LC-MS Bioanalysis of Antibody Drugs Using Fab-Selective Proteolysis nSMOL - Part 5 – Instrument comparison of precision and accuracy –

Importance of the Topic

Monoclonal antibody therapeutics require reliable bioanalysis methods to support pharmacokinetic studies and regulatory submissions. The nSMOL proteolysis workflow offers Fab‐selective digestion of antibodies, enabling reproducible LC-MS quantification that adheres to stringent bioanalytical validation criteria.

Objectives and Study Overview

This study compares the performance of the Shimadzu LCMS-8050 with a competing triple quadrupole mass spectrometer for quantifying bevacizumab in human plasma. Both platforms utilize the nSMOL Antibody BA Kit for sample pretreatment. Key metrics of precision, accuracy, and sensitivity are assessed under identical MRM acquisition conditions.

Methodology and Instrumentation

The nSMOL workflow involves two main steps: immunoglobulin G capture on a 100 nm pore resin, followed by site‐selective proteolysis of the Fab region by trypsin‐immobilized nanoparticles (200 nm FG beads). After reaction quenching, peptides are isolated via spin filtration and subjected to LC-MS analysis without further cleanup.

MRM parameters include dwell and pause times optimized for each instrument (10 ms dwell/3 ms pause for the LCMS-8050 versus 15 ms/5 ms for the competitor). Chromatography is performed on a Shim-pack GISS C18 column (50 × 2.1 mm, 1.9 µm) with a 5 min gradient (1–35 % acetonitrile) at 0.4 mL/min and 50 °C.

Instrumentation

• Shimadzu Nexera X2 UHPLC system
• Shimadzu LCMS-8050 and LCMS-8060 triple quadrupole mass spectrometers
• Shim-pack GISS C18 column (50 mm × 2.1 mm, 1.9 µm)
• 100 nm IgG collection resin and 200 nm trypsin-immobilized FG beads

Main Results and Discussion

The LCMS-8050 achieved an average accuracy of 100.7 % across 0.146–300 µg/mL bevacizumab, with low RSD (0.024). The competitor instrument showed a higher RSD (0.212) and slightly lower accuracy (average 101.3 %). The internal standard (P14R) signal remained stable on the LCMS-8050 even at low analyte levels. Enhanced sensitivity and faster cycle times contributed to superior quantitation of trace concentrations.

Benefits and Practical Applications

nSMOL proteolysis combined with the LCMS-8050 platform delivers:

• High selectivity for Fab‐derived peptides, minimizing matrix interferences
• Robust precision and accuracy compliant with regulatory guidelines
• Simplified maintenance (tool-free ESI capillary and desolvation line replacement) and cost-effective consumables
• Streamlined workflow applicable to various antibody drugs without method redesign

Future Trends and Applications

Broad adoption of Fab-selective proteolysis is expected in targeted proteomics for biotherapeutics. Advances may include automation of bead handling, expansion to bispecific antibodies, and integration with high-resolution MS for multiplexed assays. Data processing algorithms will evolve to support large‐scale PK/PD studies and immunogenicity monitoring.

Conclusion

The study demonstrates that the Shimadzu LCMS-8050, when paired with the nSMOL Antibody BA Kit, outperforms a leading competitor in sensitivity, accuracy, and operational efficiency for antibody bioanalysis. This platform supports rigorous validation requirements and simplifies routine quantitation of therapeutic antibodies.

References

• Iwamoto N et al., Analyst, DOI:10.1039/c3an02104a
• Iwamoto N et al., Drug Metab Pharmacokinet, DOI:10.1016/j.dmpk.2015.11.004