LC-MS Bioanalysis of Antibody Drugs Using Fab-Selective Proteolysis nSMOL - Part 5 – Instrument comparison of precision and accuracy –

Significance of the Topic

Accurate quantitation of monoclonal antibodies in biological matrices is essential for pharmacokinetics, therapeutic monitoring, and quality control in drug development. The nSMOL proteolysis approach selectively targets the Fab region of antibodies, minimizing matrix effects and ensuring consistent, guideline-compliant bioanalysis.

Objectives and Study Overview

This study evaluates the analytical performance of the Shimadzu LCMS-8050 against a competing triple quadrupole hybrid mass spectrometer (Company A) for quantifying bevacizumab in human plasma using the nSMOL sample-preparation workflow. Key metrics include precision, accuracy, sensitivity, and compliance with bioanalytical validation criteria.

Methodology and Instrumentation

• Sample Preparation Workflow: Capture of total IgG on 100 nm resin, removal of non-IgG components, proteolysis of Fab fragments using 200 nm trypsin-immobilized FG beads, reaction quenching, and removal of beads/resin by spin filtration.
• MRM Quantitation: Signature peptides for bevacizumab monitored via MRM transitions; P14R peptide used as internal standard for normalization.
• Liquid Chromatography: Nexera X2 system; Shim-pack GISS C18 column (50 × 2.1 mm, 1.9 µm); gradient elution (0.1% formic acid in water/acetonitrile) over 5 min at 0.4 mL/min; column temperature 50 °C; injection volume 10 µL.
• Mass Spectrometry: ESI positive mode; DL 250 °C; block heater 400 °C; interface 300 °C; gas flows: nebulizer 3 L/min, drying/heating 10 L/min; probe position 2 mm.

Main Results and Discussion

• MRM Timing: LCMS-8050 achieved a shorter cycle time (182 ms) with 10 ms dwell and 3 ms pause, compared to 280 ms (15 ms dwell, 5 ms pause) on the competitor instrument.
• Quantitation Range and Accuracy: Linear range from 0.146 to 300 µg/mL in plasma; averaged accuracy of 100.7% across levels using LCMS-8050.
• Sensitivity and Precision: Shimadzu system delivered higher sensitivity at low concentrations and a lower variability (%RSD ~2.4%) of the internal standard signal versus ~21.2% for the competitor.
• Instrument Design Impact: Differences in collision gas type and spectrometer architecture likely contributed to improved accuracy and precision on the LCMS-8050.

Benefits and Practical Applications

• Universal Protocol: Single pretreatment method applicable to diverse antibody therapeutics.
• High Selectivity: Reduced matrix contaminants preserve chromatography column and MS ion source integrity, extending uptime.
• Streamlined Maintenance: Tool-free ESI capillary replacement and vacuum-free desolvation line swaps on LCMS-8050 simplify upkeep.
• Cost Efficiency: Lower replacement part costs relative to competing systems.

Future Trends and Applications

• Broadening the nSMOL approach to additional antibody classes and biosimilars.
• Integration with high-throughput automated platforms for large-scale PK/PD studies.
• Advancements in nanoparticle-based proteolysis for enhanced digestion efficiency.
• On-line coupling of sample preparation with LC-MS for fully automated workflows.

Conclusion

Combining nSMOL proteolysis with the Shimadzu LCMS-8050 system provides a robust, high-throughput workflow that meets regulatory bioanalytical requirements while delivering superior sensitivity, accuracy, and operational efficiency for antibody drug quantitation.

References

• Iwamoto N et al. Analyst. DOI:10.1039/c3an02104a
• Iwamoto N et al. Drug Metab Pharmacokinet. DOI:10.1016/j.dmpk.2015.11.004