Microfow LC-MS/MS analysis of monoclonal antibody in human plasma at ng/mL level with nSMOL proteolysis

Significance of the Topic

Quantitative analysis of therapeutic monoclonal antibodies in human plasma at nanogram-per-milliliter levels is essential for pharmacokinetic and preclinical studies. High specificity, wide dynamic range, and rapid assay development accelerate biologics research and support clinical decision making.

Objectives and Study Overview

This work aimed to establish a highly sensitive and robust workflow for measuring trastuzumab in human plasma using nano-surface and molecular-orientation limited (nSMOL) proteolysis alongside a newly developed microflow LC-MS/MS platform. The goal was to achieve low ng/mL limits of quantitation with rapid analysis.

Methodology and Experimental Workflow

The workflow combined nSMOL proteolysis—which selectively digests the Fab region of IgG to generate signature CDR peptides—with microflow LC-MS/MS. Human plasma samples were spiked with trastuzumab across a broad concentration range, stored at –80 °C, then processed using the nSMOL Antibody BA Kit. Tryptic peptides were separated on a microflow C18 column and analyzed by triple-quadrupole MS in positive electrospray mode.

Used Instrumentation

• Shimadzu Nexera Mikros microflow LC system with 1–500 µL/min pump
• UF-Link direct column-to-source connection for minimal dead volume
• Shim-Pack MC C18 column (0.175 mm ID × 50 mm)
• Shimadzu Micro ESI-8060 ion source and LCMS-8060 mass spectrometer
• nSMOL™ Antibody BA Kit for Fab-selective proteolysis

Main Results and Discussion

The assay displayed excellent linearity (R² = 0.9998) over 0.00763–62.5 µg/mL, with a lower limit of quantitation of 7.6 ng/mL—nearly an order of magnitude improvement over previous methods. Chromatographic peaks exhibited narrow widths (~3.7 s), reflecting minimal post-column dispersion. Intra-day precision and accuracy met FDA criteria (RSD < 20% at LLOQ and < 15% at higher levels; accuracy within 91–106%).

Benefits and Practical Applications

This approach offers rapid assay development (days instead of months compared to ligand binding assays), high specificity through signature peptide selection, and multiplexing capability. Microflow operation enhances ionization efficiency and reduces sample consumption, while the UF-Link connection ensures robust routine performance.

Future Trends and Possibilities of Use

Extending this microflow LC-MS/MS strategy with nSMOL proteolysis to other therapeutic antibodies could standardize bioanalysis across multiple targets. Integration with automated sample preparation and multi-analyte panels promises further workflow acceleration and comprehensive pharmacokinetic profiling.

Conclusion

The combined nSMOL and Nexera Mikros platform delivers sensitive (single-digit ng/mL) and reliable quantitation of trastuzumab in human plasma within an 11-minute runtime. This methodology is well suited for both preclinical and clinical pharmacokinetic studies and is adaptable to a wide range of monoclonal antibodies.

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