Intact protein characterization using mass spectrometry

Intact protein characterization using mass spectrometry

Comprehensive characterization of biotherapeutics is necessary to satisfy safety standards set by regulatory agencies and helps to ensure protein drug efficacy. Intact protein analysis using a combination of liquid chromatography (LC) and mass spectrometry (MS) provides information on the accurate mass of the protein and the relative abundance of its isoforms. The analysis of these complex heterogeneous protein biotherapeutics benefits from high resolution accurate mass (HRAM) instrumentation. Combining HRAM with powerful data deconvolution algorithms facilitates structural confirmation and accurate identification of protein modifications.


Intact mass analysis and native mass spectrometry

Biopharmaceutical characterization is required throughout all stages of drug development and manufacturing. During drug development, liquid chromatography–mass spectrometry (LC-MS) approaches are used to structurally characterize monoclonal antibodies (mAbs) produced using cell lines. The main objective is to determine monoclonal cell populations producing the desired mAb with a high degree of fidelity and activity for a specific antigen. The analysis of intact biologics is continued throughout manufacturing to assess purity and heterogeneity characteristics for lot release purposes.

Multiple analytical approaches can be utilized to assess biotherapeutic molecules in their intact state. Information on accurate protein mass and heterogeneity can be provided by MS analysis following a short desalting step and is important for quantification of the various glycoforms and protein variant analysis, important considerations for therapeutic drug formulation and storage. Biotherapeutic drugs can be analysed at their subunit level following reduction and/or digestion. Analysis of therapeutic antibody subunits facilitates exact molecular weight analysis of their heavy and light chains and further top down analysis can provide sequence confirmation.

Protein analysis in native or native-like conditions decreases charge state value resulting in mAb detection at higher m/z ranges with more spatial resolution. For example, native mass spectrometry conditions also allow the preservation of structurally-critical non-covalent bonds which may be necessary to the analysis of cysteine-linked antibody-drug conjugates (ADC)


Featured intact protein analysis learning content

Poster video

Join mass spectrometry expert Dr. Stephane Houel as he presents this poster on the characterization of antibody drug conjugates using intact protein mass spectrometry, at the American Association of Pharmaceutical Scientists (AAPS) convention.

Poster video

Join biopharmaceutical chromatography expert Dr. Shanhua Lin as she discusses a poster detailing novel approaches for antibody subunit characterization, at the HPLC 2016 convention.

Poster note

In this scientific poster monoclonal antibodies (mAbs) are broken down into several large subunit fragments using reduction reagent and IdeS enzyme. A fast LC/MS separation method is employed for two applications: confirming complete deglycosylation, and monitoring charge plus oxidation variants.

Poster video

Join chromatography expert Dr. Shane Bechler as he presents a simple method for rapid purification of therapeutic proteins by mass spectrometry from high salt solutions in this fast and reproducible workflow.

Poster note

Mass spectrometry is an essential tool in the characterization of mAbs. However, the sample matrix typically comprises a variety of salts, stabilizers, detergents, and other adduct forming ions. Find out how to rapidly remove these interfering species in this poster.


