Triple Quadrupole Mass Spectrometry (Xevo TQ-XS) for the Quantification of Monoclonal Antibody Light Chains in Plasma

Importance of the Topic

Monoclonal antibodies and their subunits are vital in modern therapeutics but present analytical challenges due to high molecular weight and similarity to endogenous immunoglobulins. Traditional surrogate peptide approaches often face limitations in sensitivity and specificity at low concentrations. Direct subunit quantification by LC MS MS provides a streamlined route to measure larger protein constructs and improves selectivity for variable regions of interest with minimal sample manipulation.

Objectives and Study Overview

This work aims to establish a robust, sensitive workflow for quantifying adalimumab light chains in rat plasma using a triple quadrupole mass spectrometer. Key goals include developing a reproducible sample preparation method, achieving high chromatographic resolution of subunits, and validating method performance across a wide dynamic range.

Methodology and Instrumentation

Sample Preparation

• Immunopurification via biotinylated anti human Fc antibody on streptavidin magnetic beads
• Partial reduction of mAbs with dithiothreitol followed by alkylation with iodoacetamide
• Elution in low percentage formic acid and neutralization with ammonium bicarbonate

Chromatography

• ACQUITY UPLC I Class PLUS system with BioResolve RP mAb Polyphenyl column at 80 degrees C
• Short 8.5 minute gradient with formic acid in water and acetonitrile

Mass Spectrometry

• Xevo TQ XS triple quadrupole operated in MRM mode
• Key transitions 1329.85 m/z (y96) and 1554.39 m/z (b115) for adalimumab light chain
• Low resolution setting (1.0 Da FWHM) to enhance sensitivity by ~1.5X

Instrumentation

• ACQUITY UPLC I Class PLUS system
• BioResolve RP mAb Polyphenyl column 450 A, 2.7 micrometer, 2.1 x 50 mm
• Xevo TQ XS triple quadrupole mass spectrometer
• MassLynx 4.2 and TargetLynx software

Main Results and Discussion

Chromatographic methods achieved baseline separation of adalimumab and cetuximab light chains within an 8.5 minute cycle. The assay delivered a lower limit of quantification of 25 ng per mL from only 10 microliters of rat plasma. Calibration curves were linear (r2 > 0.99) over four orders of magnitude, with accuracy within 87 to 109 percent and precision better than 7 percent RSD. High sensitivity MRM transitions were confirmed by high resolution MS and transferred to the triple quadrupole platform with optimized cone voltages and collision energies.

Benefits and Practical Applications

• Eliminates need for surrogate peptide selection and complex digestion workflows
• Provides direct measurement of mAb subunits including variable regions
• Uses ubiquitous triple quadrupole instrumentation familiar in bioanalytical labs
• Enables high throughput and reproducible PK studies in preclinical matrices

Future Trends and Applications

Future directions include extension to other mAb subunits such as Fc fragments, integration with automated immunocapture platforms for increased throughput, and application in clinical therapeutic monitoring and QA QC environments. Advances in column chemistries and mass spectrometer designs will further enhance sensitivity and reduce cycle times.

Conclusion

This study demonstrates a complete workflow for rapid, sensitive quantification of monoclonal antibody light chains in plasma using triple quadrupole mass spectrometry. The optimized immunopurification, chromatographic separation, and MRM-based detection achieve robust performance suitable for large bioanalytical campaigns.

References

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