In-Depth Characterization of Monoclonal Antibodies Using Intact Mass Analysis and Middle-down Approaches on Orbitrap Ascend BioPharma Tribrid Mass Spectrometer

Significance of the Topic

The detailed profiling of monoclonal antibodies (mAbs) is crucial for ensuring the safety, efficacy and quality of biotherapeutics. Advanced mass spectrometric techniques deliver precise mass measurements and comprehensive sequence information to characterize intact molecules and their subunits, enabling detection of post-translational modifications and structural variants that impact clinical performance.

Objectives and Study Overview

This study evaluates the capability of the Thermo Scientific Orbitrap Ascend BioPharma Tribrid mass spectrometer to perform both intact mass analysis and middle-down sequencing of the therapeutic antibody trastuzumab. The goals include assessing mass accuracy under native and denaturing conditions and achieving exhaustive sequence coverage of antibody subdomains through various fragmentation strategies.

Methodology

Sample preparation involved dilution of trastuzumab for intact mass experiments in native buffer and in 0.1% formic acid for denaturing assays. Subunit analysis was performed after IdeS digestion and reduction with TCEP/DTT to generate Fc/2, light chain (LC) and Fd' fragments or by direct reduction to LC and heavy chain (HC).

Middle-down MS utilized electron transfer dissociation (ETD), electron transfer higher energy collision dissociation (EThcD), ultraviolet photodissociation (UVPD) and proton transfer charge reduction (PTCR) in combination, optimizing reaction times for maximal fragmentation.

Data were acquired with high resolving power (up to 240,000 at m/z 200) and processed using Thermo Fisher’s BioPharma Finder software (v5.2) employing ReSpect, Xtract and sliding window algorithms.

Used Instrumentation

• Thermo Scientific Orbitrap Ascend BioPharma Tribrid Mass Spectrometer with Native MS option
• Thermo Scientific Vanquish Flex UHPLC system
• MAbPac reverse phase column
• SEC-1 size exclusion column

Key Results and Discussion

Intact mass analysis under denaturing and native conditions yielded mass accuracy better than 3 ppm for the full antibody and its subunits, confirming high calibration precision. Sequence coverage for middle-down fragments reached nearly 100% for Fc/2 and LC, 97% for Fd’ and 73% for HC when combining EThcD, UVPD and PTCR data.

EThcD and UVPD outperformed ETD alone due to generation of additional fragment ion types. PTCR further enhanced sequence coverage by simplifying complex MS2 spectra and enabling higher confidence in residue assignment.

Benefits and Practical Applications

• Accurate intact mass measurement supports rapid quality control of mAb batches.
• Deep sequence coverage of antibody subunits facilitates identification of sequence variants and post-translational modifications.
• Integration of multiple dissociation methods enables nearly complete mapping of large proteoforms in biopharma research and development.

Future Trends and Opportunities

As mass spectrometer resolution and advanced fragmentation techniques evolve, routine top-down and middle-down workflows will become more robust, enabling real-time monitoring of biotherapeutic integrity. Integration of AI-driven data analysis and automated sample handling promises to accelerate high-throughput characterization and support regulatory compliance.

Emerging photodissociation and charge reduction strategies will further improve coverage for challenging domains, particularly for heavily glycosylated antibodies and bispecific formats.

Conclusion

The Orbitrap Ascend BioPharma Tribrid system provides exceptional mass accuracy and resolution for intact mAbs and subunit characterization. Combining EThcD, UVPD and PTCR achieves near-complete sequence coverage of ~20–25 kDa fragments and substantial coverage of ~50 kDa chains, demonstrating a powerful platform for biopharmaceutical analysis.

References

1. Srzentić K, et al. Complete characterization of monoclonal antibodies under native and denaturing conditions. Thermo Fisher Sci Applicate Note 73885;2021.
2. Scheffler K, Damoc E. Full characterization of heterogeneous antibody samples under denaturing and native conditions. Thermo Fisher Sci Applicate Note 72348;2017.
3. Kline JT, et al. Sequential Ion–Ion Reactions for Enhanced Gas-Phase Sequencing of Large Intact Proteins. JASMS.2021;32(9):2334–2345.
4. Huguet R, et al. Proton Transfer Charge Reduction Enables High-Throughput Top-Down Analysis of Large Proteoforms. Anal Chem.2019;91(24):15732–15739.
5. Scheffler K, et al. Enabling Mass Spectrometric Analysis of Intact Proteins in Native Conditions of A Hybrid Quadrupole-Orbitrap Mass Spectrometer. ASMS Poster;2016.