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

Orbitrap-Based Mass Spectrometric Antibody Drug Conjugate Characterization

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 note

In this poster note we report the characterization of ADCs with enzymatic labeled antibody N-glycans, using the Thermo Scientific Q Exactive Plus and Orbitrap Fusion mass spectrometers.

Poster video

Middle-Down Approach for Monitoring Monoclonal Antibody Variants & Deglycosylation

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

Fast Online Desalting of mAbs Using a Reversed-Phase Desalting Cartridge for LC-MS Analysis

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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Application Type Title Product Focus
ADCs Application Note High-Resolution Separation of Cysteine-Linked Antibody-Drug Conjugate Mimics Using Hydrophobic Interaction Chromatography Chemistries and Consumables
ADCs Poster Note Analysis of Monoclonal Antibodies and Antibody-Drug Conjugates Using New Hydrophobic interaction Chromatography (HIC) Columns Chemistries and Consumables
ADCs Poster Note Complete Characterization of a Cysteinelinked Antibody-Drug Conjugate Performed on a Hybrid Quadrupole-Orbitrap Mass Spectrometer with High Mass Range Life Science Mass Spec
ADCs Poster Note Complete Characterization of a Cysteinelinked Antibody-Drug Conjugate Performed on a Hybrid Quadrupole-Orbitrap Mass Spectrometer with High Mass Range Life Science Mass Spec
ADCs Poster Note Orbitrap Based Mass Spectrometric Characterization of Antibody Drug Conjugates Engineered through Antibody Glycans Life Science Mass Spec
ADCs Presentation New chromatographic workflows for charge variant profiling, intact mAb analysis and DAR determination Workflow
ADCs Webinar Overcoming Challenges in ADC Bioanalysis MSIA
Fragment analysis Application Note Fast Analysis of Therapeutic Monoclonal Antibody Fragments Using a Supermacroporous, Reversed-Phase Chromatography Chemistries and Consumables
Fragment analysis Application Note LC-MS Analysis of Digested mAbs Using a Reversed-Phase Desalting Cartridge and High-Resolution, Accurate Mass Spectrometry (HRAMS) Life Science Mass Spec
Fragment analysis Application Note Separation of mAb Fragments on a High-Resolution HIC Column Chemistries and Consumables
Fragment analysis Poster Middle-down Approach for Monitoring Monoclonal Antibody Variants and Deglycosylation Chemistries and Consumables
Fragment analysis Poster PD Biomarker Measurement by LC/MS/MS – Resolving Sensitivity and Throughput Challenges Using High Resolution Mass Spectrometry Life Science Mass Spec
Fragment analysis Poster Note Middle-down Approach for Monitoring Monoclonal Antibody Variants and Deglycosylation Chemistries and Consumables
Intact Mass Application Note Analysis of Monoclonal Antibodies and Their Fragments by Size Exclusion Chromatography Coupled with an Orbitrap Mass Spectrometer Life Science Mass Spec
Intact Mass Application Note Analysis of Monoclonal Antibodies and Their Fragments by Size-Exclusion Chromatography Coupled with an Orbitrap Mass Spectrometer Life Science Mass Spec
Intact Mass Application Note Analysis of Monoclonal Antibodies, Aggregates, and Their Fragments by Size Exclusion Chromatography Coupled with an Orbitrap Mass Spectrometer Life Science Mass Spec
Intact Mass Application Note Monoclonal Antibody and Related Product Characterization Under Native Conditions Using a Benchtop Mass Spectrometer Life Science Mass Spec
Intact Mass Application Note SEC-MS with Volatile Buffers for Characterization of Biopharmaceuticals HPLC / UHPLC
Intact Mass Brochure Protein quantitation using mass spectrometry: Specialized reagents for discovery and targeted analysis Life Science Mass Spec
Intact Mass Brochure Q Exactive BioPharma Life Science Mass Spec
Intact Mass Brochure Targeted protein quantification with high-resolution, accurate-mass MS Life Science Mass Spec
Intact Mass Poster Improving intact antibody characterization by Orbitrap mass spectrometry Life Science Mass Spec
Intact Mass Poster Note Characterization of Monoclonal Antibodies and ADCs using a Benchtop Orbitrap Mass Spectrometer Life Science Mass Spec
Intact Mass Poster Note Enabling Mass Spectrometric Analysis of Intact Proteins in Native Conditions on A Hybrid Quadrupole-Orbitrap Mass Spectrometer Life Science Mass Spec
