LCMS Bioanalysis of Antibody Drugs Using Fab-Selective Proteolysis nSMOL - Part 3 - Nivolumab analysis -

Importance of the topic

Targeted bioanalysis of monoclonal antibodies is essential for both preclinical and clinical pharmacokinetic (PK) monitoring. Nivolumab, an immune checkpoint inhibitor that blocks PD-1 signaling, requires sensitive, selective quantitation to guide dose selection, evaluate therapeutic exposure, and support pharmacodynamic studies. The nSMOL technology represents an innovative approach to address the challenges of highly homologous endogenous immunoglobulins and streamline method development across diverse antibody therapeutics.

Objectives and study overview

This study demonstrates the application of Shimadzu’s nSMOL Antibody BA Kit for quantitative LC-MS/MS analysis of nivolumab in human plasma. Key aims include establishing a uniform sample processing protocol, validating assay performance according to regulatory bioanalytical guidelines, and confirming the suitability of signature peptides for accurate quantitation from preclinical through clinical settings.

Methodology and instrumentation

The workflow combines Fab-selective proteolysis with targeted MRM detection. Sample processing follows the nSMOL protocol previously optimized for trastuzumab, requiring no drug-specific modifications.

• LC system: Shimadzu Nexera X2 with Shim-pack GISS C18 column (50 × 2.1 mm, 50 °C)
• Mobile phase: 0.1 % formic acid in water (A) and acetonitrile (B); gradient from 1 % to 95 % B; 0.4 mL/min
• MS detector: Shimadzu LCMS-8050/8060 in positive ESI mode; DL 250 °C; heat block 400 °C; interface 300 °C; nebulizer 3 L/min; drying/heating gas 10 L/min
• Quantitation peptides: ASGITFSNSGMHWVR (y11++ transition 550.8>661.5) and P14R peptide as internal standard (512.1>292.3)

Main results and discussion

The assay exhibited a calibration range of 0.15–300 μg/mL with average accuracy of 100.4 %. Precision and accuracy at QC levels (2.93 and 200 μg/mL) met acceptance criteria (accuracy ~101 %, CV <8 %). Stability tests covering freeze–thaw, long-term storage at –20 °C, and 48 h processed sample stability at 5 °C confirmed reliable peptide integrity and reproducibility. Among eight candidate peptides, only ASGITFSNSGMHWVR correlated linearly with concentration, underscoring the importance of selecting unique sequences in fully human antibodies due to high homology with endogenous IgGs.

Benefits and practical applications

• Universal sample processing applicable to multiple antibody drugs, reducing method development time
• High selectivity via Fab-region proteolysis minimizes matrix interference from endogenous immunoglobulins
• Regulatory compliance with Japanese bioanalytical method validation guidelines
• Sensitivity down to 0.15 μg/mL supports early-phase PK studies and therapeutic drug monitoring

Future trends and potential applications

Continued expansion of nSMOL toward bispecific antibodies, antibody–drug conjugates, and biosimilars is anticipated. Integration with high-resolution MS and automation could further enhance throughput. Personalized medicine approaches, including real-time therapeutic monitoring and immunogenicity assessment, may benefit from refined peptide selectivity and multiplexed quantitation.

Conclusion

The nSMOL Antibody BA Kit enables robust, sensitive, and selective quantitation of nivolumab in human plasma with a streamlined workflow that can be extended to diverse antibody therapeutics. The technology meets stringent validation criteria and supports a seamless transition from preclinical to clinical bioanalysis.

References

• Ishida Y, Agata Y, Shibahara K, Honjo T. EMBO J. 1992;11(11):3887.
• Iwamoto N et al. Analyst. 2014; DOI:10.1039/c3an02104a.
• Iwamoto N et al. J Chromatogr B. 2016; DOI:10.1016/j.jchromb.2016.04.038.