A Complete Workflow Solution for Intact Monoclonal Antibody Characterization Using a New High-Performance Benchtop Quadrupole-Orbitrap LC-MS/MS

Significance of the Topic

Monoclonal antibodies (mAbs) represent a cornerstone of modern therapeutic and diagnostic applications. Their complex structure, glycosylation heterogeneity, and sensitivity to processing demand robust analytical approaches. High-resolution mass spectrometry (MS) workflows enable precise determination of intact mass, glycoform distribution, and sequence verification, supporting quality control and accelerating biopharmaceutical development.

Objectives and Study Overview

This study presents a comprehensive LC-MS/MS workflow for intact mAb characterization using a benchtop Quadrupole-Orbitrap mass spectrometer. The goals were to achieve mass measurement accuracy below 10 ppm for intact and reduced antibodies, reproducible glycoform profiling, and on-line top-down sequencing to confirm primary structure and post-translational modifications.

Methodology and Instrumentation

Sample Preparation and Chromatography:

• Intact and reduced mAb samples (4 different antibodies) were analyzed; reduction was performed in 6 M guanidine-HCl with 5 mM DTT at 60 °C for 1 h.
• Separation employed a ProSwift RP-10R monolithic column (1 × 50 mm) at 80 °C with a 15 min gradient (20–98% acetonitrile containing 0.1% formic acid) at 60 µL/min.

Mass Spectrometry and Data Processing:

• Thermo Scientific Q Exactive™ Quadrupole-Orbitrap was used for intact mass measurement (full MS at 17,500–140,000 resolution) and top-down MS/MS via multiplexed HCD (msx HCD) at 140,000 resolution.
• Instrument settings: spray voltage 4 kV, capillary 275 °C, sheath gas 10, auxiliary gas 5, in-source CID 45 eV, AGC targets of 1e6 (MS) and 2e5 (MS/MS), max injection time 250 ms.
• Proteoform deconvolution applied Protein Deconvolution 1.0 with the ReSpect™ algorithm (m/z 2000–4000, mass 140–160 kDa, 8 charge states minimum).
• Top-down spectra were interpreted using ProSightPC™ 2.0 in single-protein mode (5 ppm fragment tolerance).

Instrumentation Used

• Q Exactive Quadrupole-Orbitrap MS with HCD/C-Trap and spectrum multiplexing.
• Thermo Scientific ProSwift RP-10R monolithic HPLC column.
• ProSightPC™ software for top-down data interpretation.
• Protein Deconvolution 1.0 with ReSpect™ algorithm.

Main Results and Discussion

Mass Accuracy and Reproducibility:

• Intact mAb mass errors consistently <10 ppm; average error 6.9 ppm (SD 6.4 ppm) across multiple runs and instruments.

Glycoform Profiling:

• Five major glycoforms baseline-resolved; relative abundance reproducibility within a few percent.

Top-Down Sequencing:

• Online msx HCD achieved >40% sequence coverage for the light chain, including variable region, with fragment mass errors <5 ppm.

Benefits and Practical Applications

This workflow offers:

• High-throughput, robust intact mass measurement for QC of mAb products.
• Accurate glycosylation profiling to monitor critical quality attributes.
• Rapid on-line top-down sequencing for sequence confirmation and PTM analysis.

Future Trends and Potential Applications

Emerging directions include integration of automated data pipelines, enhanced multiplexed fragmentation methods, deeper coverage of heavy and light chains, characterization of novel biotherapeutic formats (bispecifics, ADCs), and real-time process monitoring in biomanufacturing.

Conclusion

The described benchtop Quadrupole-Orbitrap LC-MS/MS workflow combines fast monolithic chromatography, high-resolution mass analysis, multiplexed HCD and advanced data processing to deliver precise, reproducible intact mAb characterization and reliable top-down sequencing. This solution accelerates biopharmaceutical R&D and quality control efforts.