Top-down Characterization of Monoclonal Antibody on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer

Importance of the Topic

The detailed molecular characterization of monoclonal antibodies (mAbs) is critical to ensure safety, efficacy and consistency in therapeutic development. Top-down mass spectrometry approaches enable intact mass profiling, subunit-level fragmentation and site-specific modification analysis, offering a direct view of sequence integrity and glycosylation heterogeneity without extensive bottom-up digestion.

Objectives and Study Overview

This study demonstrates a comprehensive top-down LC-MS workflow using the Orbitrap Fusion Lumos™ Tribrid Mass Spectrometer to characterize an intact therapeutic mAb. It assesses multiple fragmentation modes—electron transfer dissociation high-capacity (ETD HD), ultraviolet photodissociation (UVPD) and collision-induced dissociation (CID)—on intact mAb, IdeS-generated subunits (Fc/2, light chain, Fd) and the reduced heavy chain to achieve high sequence coverage and accurate mass measurements.

Methodology and Instrumentation

Sample preparation involved deglycosylation (PNGase F), IdeS protease digestion to generate subunits and reduction to isolate light and heavy chains. A Thermo Scientific™ UltiMate™ 3000 RSLC system with a MabPac™ RP column (2.1 × 100 mm) operated at 400 µL/min and 80 °C facilitated reversed-phase separation with organic gradients.

Used Instrumentation

• Orbitrap Fusion Lumos™ Tribrid Mass Spectrometer
• 213 nm Nd:YAG diode-pumped solid-state UV laser for UVPD
• Electron transfer dissociation high-capacity (ETD HD) module
• BioPharma Finder™ 1.0 SP1, ReSpect and Xtract deconvolution
• ProSight Lite for fragment mapping

Main Results and Discussion

Intact mass analysis resolved key mAb glycoforms with better than 1 ppm accuracy. ETD HD fragmentation of subunits yielded sequence coverages of 76 % (Fc/2), 80 % (light chain) and 73 % (Fd). UVPD provided complementary a/x/b/y ions, enhancing coverage when combined with ETD HD and CID. Deglycosylated heavy chain achieved isotopic resolution at 240 K Orbitrap setting and, across six LC-MS runs incorporating ETD HD, UVPD and CID, 40 % sequence coverage was attained with mass errors within 2 ppm.

Benefits and Practical Applications

This top-down approach delivers rapid screening of intact mAb variants, confirmation of primary sequence, glycoform distribution and site-specific modifications. It streamlines quality control workflows in biopharmaceutical R&D, facilitating biosimilar comparability studies, post-translational modification analysis and antibody–drug conjugate characterization.

Future Trends and Potential Applications

Advances in fragmentation technology and data analysis are expected to push sequence coverage higher and reduce acquisition time. Emerging methods may integrate multi-dimensional separations, improved UVPD lasers and AI-driven spectral interpretation to enable routine high-throughput top-down workflows for complex biologics including ADCs, bispecifics and glycoproteins.

Conclusion

The combined application of ETD HD, UVPD and CID on an Orbitrap Fusion Lumos enables detailed top-down characterization of mAb intact forms, subunits and reduced heavy chains. This workflow achieves reliable mass accuracy, extensive sequence coverage and efficient glycoform profiling, supporting rigorous biopharmaceutical development.