IMPROVE SPEED AND ACCURACY OF MONOCLONAL ANTIBODY BIOANALYSIS USING NANOTECHNOLOGY AND LCMS

Importance of the Topic

Monoclonal antibodies (mAbs) are among the top‐selling therapeutics for treating autoimmune disorders and various cancers. Accurate bioanalysis of mAbs is essential to determine pharmacokinetic and pharmacodynamic profiles, optimize dosing regimens, and ensure safety and efficacy during drug development and clinical monitoring.

Goals and Study Overview

This work introduces nSMOL (nano‐surface and molecular‐orientation limited proteolysis), a nanotechnology‐enabled method combined with liquid chromatography–mass spectrometry (LC–MS/MS) to accelerate and enhance the precision of mAb bioanalysis. The approach aims to simplify sample preparation, increase selectivity for antibody fragments, and enable multiplex quantitation of multiple therapeutic antibodies in plasma.

Methodology and Instrumentation

nSMOL employs an immunoglobulin‐binding resin coated with Protein A ligands to capture intact antibodies from biological matrices. Following two wash cycles, trypsin immobilized on nanoparticles is added; its size restricts digestion to the Fab region protruding from nano‐wells, yielding signature peptides.

Instrumentation

• Triple quadrupole LC–MS/MS system (Shimadzu LCMS-8060)
• Immunoglobulin collection resin with Protein A ligands
• Trypsin‐immobilized nanoparticles

Peptides are directly injected into the LC–MS/MS for multiple reaction monitoring (MRM). The entire workflow, from digestion to data acquisition, can be completed in about five minutes per sample.

Key Results and Discussion

nSMOL demonstrated:

• High reproducibility and quantitative repeatability due to selective Fab‐only digestion
• Significant reduction of background noise and ion suppression compared to traditional LC–MS/MS workflows
• Elimination of denaturation, reduction, alkylation, and solid‐phase extraction steps
• Full method validation for Trastuzumab, Nivolumab, Bevacizumab, and an antibody‐drug conjugate (Brentuximab vedotin)
• Successful multiplex analysis of at least three mAbs in a single plasma sample

Benefits and Practical Applications

nSMOL offers:

• Streamlined sample preparation without capture antibodies or ligands
• Rapid method development at lower cost
• Compliance with FDA and Japan MHLW bioanalytical guidelines
• Capability to perform multiplex PK/PD studies in preclinical and clinical settings

Future Trends and Opportunities

Emerging directions include:

• Application to bispecific and next‐generation antibody formats
• Integration with high‐resolution mass spectrometry for top‐down proteomics
• Automation and high‐throughput sample processing in regulated labs
• Real‐time monitoring of therapeutic antibodies in clinical trials

Conclusion

The nSMOL approach, combining nanotechnology and LC–MS/MS, offers a universal, fast, and highly selective bioanalysis platform for mAbs and related biotherapeutics. It addresses limitations of ligand binding assays and conventional LC–MS workflows and supports robust PK/PD and ADC quantification in drug development.

References

1. LC–MS Bioanalysis of Trastuzumab using Fab‐selective proteolysis nSMOL, Shimadzu application note
2. LC–MS Bioanalysis of Nivolumab using Fab‐selective proteolysis nSMOL, Shimadzu application note
3. LC–MS Bioanalysis of Bevacizumab using Fab‐selective proteolysis nSMOL, Shimadzu application note
4. Multiplex LC–MS Bioanalysis of Antibody Drugs using Fab‐selective proteolysis nSMOL, Shimadzu application note