LC/MS Analysis of the Monoclonal Antibody Rituximab Using the Q Exactive Benchtop Orbitrap Mass Spectrometer

Importance of the Topic

Monoclonal antibodies (mAbs) are critical biotherapeutics used in cancer, inflammation, autoimmune and infectious diseases.
Their complex structure and microheterogeneity demand thorough analytical characterization to ensure safety and efficacy.
High-resolution mass spectrometry coupled with efficient separations accelerates mAb development and quality control.

Study Objectives and Overview

This study presents a comprehensive LC-MS workflow for intact and reduced rituximab characterization using Thermo Scientific™ Q Exactive™ Orbitrap mass spectrometry.
Key aims:

• Accurate mass measurement of intact and subunit forms
• Glycoform profiling and relative quantitation
• Sequence confirmation via top-down and bottom-up approaches
• Evaluation of sensitivity and chromatographic performance

Methodology and Instrumentation

Sample Preparation:

• Rituximab dialyzed to remove surfactants
• Reduction with TCEP for subunit analysis
• Alkylation with IAA and trypsin digestion for peptide mapping

Chromatography:

• Monolithic PS-DVB columns: PepSwift 250×0.2 mm (1 µL/min) and ProSwift RP-10R 50×1 mm (60 µL/min)
• Gradient optimization for intact, reduced, and digested forms
• Column temperatures at 55 °C

Mass Spectrometry:

• Q Exactive Orbitrap with NanoFlex or IonMax HESI sources
• Full MS at resolutions up to 140 000
• All-Ion Fragmentation (AIF) and targeted MS2 (5-plex) for top-down
• Data deconvolution with Protein Deconvolution, Xtract and ReSpect algorithms
• Peptide identification using ProSightPC and Proteome Discoverer (SEQUEST)

Main Results and Discussion

Intact Mass Analysis:

• Baseline resolution of major glycoforms (G0F/G0F to G2F/G2F) with mass errors below 5 ppm
• Limit of detection ~500 pg on the PepSwift column; ~1 ng on ProSwift

Subunit Analysis:

• Efficient separation of light (23 kDa) and heavy (50 kDa) chains
• Isotopic resolution of light chain at 140 k resolution; heavy chain glycoforms resolved at 17 500 resolution

Top-Down Sequencing:

• AIF and multiplexed targeted MS2 yielded 15–28 % fragment coverage of light chain termini
• Heavy chain fragmentation proved more challenging; limited to N- and C-terminal fragments

Bottom-Up Peptide Mapping:

• Trypsin digest achieved 100 % sequence coverage of light chain and 99.5 % of heavy chain (including glycopeptides)
• Glycopeptide profiling confirmed heterogeneity at Asn301

Benefits and Practical Applications

• Rapid intact mass and glycoform profiling for lot release and comparability studies
• High-confidence subunit and peptide mapping to confirm primary structure
• Low sample consumption and high throughput via monolithic columns
• Robust platform for biosimilar characterization and QA/QC

Future Trends and Opportunities

• Integration of ion mobility and advanced fragmentation for deeper top-down coverage
• Automated data interpretation workflows for rapid biotherapeutic screening
• Expansion to higher order structure and charge variant analysis

Conclusion

An optimized LC-MS workflow using monolithic columns and Q Exactive Orbitrap enables sensitive, accurate characterization of intact, subunit, and digested mAbs.
The approach delivers comprehensive glycoform profiling, sequence confirmation, and quantitation with minimal sample input.
This strategy supports accelerated development and quality control of innovative biopharmaceuticals.

Reference

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