LCMS Bioanalysis of Antibody Drugs Using Fabselective Proteolysis "nSMOL Method" - Part 6 - Improvement of Reaction Conditions for Automated Analyses -

Importance of the Topic

Antibody-based therapeutics are increasingly used in clinical practice, requiring robust bioanalytical methods to support pharmacokinetics, toxicology, and clinical trials. The nSMOL (nano-surface and molecular-orientation limited) technique selectively digests the Fab region of immunoglobulin G (IgG), enabling targeted peptide quantitation by LC–MS/MS. Simplifying this protocol by eliminating the need for continuous shaking enhances throughput and automation, addressing key bottlenecks in antibody drug analysis.

Study Objectives and Overview

This study evaluates the feasibility and performance of nSMOL proteolysis under static conditions (no continuous agitation) and validates the method against Japanese bioanalytical guidelines for small molecules. Four monoclonal antibodies—Trastuzumab, Bevacizumab, Rituximab, and Nivolumab—were analyzed to assess digestion efficiency, accuracy, and precision.

Methodology

Key steps in the static nSMOL workflow:

• IgG capture: Plasma samples are exposed to a 100 nm pore resin that selectively binds IgG.
• Washing: Non-IgG matrix components are removed by filtration and rinsing.
• Proteolysis: FG beads (200 nm trypsin-immobilized nanoparticles) are added, mixed for 10 seconds, and incubated at 50 °C without further shaking.
• Quenching and cleanup: A stop solution is added, then beads and resin are removed by spin filtration.
• LC-MS/MS analysis: Peptides are separated on a Shim-pack GISS C18 column (50 × 2.1 mm, 1.9 μm) using a gradient of 0.1 % formic acid in water and acetonitrile, followed by MRM detection on an LCMS-8050 triple quadrupole mass spectrometer.

Used Instrumentation

• Nexera X2 UHPLC system
• Shim-pack GISS C18 column (50 × 2.1 mm, 1.9 μm)
• LCMS-8050 triple quadrupole mass spectrometer with ESI in positive mode
• FG beads: 200 nm trypsin-immobilized nanoparticles
• IgG collection resin with 100 nm pores

Main Results and Discussion

Comparative analysis showed that static digestion achieved 84.6 % efficiency relative to shaken conditions for Trastuzumab and 95.7–102 % for the other antibodies, with no significant impact on quantitation values. Validation at low and high concentration levels (0.24–200 μg/mL for Trastuzumab; 0.44–240 μg/mL for Bevacizumab) demonstrated accuracy within 95–104 % and precision (CV) below 15 %, meeting regulatory criteria. The consistent performance under static conditions is attributed to Brownian motion of the FG beads, obviating the need for mechanical agitation.

Benefits and Practical Applications

The static nSMOL workflow offers:

• Elimination of shaking incubators, reducing equipment footprint.
• Simplified sample preparation compatible with automation.
• Robust quantitation across a wide concentration range.
• Applicability to pharmacokinetic studies in preclinical and clinical settings.

Future Trends and Applications

Potential developments include:

• Integration into high-throughput robotic platforms for large-scale studies.
• Extension to other antibody formats and biotherapeutics, such as ADCs and PEGylated proteins.
• Real-time or near-real-time bioanalysis workflows for adaptive dosing.
• Enhanced data analytics using machine learning for peptide selection and quantitation.

Conclusion

nSMOL proteolysis under static conditions is validated as an effective, accurate, and precise method for antibody bioanalysis. By removing the requirement for continuous shaking, this approach streamlines sample processing, facilitates automation, and maintains compliance with bioanalytical guidelines.

Reference

1. Iwamoto N et al. Analyst. DOI: 10.1039/c3an02104a
2. Iwamoto N et al. Anal Methods. DOI: 10.1039/C5AY01588J
3. Iwamoto N et al. Drug Metab Pharmacokinet. DOI: 10.1016/j.dmpk.2015.11.004
4. Iwamoto N et al. Biol Pharm Bull. DOI: 10.1248/bpb.b16-00230
5. Iwamoto N et al. J Chromatogr B Analyt Technol Biomed Life Sci. DOI: 10.1016/j.jchromb.2016.04.038