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

Significance of the Topic

The precise measurement of therapeutic monoclonal antibodies (t-mAbs) such as infliximab is essential for personalized dosing, cost management, and optimized patient outcomes. Traditional ligand binding assays can suffer from cross-reactivity, limited dynamic range, and high per-sample cost when processing small batches. LC-MS/MS presents a compelling alternative, delivering high specificity, broad quantification range, and streamlined workflows for clinical and research laboratories.

Objectives and Study Overview

This work assessed a ready-to-use mAbXmise commercial kit combined with LC-MS/MS for the quantification of infliximab in human plasma. The study aimed to evaluate analytical sensitivity, linearity, precision, matrix effects, and practical implementation on two tandem MS platforms.

Methodology and Instrumentation

Sample preparation employed the mAbXmise Inflammation Kit containing calibrators (2–100 µg/mL), quality controls at 4 and 25 µg/mL, and stable isotope-labeled internal standards.

• Immunocapture and wash steps minimized matrix interferences.
• Proteolytic digestion using CutXmise generated signature tryptic peptides overnight.
• LC separation was performed on an ACQUITY UPLC I-Class FL with an XSelect Premier HSS T3 column (2.1×50 mm, 2.5 µm) using a 4.5 min gradient.
• MRM detection targeted infliximab peptides and internal standards on Xevo TQ-XS and Xevo TQ-S micro mass spectrometers.
• Evaluation metrics included calibration linearity, QC precision, matrix effects in individual serum samples, and analysis of 29 anonymized clinical plasma samples.

Main Results and Discussion

• Calibration curves exhibited excellent linearity (r²>0.998) over the 2–100 µg/mL range.
• The SIN tryptic peptide provided the highest sensitivity at 2 µg/mL, followed by the ASQ peptide.
• Inter- and intra-day precision at QC levels showed ≤9.5 % CV, with accuracy between 95–108 % of nominal concentrations.
• Matrix effect evaluation across six serum samples indicated minimal signal variability and consistent response ratios.
• Analysis of 29 plasma samples revealed peptide reproducibility of CV 29 %; excluding the SIN peptide (prone to deamidation at an N-S motif causing a 0.98 Da mass shift) improved reproducibility to CV 10 %.

Benefits and Practical Applications

The mAbXmise-LC-MS/MS workflow offers:

• High analytical performance with sensitivity down to 2 µg/mL.
• Low sample volume requirement (20 µL plasma).
• Pre-formatted reagents enabling manual or automated operation.
• Adaptability to multiple MS platforms for both routine and research settings.

Future Trends and Potential Applications

• Extension of MS-based quantification to other therapeutic antibodies and multiplex assays.
• Increased automation and high-throughput capabilities for large-scale clinical studies.
• Implementation of real-time therapeutic drug monitoring in clinical practice.
• Regulatory standardization of MS-based bioanalytical methods to support widespread clinical adoption.

Conclusion

This study demonstrates that LC-MS/MS quantification of infliximab using the mAbXmise kit is robust, sensitive, and straightforward to implement. The approach overcomes limitations of ligand binding assays by delivering precise, reproducible data with minimal sample volumes and simplified workflows, making it well suited for both research and clinical pharmacology applications.

Used Instrumentation

• ACQUITY UPLC I-Class FL System (Waters).
• XSelect Premier HSS T3 column (2.1×50 mm, 2.5 µm).
• Xevo TQ-XS and Xevo TQ-S micro tandem mass spectrometers.