生物药物中单克隆抗体检测方案(毛细管电泳仪)

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Authors Suresh Babu CV and Ravindra GudihalAgilent Technologies Pvt LtdBangalore，India Characterization of MonoclonalAntibodies Using CapillaryElectrophoresis-Electrospraylonization-Mass Spectrometry(CE-ESI-MS) Application Note Abstract This Application Note demonstrates the utility of capillary electrophoresis/massspectrometry (CE/MS) for the analysis of intact monoclonal antibodies (mAbs).The Agilent 7100 CE System coupled to an Agilent 6530 Q-TOF LC/MS wasused to study the intact and reduced forms of mAbs. The combination of massmeasurements obtained by CE/MS and the data processing capabilities ofAgilent MassHunter and BioConfirm Software enables the identification ofheterogeneity in an intact mAb and its fragments. The result demonstrates theutility of CE/MS as a complementary technique to liquid chromatography/massspectrometry (LC/MS) for the analysis of mAbs. Introduction Therapeutic proteins such as monoclonalantibody (mAb) are gaining muchattention in the biopharmaceuticalindustry. mAbs are susceptible tochemical modification and degradationduring production， formulation， andstorage. Hence， it is important tomonitor the manufacturing consistency，purity， and molecular weight. Capillaryelectrophoresis-sodium dodecyl sulfate(CE-SDS) with UV and fluorescencedetection is a technique routinely used forquality control testing of mAbs. However，there is growing interest in exploring CEcoupled to mass spectrometry (MS) forhigher sensitivity and better compoundidentification with accurate massmeasurements. The reagents and sample preparationprocedures used for CE-SDS separationare not compatible with MS (mainly dueto ion suppression issues). In addition，the assignment of peaks correspondingto post-translational modification ischallenging in SDS-protein capillaryelectrophoresis. Although liquidchromatography/mass spectrometry(LC/MS) can be regarded as the goldstandard for recombinant proteincharacterization， CE/MS is an emergingtechnology in this field2. For the analysisof peptides， CE/MS is an attractivecomplementary method to LC/MS， dueto the efficient analysis of hydrophiliclow molecular weight peptides34. Further，CE/MS has the additional advantage ofhaving low sample volume requirements(1-20 nL) compared to LC/MS. Inthis context， this Application Notedemonstrates a CE/MS method for theprimary characterization of mAbs. Intact，heavy， and light chain fragments of mAbswere analyzed with the Agilent CE/MSsolution for two of the mAb samples.The results of this work are contrastedto a recent publication that shows theanalysis of the same samples by LC/MS. MassHunter Acquisition Software (B.06)for data acquisition and BioConfirmsoftware for data analysis. The rawMS spectra were subjected to pModalgorithms to generate the zero chargestate spectra. The CE/MS analysis was performedusing the 7100 CE System with a CE/MScapillary cassette (G1603A) coupled tothe 6530 Accurate-Mass Q-TOF equippedwith electrospray source and orthogonalcoaxial sheath liquid interface (G1607B).The sheath liquid was delivered byan Agilent 1200 series isocratic pumpequipped with a 1：100 flow splitter.TTahlfable 1 shows the CE/MS parameters. Table 1. Instrumental conditions. Capillary electrophoresis (CE) CE Agilent 7100 CE Sample mAbs (3 pg/pL) Injection 10 seconds and 20 seconds at 50 mbar Capillary PVA， total length 70 cm， 50 pm id Buffer 50 mM acetic acid Voltage 30 kV Internal pressure 20 mbar lemperature 20°C Mass spectrometry (MS) MS Agilent 6530 Accurate-Mass Q-TOF LC/MS lonization mode ESI Acquisition mode MS (mass range 1，000-6，000m/z) Sheath liquid 0.5% acetic acid in 50 % methanol， 5 pL/min Drying gas flow 5 L/min Nebulizer 10 psi Drying gas temperature 250°C Fragmentor 350 V Vcap 3，500V Results and Discussion Intact mAb analysis Figure 1 shows the CE/MS trace for themAbs at intact levels. The mAbs weredetected between 12 and 18 minutes asa broad peak (Figure 1A and 1B). Theaverage mass spectrum