IMSC: Full Characterization of Heterogeneous Antibody Samples Under Denaturing and Native Conditions on a Hybrid Quadrupole-Orbitrap Mass Spectrometer

Significance of the Topic

The molecular characterization of monoclonal antibodies (mAbs) is essential in biopharmaceutical research and manufacturing. Subtle modifications, glycosylation patterns and disulfide bonds can alter efficacy, stability and safety. Mass spectrometry (MS) at both intact-protein and peptide levels offers detailed insight into antibody heterogeneity, enabling quality control and accelerating therapeutic development.

Objectives and Study Overview

This study evaluated three complementary MS workflows for full characterization of four therapeutic antibodies (Trastuzumab, Trastuzumab–Emtansine, Infliximab and Bevacizumab):

• Intact mass analysis under native and denaturing conditions
• Subunit analysis following IdeS digestion and reduction
• Peptide mapping, including glycoform and disulfide-bond profiling using SMART Digest chemistry

The aim was to compare offline desalting versus online desalting via size-exclusion or reversed-phase chromatography and to assess the benefits of a high-mass-range Orbitrap configuration extending to m/z 8000.

Methodology and Instrumentation

Sample Preparation:

• Native intact analysis: Direct SEC–LC–MS injection in ammonium acetate buffer
• Denaturing intact analysis: Offline desalting on Bio-Rad P6 columns and 50% ACN/0.1% formic acid dilution
• Subunit analysis: IdeS (FabRICATOR®) digestion ± TCEP reduction
• Peptide mapping: Thermo Scientific SMART Digest™ enzyme, followed by LC–MS/MS

Chromatography and Desalting:

• Size-exclusion (MAbPAC SEC) for native desalting
• Reversed-phase (MAbPAC RP, Acclaim C18) for denatured and peptide runs

Mass Spectrometry:

• Thermo Scientific Q Exactive™ Biopharma Orbitrap with modified control software for enhanced high-mass transmission (m/z ≤8000)
• Acquisition modes: Protein, Enhanced Resolution, High Mass Range
• Data analysis: BioPharma Finder™ 1.0 SP1, ProSight Lite, Xtract deconvolution

Main Results and Discussion

Intact Mass Analysis:

• Native conditions reduced charge states, shifting peaks to higher m/z and improving mass accuracy and peak separation
• Denaturing conditions yielded higher charge envelopes but lower spatial resolution
• Drug-conjugated Trastuzumab-Emtansine displayed distinct mass shifts and intact mass profiles under native SEC–MS

Subunit Analysis:

• IdeS digestion produced consistent Fc, light-chain (LC) and Fd′ fragments
• Non-reduced native subunits preserved disulfide linkages, offering higher m/z resolution
• Top-down MS/MS of reduced subunits confirmed sequence and post-translational modifications

Peptide Mapping and Disulfide Profiling:

• 100% sequence coverage achieved for all antibodies in individual and mixed runs
• Comprehensive glycopeptide identification revealed common G0F/G1F variants
• Disulfide mapping confirmed eight expected bonds and uncovered minor scrambled or unexpected linkages
• Infliximab exhibited C-terminal Lys truncation, validated at intact, subunit and peptide levels

Benefits and Practical Applications

• Unified MS platform supports intact, subunit and peptide characterization in a single laboratory workflow
• High mass range enables native analysis of large biopharmaceutical complexes and antibody–drug conjugates
• Automated data processing streamlines identification of glycoforms, truncations and disulfide patterns
• Robust sequence confirmation enhances regulatory compliance and comparability studies

Future Trends and Possible Applications

• Integration of native MS with ion mobility for conformational and higher-order structure analysis
• Deeper glycoform profiling using advanced fragmentation (e.g., ETD, EThcD)
• Machine-learning tools for automated spectral interpretation and anomaly detection
• Real-time process monitoring in biomanufacturing through rapid LC–MS assays
• Expansion to multispecific antibodies, fusion proteins and complex conjugates

Conclusion

This work demonstrates the versatility of a modified Orbitrap MS platform for comprehensive antibody characterization. The combined intact, subunit and peptide mapping workflows deliver high mass accuracy, complete sequence coverage and detailed insight into modifications. Such an integrated approach accelerates biopharmaceutical development, quality control and comparability assessments.

References

• Scheffler K. et al. Full Characterization of Heterogeneous Antibody Samples Under Denaturing and Native Conditions on a Hybrid Quadrupole-Orbitrap Mass Spectrometer, Thermo Fisher Scientific Application Note (2016).
• Genovis AB. FabRICATOR® (IdeS) Enzyme Protocol, Genovis White Paper.
• Thermo Fisher Scientific. Q Exactive™ Biopharma Mass Spectrometer Operator’s Manual.