Intact protein analysis literature library

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ApplicationTypeTitleProduct Focus
ADCsApplication NoteHigh-Resolution Separation of Cysteine-Linked Antibody-Drug Conjugate Mimics Using Hydrophobic Interaction ChromatographyChemistries and Consumables
ADCsPoster NoteAnalysis of Monoclonal Antibodies and Antibody-Drug Conjugates Using New Hydrophobic interaction Chromatography (HIC) ColumnsChemistries and Consumables
ADCsApplication NoteQuick screening of intact antibody and antibody-drug conjugates with integrated microfluidic capillary electrophoresis and mass spectrometryLife Science Mass Spec
ADCsPoster NoteComplete Characterization of a Cysteinelinked Antibody-Drug Conjugate Performed on a Hybrid Quadrupole-Orbitrap Mass Spectrometer with High Mass RangeLife Science Mass Spec
ADCsPoster NoteComplete Characterization of a Cysteinelinked Antibody-Drug Conjugate Performed on a Hybrid Quadrupole-Orbitrap Mass Spectrometer with High Mass RangeLife Science Mass Spec
ADCsPoster NoteOrbitrap Based Mass Spectrometric Characterization of Antibody Drug Conjugates Engineered through Antibody GlycansLife Science Mass Spec
ADCsPresentationNew chromatographic workflows for charge variant profiling, intact mAb analysis and DAR determinationWorkflow
ADCsWebinarOvercoming Challenges in ADC BioanalysisMSIA
ADCsCase StudyCharacterizing Therapeutic Proteins for Drug Discovery and DevelopmentWorkflow
Fragment analysisApplication NoteFast Analysis of Therapeutic Monoclonal Antibody Fragments Using a Supermacroporous, Reversed-Phase ChromatographyChemistries and Consumables
Fragment analysisApplication NoteLC-MS Analysis of Digested mAbs Using a Reversed-Phase Desalting Cartridge and High-Resolution, Accurate Mass Spectrometry (HRAMS)Life Science Mass Spec
Fragment analysisApplication NoteSeparation of mAb Fragments on a High-Resolution HIC ColumnChemistries and Consumables
Fragment analysisPosterMiddle-down Approach for Monitoring Monoclonal Antibody Variants and DeglycosylationChemistries and Consumables
Fragment analysisPosterPD Biomarker Measurement by LC/MS/MS – Resolving Sensitivity and Throughput Challenges Using High Resolution Mass SpectrometryLife Science Mass Spec
Fragment analysisPoster NoteMiddle-down Approach for Monitoring Monoclonal Antibody Variants and DeglycosylationChemistries and Consumables
Intact MassApplication NoteAnalysis of Monoclonal Antibodies and Their Fragments by Size Exclusion Chromatography Coupled with an Orbitrap Mass SpectrometerLife Science Mass Spec
Intact MassApplication NoteAnalysis of Monoclonal Antibodies and Their Fragments by Size-Exclusion Chromatography Coupled with an Orbitrap Mass SpectrometerLife Science Mass Spec
Intact MassApplication NoteAnalysis of Monoclonal Antibodies, Aggregates, and Their Fragments by Size Exclusion Chromatography Coupled with an Orbitrap Mass SpectrometerLife Science Mass Spec
Intact MassApplication NoteMonoclonal Antibody and Related Product Characterization Under Native Conditions Using a Benchtop Mass SpectrometerLife Science Mass Spec
Intact MassApplication NoteSEC-MS with Volatile Buffers for Characterization of BiopharmaceuticalsHPLC / UHPLC
Intact MassBrochureProtein quantitation using mass spectrometry: Specialized reagents for discovery and targeted analysisLife Science Mass Spec
Intact MassBrochureQ Exactive BioPharmaLife Science Mass Spec
Intact MassBrochureTargeted protein quantification with high-resolution, accurate-mass MSLife Science Mass Spec
Intact MassPosterImproving intact antibody characterization by Orbitrap mass spectrometryLife Science Mass Spec
Intact MassPoster NoteCharacterization of Monoclonal Antibodies and ADCs using a Benchtop Orbitrap Mass SpectrometerLife Science Mass Spec
Intact MassPoster NoteEnabling Mass Spectrometric Analysis of Intact Proteins in Native Conditions on A Hybrid Quadrupole-Orbitrap Mass SpectrometerLife Science Mass Spec
Intact MassPoster NoteFull Characterization of Heterogeneous Antibody Samples Under Denaturing and Native Conditions on a Hybrid Quadrupole-Orbitrap Mass SpectrometerLife Science Mass Spec
Intact MassPoster NoteRapid Online Desalting Using a Supermacroporous Reversed Phase Cartridge for Mass Spectrometry Analysis of a Monoclonal AntibodyChemistries and Consumables
Intact MassPoster NoteThe Sliding Window Algorithm for the Analysis of LC/MS Intact Protein DataLife Science Mass Spec
Intact MassPress ReleaseNew Technology Enables Comprehensive Biopharmaceutical Characterization Workflow with a Single PlatformLife Science Mass Spec