of both intact mAbs are shown in Figures 1C and1D. The charge state envelope spreadbetween 2，400 m/z-5，100 m/z， higher inm/z than a typical LC/MS analysis using0.1 % formic acid. The mass spectrumwas deconvoluted using the peakmodeling (pMod) deconvolution algorithmin Agilent MassHunter BioConfirm Software， and the deconvolutedspectra are shown in Figures 1E and1F. The deconvoluted spectra for bothmAbs showed four major mass peakscorresponding to different glycoformspecies， which is in good agreement withLC/MS measurements5. Reduced mAb analysis To study the mAb fragments， reductionof disulfides (with DTT) was performedto produce light and heavy chains ofmAbs5. The total ion electropherogramand the deconvoluted spectra of thelight chain (LC) and heavy chain (HC) ofboth mAbs are shown in Figures 2A-2F respectively. The CE/MS method showedreasonably good separation betweenLC and HC. The light chains of bothlgG1 and IgG2 showed a single masspeak at 22，930.44 Da and 22，929.19 Darespectively (Figures 2C and 2D). LC/MSanalysis of the same samples showedvarious satellite peaks near the light chain signal， corresponding to saltadducts. Inspection of the heavy chainCE/MS deconvoluted spectrum showsmultiple peaks assigned to glycanmodifications (Figures 2E and 2F)， whichare identical to the glycoforms observedin the intact level， further validatingthe glycan assignment at the intactmass level. Figure 2. CE/MS of reduced mAbs. A and B) total ion electropherograms of reduced lgG1 and IgG2， C and D) deconvoluted masses of light chain oflgG1 andlgG2， E and F) deconvoluted masses of heavy chain of lgG1 and lgG2. *n-acetyl glucosamine. The CE/MS deconvoluted spectra of lgG1heavy chain showed a peak with a massdifference of 203 Da， corresponding ton-acetyl glucosamine modification (*)，which was absent in the LC/MS run. Theresults obtained are in good agreementwith LC/MS measurements. Conclusion The characterization of mAbs usingan Agilent 7100 CE System coupled toan Agilent 6530 Accurate-Mass Q-TOFhas been showcased. The usefulnessof CE/MS for the analysis of intactantibodies and its fragments wasdemonstrated by using two mAbs in thepresent study. This CE/MS method canbe used as a complement to LC/MS forthe routine analysis of mAbs. References 1. Optimization and validation of aquantitative capillary electrophoresissodium dodecyl sulfate method forquality control and stability monitoringof monoclonal antibodies. Anal. Chem.2006， 78(18)， pp 6583-6594. 2. Characterization of TherapeuticAntibodies and Related Products.Anal. Chem. 2012， 85(2)， pp 715-736. 3. Applications of capillaryelectrophoresis in characterizingrecombinant protein therapeutics.Electrophoresis 2014， 35 (1)， pp 96-108. ( 4.C CE/MS and LC/MS Synergy，Complementary Solutions for PeptideMapping， Agilent Technologies， publication number 5991-2583EN. ) 5. Analysis of Monoclonal Antibody(mAb) Using Agilent 1290 InfinityLC System Coupled to Agilent6530 Accurate-Mass QuadrupoleTime-of-Flight (Q-TOF)， AgilentTechnologies， publication number5991-4266EN. 6. Primary Characterization of aMonoclonal Antibody Using AgilentHPLC-Chip Accurate-Mass LC/MSTechnology， Agilent Technologies，publication number 5990-3445EN. This information is subject to change without notice. C Agilent Technologies， Inc.， 2014 Published in the USA， October 1， 2014 5991-5212EN Agilent Technologies This Application Note demonstrates the utility of capillary electrophoresis/mass spectrometry (CE/MS) for the analysis of intact monoclonal antibodies (mAbs).The Agilent 7100 CE System coupled to an Agilent 6530 Q-TOF LC/MS was used to study the intact and reduced forms of mAbs. The combination of mass measurements obtained by CE/MS and the data processing capabilities of Agilent MassHunter and BioConfi rm Software enables the identifi cation of heterogeneity in an intact mAb and its fragments. The result demonstrates the utility of CE/MS as a complementary technique to liquid chromatography/mass spectrometry (LC/MS) for the analysis of mAbs.