, chromatographic condition is:The series of high efficiency liquid chromatographic systems of U.S. match Mo Feishier company Ultimate 3000;Chromatogram
Post:Make peptide C18 capillary chromatographic columns, 15cm × 75 μm I.D., 5 μm of particle diameters by oneself;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 300nL/min;
Described gradient elution, its program can be found in table 1.
2nd, Mass Spectrometry Conditions are:Q-Exactive Orbitrap level Four bar-orbit trap liquid chromatography-tandem mass spectrometry instrument, is furnished with
Nanoliter level electro-spray ionization source and the data processing softwares of Xcalibur 2.1;Ion gun:Nanoliter level electro-spray ionization source;Just
Ionic means detect;Scan mode monitors for parallel reaction;Full resolution ratio of sweeping is 70000, and targeted scans resolution ratio is 35000,
Maximum injection length is 200ms.
The peptide fragment parallel reaction monitoring and setting value of table 2
Embodiment 2:The quantitative analysis of H5N2 glycopeptides after rat plasma sample enrichment
Part I:Rat vein is administered
A, rat vein administration process
(1) it is mono- male rat of 200g ± 10g to choose body weight, free diet;
(2) commercially available Bevacizumab parenteral solutions, concentration 25mg/mL are bought;
(3) according to clinical administration dosage conversions, Rat monoclonal is given by 50mg/kg dosages using intravenous injection mode
Antibody drug;
B, the preparation of rat plasma sample
Different time point (0,30min, 1h, 2h, 4h, 8h, 12h, 24h, 32h) is selected respectively, from venous collection rat
Whole blood, 13000g, 5min centrifuge serum, and -80 DEG C of refrigerators preserve.
Part II:The assay of monoclonal antibody medicine Bevacizumab H5N2 glycopeptides after rat plasma sample enrichment
A, the glycopeptide enrichment of plasma sample
(1) reduction of plasma sample,
The plasma sample of 2 μ L rats is added in polyethylene pipe,
The pH buffer solutions that 96 μ L contain denaturant are added, vortex mixes,
2 μ L reducing agents are added, vortex mixes,
60 DEG C are incubated 1 hour, obtain reaction solution;
(2) closing of cysteine,
0.75mg solid cysteine sealers are added in reaction solution, vortex mixes;
25 DEG C of lucifuges are incubated 40min;
(3) the pancreatin digestion of plasma sample,
700 μ L digestion buffer solutions are added in reaction solution through cysteine closing, vortex mixing, it is molten to add 3 μ L pancreatin
Liquid, vortex mixing, 37 DEG C are incubated 12~16h and obtain plasma sample enzymolysis liquid, add 5 μ L formic acid terminating reactions;
(4) SPE of blood plasma enzymolysis liquid,
1mL acetonitrile solutions are added in solid-phase extraction column, have been flowed naturally to liquid,
1mL eluent solutions are added in solid-phase extraction column, have been flowed naturally to liquid,
The enzymolysis liquid of plasma sample is taken, has been flowed naturally to liquid,
1mL eluent solutions are added in solid-phase extraction column, have been flowed naturally to liquid, are washed for three times,
1mL elution solution is added in solid-phase extraction column, has been flowed naturally to liquid, has obtained plasma sample eluent, be placed in poly-
In ethylene tube;
(5) concentration, the redissolution of plasma sample,
The plasma sample eluent traditional vacuum obtained in above-mentioned steps is evaporated, adds acetonitrile/1% 3 of 50 μ L 80%
Fluoroacetic acid, vortex mix, and obtain the enrichment solution of plasma sample.
(6) enrichment of plasma sample
10 μ L liquid-transfering gun pipette tips tips are blocked with appropriate absorbent cotton, the hydrophilic enrichment glycopeptide materials of 5mg are dissolved in 50 μ L
The trifluoroacetic acid solution of 80% acetonitrile/1%, inserts pipette tips, and 700g is centrifuged to liquid from dry, and tip posts are made,
In the enrichment solution that plasma sample is added in tip posts, 700g is centrifuged off liquid,
In adding the trifluoroacetic acid solution of acetonitriles of 50 μ L 80%/1% in tip posts, 700g is centrifuged off liquid, washes for three times,
In adding the acetonitrile solutions of 50 μ L 10% in tip posts, 700g is centrifuged to liquid from dry, is washed for two times, collects plasma sample
Enrichment eluent;
(7) concentration, the redissolution of blood plasma enriched sample
The enrichment eluent traditional vacuum of the plasma sample obtained in above-mentioned steps is evaporated, adds the first of 20 μ L 0.1%
Acid, vortex mix, and obtain the redissolution liquid of blood plasma enriched sample.
