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

Significance of the Topic

The selective quantification of monoclonal antibodies is a critical step in drug discovery and clinical pharmacokinetic studies. Nivolumab, an immune checkpoint inhibitor targeting PD-1, requires robust bioanalytical methods to monitor its concentration in human plasma. Shimadzu’s nSMOL™ technology provides Fab‐selective proteolysis, simplifying sample preparation and enabling highly specific LC‐MS/MS assays across a range of antibody therapeutics.

Study Objectives and Overview

This application note describes the development and full validation of an nSMOL‐based LC‐MS/MS method for quantifying Nivolumab. Key goals include establishing a universal sample processing workflow, selecting signature peptides for specificity, and demonstrating accuracy, precision, and stability in human plasma over a clinically relevant concentration range.

Methodology and Instrumentation

The nSMOL workflow enriches Fv‐region peptides from digested antibody using immobilized protease and protein A resin. A consistent sample preparation protocol ensures compatibility with various monoclonal antibodies.

Used Instrumentation

• LC System: Nexera X2 with Shim-pack GISS C18 column (50 mm × 2.1 mm), column oven at 50 °C
• Mobile Phases: 0.1 % formic acid in water (A) and acetonitrile (B); gradient from 1 % to 95 % B
• Flow Rate: 0.4 mL/min; Injection Volume: 10 μL
• MS: LCMS-8050/8060, positive ESI; interface 300 °C; heat block 400 °C; DL 250 °C; nebulizer gas 3 L/min; drying/heating gas 10 L/min

Key Findings and Discussion

Candidates from eight signature peptides were tested; only ASGITFSNSGMHWVR correlated reliably with Nivolumab concentration, highlighting the challenge of homology with endogenous IgGs. MRM transitions for quantitation and structural confirmation were optimized. The calibration curve displayed linearity between 0.15 and 300 μg/mL in human plasma.

Validation data at low (2.93 μg/mL) and high (200 μg/mL) concentrations demonstrated:

• Precision (CV): 6.75–7.51 %
• Accuracy: 95.6–107 %
• Freeze‐thaw, long‐term (–20 °C), and 48 h processed‐sample stability (5 °C) met acceptance criteria.

Benefits and Practical Applications

The nSMOL approach offers a single, streamlined protocol for multiple antibody drugs, reducing method development time. Its Fv‐selectivity enhances specificity in complex matrices, and the validated LLOQ of 0.15 μg/mL covers preclinical to clinical dosing levels. This makes the assay suitable for pharmacokinetic monitoring, immunogenicity studies, and biomarker research.

Future Trends and Opportunities

As biologics pipelines expand, multiplexed monitoring of antibody therapeutics and anti‐drug antibodies will become important. Integration of automated nSMOL sample preparation and high‐throughput LC‐MS platforms could accelerate large‐scale studies. Further refinement of signature peptide selection may improve sensitivity and broaden applicability to biosimilar and next‐generation antibody formats.

Conclusion

The validated nSMOL‐LC-MS/MS method for Nivolumab provides a robust, accurate, and precise assay across a wide concentration range in human plasma. It fulfills regulatory guidance for bioanalytical validation and supports translational research from early preclinical models to clinical trials.

Reference

• 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.