Microflow LC-MS/MS Analysis of Monoclonal Antibody in Human Plasma at ng/mL Level with nSMOLTM Proteolysis

Microflow LC-MS/MS Analysis of Monoclonal Antibody in Human Plasma Using nSMOL Proteolysis

Significance of the Topic:
The quantification of therapeutic monoclonal antibodies in human plasma is vital for pharmacokinetic and clinical research. Traditional ligand binding assays require extensive development time and may lack specificity. Microflow LC-MS/MS combined with nSMOL proteolysis offers rapid method deployment, high specificity through signature peptide selection, and enhanced sensitivity for low ng/mL detection in complex biological matrices.

Objectives and Study Overview:

• Establish a bioanalytical method for Trastuzumab quantification in human plasma at sub-μg/mL levels.
• Integrate nSMOL sample preparation with the Nexera Mikros microflow LC-MS/MS platform.
• Validate linearity, sensitivity (LLOQ), accuracy, and precision across calibration and QC samples.

Instrumentation:

• LC system: Nexera Mikros with Shim-Pack MC C18 column (50 mm × 0.175 mm) at 4 μL/min, 50 °C.
• Trap column: L-column 2 ODS Micro (5 mm × 0.3 mm) at 50 °C.
• Mass spectrometer: LCMS-8060 with micro-ESI in positive ion mode.

Methodology and Sample Preparation:
Human plasma was spiked with Trastuzumab over a concentration range of 7.6 ng/mL to 62.5 μg/mL for calibration and QC samples. Following storage at –80 °C, samples underwent nSMOL proteolysis using immobilized Protein A resin to selectively digest complementarity-determining regions. Resulting signature peptides (e.g., IYPTNGYTR) were analyzed by MRM transitions. Chromatographic separation employed a gradient of 0.1% formic acid in water and acetonitrile.

Key Results and Discussion:

• The assay exhibited linear response over four orders of magnitude with an LLOQ of 7.6 ng/mL, nearly tenfold improvement compared to semi-microflow systems.
• Accuracy ranged from 94% to 106% and precision (RSD) remained below 12% across QC levels on different days.
• Representative MRM chromatograms showed high signal-to-noise and clear peak resolution at low analyte concentrations.

Practical Benefits and Applications:

• Rapid assay development, reducing timeline from months to days.
• High specificity for antibody CDR peptides minimizes matrix interferences.
• Suitable for preclinical and clinical pharmacokinetic studies of monoclonal antibodies.

Future Trends and Opportunities:

• Coupling microflow LC systems with high-resolution MS to further enhance sensitivity and throughput.
• Adapting nSMOL workflows to other biotherapeutics such as antibody fragments and fusion proteins.
• Automating sample preparation and data processing to support high-throughput bioanalysis in contract research organizations and clinical laboratories.

Conclusion:
The integration of nSMOL proteolysis with microflow LC-MS/MS delivers a robust, sensitive, and efficient platform for therapeutic antibody quantification in plasma. The improved LLOQ and validated assay performance support streamlined pharmacokinetic evaluation and broad adoption in bioanalytical settings.

Reference

• Iwamoto N. et al. Analyst, DOI:10.1039/c3an02104a
• Iwamoto N. et al. Anal Methods, DOI:10.1039/c5ay01588j
• Iwamoto N. et al. Application News No. C145A