Intact Mass Poster Note Full Characterization of Heterogeneous Antibody Samples Under Denaturing and Native Conditions on a Hybrid Quadrupole-Orbitrap Mass Spectrometer Life Science Mass Spec
Intact Mass Poster Note Rapid Online Desalting Using a Supermacroporous Reversed Phase Cartridge for Mass Spectrometry Analysis of a Monoclonal Antibody Chemistries and Consumables
Intact Mass Poster Note The Sliding Window Algorithm for the Analysis of LC/MS Intact Protein Data Life Science Mass Spec
Intact Mass Press Release New Technology Enables Comprehensive Biopharmaceutical Characterization Workflow with a Single Platform Life Science Mass Spec
Intact Mass Web Page BioPharma Finder Software - Planet Orbitrap Life Science Mass Spec
Intact Protein Analysis Application Brief High Salt Gradient Analysis of Post-Translational Modifications- Deamidation Monitoring HPLC / UHPLC
Intact Protein Analysis Application Note Fast Online Desalting of mAbs Using a Reversed-Phase Desalting Cartridge for LC-MS Analysis Chemistries and Consumables
Intact Protein Analysis Application Note High-Resolution Separation of a Fusion Protein HPLC / UHPLC
Intact Protein Analysis Application Note LC/MS Analysis of the Monoclonal Antibody Rituximab Using the Q Exactive Benchtop Orbitrap Mass Spectrometer Life Science Mass Spec
Intact Protein Analysis Application Note Ligand 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 Plasma MSIA
Intact Protein Analysis Application Note Monoclonal Antibody Analysis on a Reversed-Phase C4 Polymer Monolith Column Chemistries and Consumables
Intact Protein Analysis Application Note New integrated informatics solution for protein biotherapeutics characterization Life Science Mass Spec
Intact Protein Analysis Application Note Reversed-Phase Analysis of a Monoclonal Antibody on an Accucore 150-C4 HPLC Column Chemistries and Consumables
Intact Protein Analysis Application Note Reversed-Phase Separation of Intact Therapeutic Antibodies Using the Vanquish Flex UHPLC System HPLC / UHPLC
Intact Protein Analysis Application Note Separation of Monoclonal Antibody (mAb) Oxidation Variants on a High-Resolution HIC Column Chemistries and Consumables
Intact Protein Analysis Application Note Separatoion of Intact Monoclonal Antibody Sialylation Isoforms by pH Gradient Ion-Exchange Chromatography Chemistries and Consumables
Intact Protein Analysis Poster Note Fast Analysis of Therapeutic Monoclonal Antibodies, Fragments, and Oxidation Variants Using a Super-Macro Porous Reversed-Phase Column Coupled with an Orbitrap Mass Spectrometer Chemistries and Consumables
Intact Protein Analysis Poster Note High Temperature, High Throughput Reversed Phase Separation of Intact Monoclonal Antibodies HPLC / UHPLC
Intact Protein Analysis Poster Note Separation of Bispecific mAbs Using Hydrophobic Interaction Chromatography (HIC) Chemistries and Consumables
Intact Protein Analysis Poster Note Separation of Bispecific mAbs Using Hydrophobic Interaction Chromatography (HIC) Chemistries and Consumables
Intact Protein Analysis Poster Note Structure Characterization and Differentiation of Biosimilar and Reference Products Using Unique Combination of Complementary Fragmentation Mechanisms Life Science Mass Spec
Intact Protein Analysis Presentation Characterization of mAbs Using RP UHPLC HRAM MS Life Science Mass Spec
Intact Protein Analysis Technical Note Influence of Column Temperature on Reversed-Phase Chromatography of an Intact Antibody HPLC / UHPLC
Intact Protein Analysis Webinar Intact Analysis of Protein on the Q Exactive Platform Life Science Mass Spec
Intact Protein Analysis White Paper Characterizing Therapeutic Monoclonal Antibodies HPLC / UHPLC
Top Down Poster Note Optimizing Top Down Analysis of Proteins on Orbitrap Fusion Lumos Tribrid Mass Spectrometer Life Science Mass Spec
Intact Protein Analysis Poster Note Optimization of Sliding Window Deconvolution Parameters Maximizes Quality of Protein Identification and Relative Quantitation in LC/MS Intact Protein Analysis Life Science Mass Spec
Intact Mass Poster Note Intact Mass Analysis of Lysine-Linked Antibody-Drug Conjugates using Microfluidic Chip Electrophoresis and Size Exclusion Chromatography Coupled to an Orbitrap Mass Spectrometer Life Science Mass Spec
Intact Mass Poster Note Capillary Electrophoresis – Mass Spectrometry for Intact Mass Analysis of Antibodies and Antibody-Drug-Conjugates Life Science Mass Spec

Intact protein analysis videos