(8) mass spectral analysis of blood plasma enriched sample,
Take blood plasma enriched sample to redissolve the μ L of liquid 4 and carry out liquid chromatography-tandem mass spectrometry analysis, record chromatogram, selected spy
The peak area for determining glycopeptide represents its relative amount in plasma sample;
It is as follows to be related to solution formula in above-mentioned steps:
PH buffer solutions containing denaturant, it is the 50mM HEPES cushioning liquid containing 8M urea;
Reducing agent, it is 1M dithiothreitol (DTT) solution;
Cysteine sealer, it is solid iodoacetamide reagent;
Buffer solution is digested, is 50mM HEPES cushioning liquid;
Trypsin solution, it is 1mg/mL trypsin solution;
Eluent solution, it is the solution of water/trifluoroacetic acid=100/0.1 by volume;
Solution is eluted, is the solution of acetonitrile/water/trifluoroacetic acid=80/20/0.1 by volume.
B, monoclonal antibody medicine H5N2 glycopeptide assays after plasma sample enrichment
Different time points after being handled by step B of learning from else's experience respectively redissolve the μ L of liquid 4 and carry out LC/MS/MS analyses, record chromatogram,
H5N2 glycopeptide typical case chromatograms such as Fig. 4 after plasma sample enrichment, the peak area of selected specific glycopeptide (H5N2) represent its
Relative amount in plasma sample, monoclonal antibody medicine Bevacizumab H5N2 glycopeptides after obtained rat plasma sample enrichment
Drug-time curve such as Fig. 6.
The condition for being related to monoclonal antibody Bevacizumab H5N2 glycopeptides measure in above-mentioned steps is as follows:
1st, chromatographic condition is:The series of high efficiency liquid chromatographic systems of U.S. match Mo Feishier company Ultimate 3000;Chromatogram
Post:Make peptide C18 capillary chromatographic columns, 15cm × 75 μm I.D., 5 μm of particle diameters by oneself;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 300nL/min;
Described gradient elution, its program can be found in table 1.
2nd, Mass Spectrometry Conditions are:Q-Exactive Orbitrap level Four bar-orbit trap liquid chromatography-tandem mass spectrometry instrument, is furnished with
Nanoliter level electro-spray ionization source and the data processing softwares of Xcalibur 2.1;Ion gun:Nanoliter level electro-spray ionization source;Just
Ionic means detect;Scan mode monitors for parallel reaction;Full resolution ratio of sweeping is 70000, and targeted scans resolution ratio is 35000,
Maximum injection length is 200ms;Peptide fragment parallel reaction monitoring and setting value is referring to table 2.
A kind of glycosylation modified mass spectrometric analysis method degree of accuracy of monoclonal antibody drug of the present invention is high, reappearance
Good and sensitive, reliable, glycosylation modified available for said monoclonal antibody medicine Bevacizumab pharmacokinetic analysis
Measure.
