TANDEM QUADRUPOLE MS FOR THE QUANTIFICATION OF MONOCLONAL ANTIBODY SUBUNIT LIGHT CHAINS IN PLASMA

Significance of Topic

The increasing complexity of biotherapeutics requires highly sensitive and selective methods to quantify intact monoclonal antibodies and their subunits directly in biological matrices. Traditional surrogate peptide assays are robust but may not fully capture structural variants. Direct measurement of subunit light chains complements peptide approaches by preserving higher-order information.

Study Objectives and Overview

This study demonstrates the feasibility of an optimized immunocapture and LC-MS/MS workflow on a tandem quadrupole mass spectrometer for quantifying adalimumab subunit light chains in rat plasma. The goals were to develop a rapid, selective sample preparation protocol, identify generic MS/MS transitions, and achieve a broad calibration range with high accuracy and precision.

Methodology

The workflow involves:

• Affinity purification of adalimumab from 10 µL rat plasma using biotinylated anti-human Fc antibody and streptavidin magnetic beads
• Reduction of captured antibodies with dithiothreitol to split light and heavy chains
• Alkylation with iodoacetamide for peptide stability
• Acidification and direct injection

Chromatographic separation was evaluated on a BioResolve RP mAb Polyphenyl column and a BEH C4 column under 0.1% formic acid in water and acetonitrile. A rapid 8.5-minute gradient at 80°C delivered well-resolved light chain peaks. Mass spectrometry employed a Xevo TQ-XS tandem quadrupole system in MRM mode, targeting the conserved fragment m/z transition 1236.02→1329.85.

Main Results and Discussion

The BioResolve column significantly improved peak shape and sensitivity versus the BEH C4 phase. Tandem quadrupole spectra produced charge state envelopes comparable to high-resolution instruments, confirming suitability for subunit analysis. A common y96 fragment was used across analyte and internal standard for generic quantification. Calibration was linear from 25 to 100,000 ng/mL (r2 > 0.99). Quality control samples showed accuracies of 89–112% and RSDs below 6% at multiple concentration levels.

Practical Benefits and Applications

This method offers:

• High sensitivity and specificity in small plasma volumes
• Fast throughput with an 8.5-minute cycle
• Generic application to human IgG light chains
• Compatibility with long-term preclinical and clinical studies on tandem quadrupole instruments

Used Instrumentation

• UPLC System: ACQUITY UPLC I-Class PLUS with fixed loop injector
• Columns: BioResolve RP mAb Polyphenyl (450Å, 2.7 µm, 2.1×50 mm) and Waters BEH C4 (300Å, 1.7 µm, 2.1×50 mm)
• Mass Spectrometer: Xevo TQ-XS tandem quadrupole with electrospray source

Future Trends and Applications

Expanding this workflow to additional monoclonal antibodies and antibody–drug conjugates will streamline pharmacokinetic profiling. Automation of immunocapture and sample processing can enhance throughput. Multiplexed assays and coupling with high-resolution instruments may further broaden structural characterization.

Conclusion

A rapid and robust LC-MS/MS assay on a tandem quadrupole system has been established for quantifying monoclonal antibody subunit light chains directly from plasma. The method balances sensitivity, accuracy, and throughput, making it suitable for routine biotherapeutic monitoring.

References

• Nguyen JM, Kizekai L, Walsh D, Cook J, Lauber MA. A Novel Phenyl Bonded Phase for Improved Reversed-Phase Separations of Proteins. Waters Application Note 720006169EN, January 2018.
• Dunning CM, Lame M, Wrona M, Haynes K. Tackling Non-Specific Binding of Biotherapeutics Using LC-MS Compatible QuanRecovery Sample Plates with MaxPeak High Performance Surfaces. Waters Application Note 720006528EN, March 2019.