Qualitative Characterization and Quantitative Assessment of Monoclonal Antibodies Using Protein Metrics and nSMOLcoupled with the Shimadzu LCMS-9030 QTOF

Importance of the topic

Monoclonal antibodies (mAbs) have become a cornerstone of modern biotherapeutics due to their specificity and efficacy in treating a variety of diseases. Reliable analytical methods are essential to ensure product quality, safety, and consistency during development, manufacturing, and therapeutic monitoring. Advanced mass spectrometry approaches enable detailed characterization of intact mAbs, their subunits, post-translational modifications, and quantitation in complex biological matrices.

Objectives and study overview

This study demonstrates a comprehensive workflow combining the Shimadzu LCMS-9030 quadrupole time-of-flight (QTOF) system with Protein Metrics software and Shimadzu’s nano-surface and molecular orientation limited proteolysis (nSMOL) technology. The goals are to:

• Qualitatively characterize intact NIST mAb reference standard, its heavy (HC) and light chain (LC) subunits, and peptide fragments.
• Assess glycoform heterogeneity and confirm mass accuracy against theoretical values.
• Quantitatively determine mAb concentration in human serum using targeted MRM transitions enabled by nSMOL.

Used methodology and instrumentation

The workflow integrates:

• Shimadzu Nexera X2 UHPLC coupled to the LCMS-9030 QTOF mass spectrometer.
• LC modules: two LC-30AD solvent delivery pumps, DGU-20A5R online degasser, SIL-30AC autosampler, CTO-20AC column oven, CBM-20A system controller.
• LabSolutions Ver. 5.95 software for data acquisition.
• Protein Metrics Byos for intact mass and PTM analysis.
• nSMOL technology for selective Fab-region digestion and enhanced sensitivity.
• Enzymatic deglycosylation using PNGase F and proteolytic digestion with trypsin, Lys-C, and Glu-C.

Main results and discussion

Intact mAb analysis produced deconvoluted mass spectra matching theoretical masses within 3.7 Da across multiple glycoforms. Subunit evaluation showed clear separation and accurate mass assignment for HC and LC. Peptide mapping with different proteases achieved high sequence coverage and confident identification of PTMs. nSMOL-mediated digestion followed by MRM detection of a signature peptide (FTFSLDTSK) yielded a linear calibration range of 100–10,000 ng/mL in serum with accuracy within 20% and r² of 0.998, highlighting robust quantitation capability in complex matrices.

Benefits and practical applications

The combined use of high-resolution QTOF MS and targeted nSMOL proteolysis delivers:

• Comprehensive structural and glycoform characterization in a single platform.
• Accurate mass measurement to verify product consistency.
• High-sensitivity quantitation for pharmacokinetic, pharmacodynamic, and therapeutic drug monitoring studies.
• Streamlined workflow suitable for biopharmaceutical QC and R&D environments.

Future trends and potential applications

Advances likely include further integration of MS platforms with automated sample preparation, expanded use of site-specific proteolysis reagents for accelerated PTM profiling, and adoption of real-time data processing powered by machine learning. Emerging techniques may enable deeper structural insights and higher-throughput monitoring of mAbs in clinical settings.

Conclusion

This work validates the Shimadzu LCMS-9030 QTOF and Protein Metrics workflows combined with nSMOL technology as a powerful solution for the qualitative and quantitative analysis of monoclonal antibodies. It supports rigorous characterization and reliable quantification in complex biological samples, meeting critical needs in biotherapeutic development and monitoring.