ADLM: LC-MS/MS ANALYSIS OF MABS USING A MONOCLONAL ANTIBODIES QUANTIFICATION KIT – SPOTLIGHT ON INFLIXIMAB

Significance of the Topic

Therapeutic monoclonal antibodies (t-mAbs) have transformed patient care by providing highly specific treatment options. Accurate quantification of agents such as infliximab is essential for optimizing dosing, monitoring therapeutic levels, and managing costs. Traditional ligand binding assays face limitations including narrow dynamic range, cross-reactivity, and high per-sample cost, driving interest in mass spectrometry–based alternatives.

Objectives and Study Overview

This work evaluates a commercial mAbXmise sample preparation kit coupled with LC-MS/MS for the quantification of infliximab in human plasma. Key aims include:

• Assessing assay linearity and sensitivity over a 2–100 µg/mL calibration range
• Evaluating precision and accuracy using quality control samples at 4 and 25 µg/mL
• Comparing performance on two tandem mass spectrometer platforms

Methodology

Twenty-microliter plasma aliquots, including calibrators and QCs, were processed via the mAbXmise immunocapture workflow. Captured infliximab was eluted, dried, and digested overnight with protease. Resulting tryptic peptides were separated on an ACQUITY UPLC system using a 4.5-minute acetonitrile/water/formic acid gradient and detected by MRM targeting signature infliximab peptides and stable isotope–labeled standards.

Used Instrumentation

• ACQUITY I-Class FL UPLC System with XSelect Premier HSS T3 column (2.1 × 50 mm, 2.5 µm)
• Xevo TQ-XS Mass Spectrometer
• Xevo TQ-S micro Mass Spectrometer

Main Results and Discussion

The assay demonstrated excellent linearity (r2 > 0.998) across the calibration range. The SIN and ASQ tryptic peptides provided optimal sensitivity at the 2 µg/mL level on both MS platforms. QC precision studies showed total CVs ≤ 9.5% and accuracies of 95–108%. Matrix effects were minimal across six individual serum samples. Analysis of 29 clinical specimens revealed higher variability (CV 29%) for a deamidation-prone peptide; removal of this peptide improved overall reproducibility to CV 10%.

Benefits and Practical Applications

The mAbXmise kit simplifies sample preparation, reduces matrix interferences, and enables low-volume workflows. Compatibility with manual and automated formats makes it suitable for clinical laboratories performing therapeutic drug monitoring and pharmacokinetic studies of infliximab.

Future Trends and Applications

• Development of multiplexed kits for simultaneous quantification of multiple therapeutic mAbs
• Enhanced laboratory automation to boost throughput and consistency
• Integration of high-resolution mass spectrometry for superior specificity
• Method standardization for broader regulatory acceptance in clinical settings

Conclusion

The combination of the mAbXmise kit with LC-MS/MS offers a robust, sensitive, and flexible approach for infliximab quantification. This method overcomes key limitations of ligand binding assays and supports precise therapeutic monitoring, with potential for automation and adaptation to other mAbs.