Intact MassWeb PageBioPharma Finder Software - Planet OrbitrapLife Science Mass Spec
Intact Protein AnalysisPoster NoteOptimization of Sliding Window Deconvolution Parameters Maximizes Quality of Protein Identification and Relative Quantitation in LC/MS Intact Protein AnalysisLife Science Mass Spec
Intact MassPoster NoteIntact Mass Analysis of Lysine-Linked Antibody-Drug Conjugates using Microfluidic Chip Electrophoresis and Size Exclusion Chromatography Coupled to an Orbitrap Mass SpectrometerLife Science Mass Spec
Intact MassPoster NoteCapillary Electrophoresis – Mass Spectrometry for Intact Mass Analysis of Antibodies and Antibody-Drug-ConjugatesLife Science Mass Spec
Intact MassCase StudySimple, Robust, High Quality Intact Mass Analysis—BiosimilarsLife Science Mass Spec
Intact MassApplication NoteAntibody Subunit Analysis Workflow on a Quadrupole-Orbitrap Mass Spectrometer: From Optimized Sample Preparation to Data AnalysisWorkflow
Intact Protein AnalysisApplication BriefHigh Salt Gradient Analysis of Post-Translational Modifications- Deamidation MonitoringHPLC / UHPLC
Intact Protein AnalysisApplication NoteFast Online Desalting of mAbs Using a Reversed-Phase Desalting Cartridge for LC-MS AnalysisChemistries and Consumables
Intact Protein AnalysisApplication NoteHigh-Resolution Separation of a Fusion ProteinHPLC / UHPLC
Intact Protein AnalysisApplication NoteLC/MS Analysis of the Monoclonal Antibody Rituximab Using the Q Exactive Benchtop Orbitrap Mass SpectrometerLife Science Mass Spec
Intact Protein AnalysisApplication NoteLigand Binding Mass Spectrometric Immunoassay (LB-MSIA) Workflow for Therapeutic Antibodies: A Universal Pre-Clinical Solution for the Bio-analysis of Fully Human Therapeutic Monoclonal Antibodies in PlasmaMSIA
Intact Protein AnalysisApplication NoteMonoclonal Antibody Analysis on a Reversed-Phase C4 Polymer Monolith ColumnChemistries and Consumables
Intact Protein AnalysisApplication NoteNew integrated informatics solution for protein biotherapeutics characterizationLife Science Mass Spec
Intact Protein AnalysisApplication NoteReversed-Phase Analysis of a Monoclonal Antibody on an Accucore 150-C4 HPLC ColumnChemistries and Consumables
Intact Protein AnalysisApplication NoteReversed-Phase Separation of Intact Therapeutic Antibodies Using the Vanquish Flex UHPLC SystemHPLC / UHPLC
Intact Protein AnalysisApplication NoteSeparation of Monoclonal Antibody (mAb) Oxidation Variants on a High-Resolution HIC ColumnChemistries and Consumables
Intact Protein AnalysisApplication NoteSeparatoion of Intact Monoclonal Antibody Sialylation Isoforms by pH Gradient Ion-Exchange ChromatographyChemistries and Consumables
Intact Protein AnalysisPoster NoteFast Analysis of Therapeutic Monoclonal Antibodies, Fragments, and Oxidation Variants Using a Super-Macro Porous Reversed-Phase Column Coupled with an Orbitrap Mass SpectrometerChemistries and Consumables
Intact Protein AnalysisPoster NoteHigh Temperature, High Throughput Reversed Phase Separation of Intact Monoclonal AntibodiesHPLC / UHPLC
Intact Protein AnalysisPoster NoteSeparation of Bispecific mAbs Using Hydrophobic Interaction Chromatography (HIC)Chemistries and Consumables
Intact Protein AnalysisPoster NoteSeparation of Bispecific mAbs Using Hydrophobic Interaction Chromatography (HIC)Chemistries and Consumables
Intact Protein AnalysisPoster NoteStructure Characterization and Differentiation of Biosimilar and Reference Products Using Unique Combination of Complementary Fragmentation MechanismsLife Science Mass Spec
Intact Protein AnalysisPresentationCharacterization of mAbs Using RP UHPLC HRAM MSLife Science Mass Spec
Intact Protein AnalysisTechnical NoteInfluence of Column Temperature on Reversed-Phase Chromatography of an Intact AntibodyHPLC / UHPLC
Intact Protein AnalysisWebinarIntact Analysis of Protein on the Q Exactive PlatformLife Science Mass Spec
Intact Protein AnalysisWhite PaperCharacterizing Therapeutic Monoclonal AntibodiesHPLC / UHPLC
Mass SpectrometryPoster NoteNovel Ways to Introduce the Traditional Salt Based Chromatography Techniques of Size Exclusion and Ion Exchange Chromatography of Biopharmaceutical Proteins Into High Resolution Mass SpectrometryWorkflow
Top DownPoster NoteOptimizing Top Down Analysis of Proteins on Orbitrap Fusion Lumos Tribrid Mass SpectrometerLife Science Mass Spec

Intact protein analysis videos


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