LCMS Bioanalysis of Antibody Drugs Using Fab- Selective Proteolysis nSMOL - Part 4 - Multiplex Analysis -

Importance of the Topic

The rapid expansion of antibody–based therapeutics in clinical use demands sensitive, selective, and high-throughput bioanalytical methods. Traditional peptide mapping approaches often struggle with matrix interference and labor-intensive sample preparation, especially when multiple antibodies must be monitored simultaneously. The nSMOL (nano-surface and molecular orientation limited) proteolysis platform addresses these challenges by enabling Fab-directed, selective digestion of monoclonal antibodies, streamlining downstream LC–MS quantitation.

Objectives and Study Overview

This application note demonstrates the use of the Shimadzu nSMOL Antibody BA Kit for multiplex quantification of three therapeutic antibodies—brentuximab vedotin, rituximab, and cetuximab—in human plasma. The goals were to establish a unified sample preparation protocol, develop a robust LC–MS/MS method, and validate precision, accuracy, and lower limit of quantitation (LLOQ) according to international bioanalytical guidelines.

Methodology and Instrumentation

Sample Preparation Protocol

• Capture of human IgG from plasma via Fc affinity.
• Immobilized trypsin on nanoparticles selectively cleaves the Fab region, generating signature peptides.
• Cleanup and direct injection of digests without further desalting.

LC–MS/MS Conditions

• LC System: Nexera X2 with Shim-pack GISS C18 column (50 × 2.1 mm, 50 °C).
• Mobile Phase: A = 0.1% formic acid in water; B = 0.1% formic acid in acetonitrile; gradient 1%–42% B over 4 min.
• Flow Rate: 0.4 mL/min; Injection: 10 μL.
• MS: LCMS-8050/8060, ESI positive, DL 250 °C, heat block 400 °C, interface 300 °C, nebulizer 3 L/min, drying and heating gas 10 L/min.

Main Results and Discussion

Multiplexed MRM transitions were optimized for each antibody’s quantifier and qualifier peptides. Calibration curves spanning 0.58–300 μg/mL showed linearity with accuracies between 91.5% and 115% and intra-assay precision (CV) below 12%.

• Brentuximab Vedotin: LLOQ 0.58 μg/mL, CV ≤7.6%, accuracy 99–104%.
• Rituximab: CV ≤9.7%, accuracy 101–104%.
• Cetuximab: CV ≤8.5%, accuracy 97.5–106%.

MRM chromatograms demonstrated clear peak separation and low background. Performance met the FDA and Japanese MHLW bioanalytical validation criteria for therapeutic proteins.

Benefits and Practical Applications

The nSMOL approach offers:

• Uniform protocol for diverse antibody drugs, reducing method development time.
• High selectivity for Fab peptides, minimizing matrix effects.
• Capability for multiplex analysis, ideal for combination therapies and pharmacokinetic studies.
• Applicability from preclinical to clinical stages using the same workflow.

Future Trends and Potential Applications

Advancements in nanoparticle-supported proteolysis and MS instrumentation may further lower LLOQs and increase multiplex capacity. Integration with automation platforms could accelerate large-scale PK/PD studies. Expanding nSMOL to bispecific antibodies and antibody–drug conjugates promises broader adoption in biopharma bioanalysis.

Conclusion

The Shimadzu nSMOL Antibody BA Kit combined with Nexera X2 and LCMS-8050/8060 systems enables robust, high-throughput multiplex quantitation of therapeutic antibodies in plasma. Its validated precision, accuracy, and broad dynamic range make it a versatile tool for bioanalysis across drug development phases.

Reference

• Iwamoto N et al. Analyst, 2014; DOI:10.1039/c3an02104a.
• Iwamoto N et al. Clinical Pharmacology & Biopharmaceutics, 2016; DOI:10.4172/2167-065X.1000164.