Claims (3)
1. a kind of glycosylation modified Method of Mass Spectrographic Quantitative Analysis of monoclonal antibody drug, described monoclonal antibody drug are shellfishes
Cut down pearl monoclonal antibody;The quantitative analysis of specific glycopeptide H5N2 after plasma sample enrichment is carried out by following 8 steps:
(1) reduction of plasma sample:The plasma sample of 2 μ L rats is added in polyethylene pipe, 96 μ L is added and contains denaturant
PH buffer solutions, vortex mix;2 μ L reducing agents are added, vortex mixes;60 DEG C are incubated 1 hour, obtain reaction solution;
(2) closing of cysteine:0.75mg solid cysteine sealers are added in reaction solution, vortex mixes;25 DEG C are kept away
Light is incubated 40min;
(3) the pancreatin digestion of plasma sample:700 μ L digestion buffer solutions are added in reaction solution through cysteine closing, vortex is mixed
Close, add 3 μ L trypsin solutions, vortex mixing, 37 DEG C are incubated 12~16h and obtain plasma sample enzymolysis liquid, and it is whole to add 5 μ L formic acid
Only react;
(4) SPE of blood plasma enzymolysis liquid:1mL acetonitrile solutions are added in solid-phase extraction column, have been flowed naturally to liquid;Yu Gu
1mL eluent solutions are added in phase extraction column, have been flowed naturally to liquid;Plasma sample enzymolysis liquid is taken, has been flowed naturally to liquid;Yu Gu
1mL eluent solutions are added in phase extraction column, have been flowed naturally to liquid, are washed for three times;It is molten that 1mL elutions are added in solid-phase extraction column
Liquid, flowed naturally to liquid, obtained plasma sample eluent, be placed in polyethylene pipe;
(5) concentration, the redissolution of plasma sample:The plasma sample eluent traditional vacuum obtained in upper step is evaporated, adds 50 μ
The trifluoroacetic acid of acetonitriles of L 80%/1%, vortex mix, and obtain the enrichment solution of plasma sample;
(6) enrichment of plasma sample:10 μ L liquid-transfering gun pipette tips tips are blocked with appropriate absorbent cotton, by the hydrophilic enrichment glycopeptides of 5mg
Material is dissolved in the trifluoroacetic acid solution of acetonitriles of 50 μ L 80%/1%, inserts pipette tips, and 700g is centrifuged to liquid from dry, and tip posts are made;
In the enrichment solution that plasma sample is added in tip posts, 700g is centrifuged off liquid;In added in tip posts the acetonitriles of 50 μ L 80%/
1% trifluoroacetic acid solution, 700g are centrifuged off liquid, wash for three times;In adding the acetonitrile solutions of 50 μ L 10% in tip posts, 700g from
The heart from dry, is washed for two times to liquid, collects the enrichment eluent of plasma sample;
(7) concentration, the redissolution of blood plasma enriched sample:The enrichment eluent traditional vacuum of the plasma sample obtained in upper step is steamed
It is dry, the formic acid of 20 μ L 0.1% is added, vortex mixes, and obtains the redissolution liquid of blood plasma enriched sample;
(8) mass spectral analysis of blood plasma enriched sample:Take blood plasma enriched sample to redissolve the μ L of liquid 4 and carry out liquid chromatography-tandem mass spectrometry point
Analysis, records chromatogram, and the peak area of selected specific glycopeptide represents its relative amount in plasma sample.
2. the glycosylation modified Method of Mass Spectrographic Quantitative Analysis of monoclonal antibody drug according to claim 1, its feature exist
In, the analysis of described liquid chromatography-tandem mass spectrometry,
Chromatographic condition is:The series of high efficiency liquid chromatographic systems of U.S. match Mo Feishier company Ultimate 3000;Self-control
Peptide C18 capillary chromatographic columns, 15cm × 75 μm I.D., 5 μm of particle diameters;Mobile phase is that formic acid percentage by volume accounts for 0.1%
Water and formic acid percentage by volume account for 0.1% acetonitrile, gradient elution;Flow velocity 300nL/min;
Mass Spectrometry Conditions are:Q-ExactiveOrbitrap level Four bar-orbit trap liquid chromatography-tandem mass spectrometry instrument, equipped with nanoliter level
Electro-spray ionization source and the data processing softwares of Xcalibur 2.1;Ion gun is nanoliter level electro-spray ionization source;Cation
Mode detects;Scan mode monitors for parallel reaction;Full resolution ratio of sweeping is 70000, and targeted scans resolution ratio is 35000, maximum
Injection length is 200ms.
3. the glycosylation modified Method of Mass Spectrographic Quantitative Analysis of monoclonal antibody drug according to claim 1, its feature exist
In the pH buffer solutions containing denaturant, being the 50mM 4- hydroxyethyl piperazineethanesulfonic acid cushioning liquid containing 8M urea;
Described reducing agent, it is 1M dithiothreitol (DTT) solution;Described cysteine sealer, it is solid iodoacetamide reagent;Institute
The digestion buffer solution stated, it is 50mM 4- hydroxyethyl piperazineethanesulfonic acid cushioning liquid;Described trypsin solution, it is 1mg/mL pancreas
Enzyme solutions;Described eluent solution, it is the solution of water/trifluoroacetic acid=100/0.1 by volume;Described elution solution, it is
The solution of acetonitrile/water/trifluoroacetic acid=80/20/0.1 by volume.
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