MSACL 2024 Abstract

LC-MS/MS Analysis of MAbs Using a Monoclonal Antibodies Quantification Kit – Spotlight on Infliximab

Dominic Foley (1), Dorothee Lebert (2), Lisa J Calton (1), John Vukovic (3)
(1) Waters Corporation, Stamford Avenue, Altrincham Road, Wilmslow, SK9 4AX, UK (2) Promise Proteomics, Grenoble, France (3) Waters Corporation, 34 Maple Street, Milford, MA 01757 USA

John Vukovic, MSc (Presenter) Waters Corporation

Presenter Bio: John received his Hons BSc in Biochemistry from McMaster University and his MSc in Biochemistry from Western University. He has been involved in liquid chromatographic analysis and method development for over 40 years. For more than two decades, he has been working with clinical laboratories across Canada in the installation, implementation and support of LCMS systems for a variety of Clinical Applications.

Relevant Financial Disclosures (within past 24 months, reported on Jul 11, 2025) Conflict mitigation NOT REQUIRED.

Background: Therapeutic monoclonal antibodies (t-mAbs) have been a revolution in the therapeutic tools available to clinicians for treating a variety of conditions. In the era of personalized medicine, there is increasing awareness of the need to measure mAbs for the purposes of dose optimization and cost management. The use of Ligand Binding Assay (LBA) based techniques for measuring mAbs is well established but has some limitations, including poor performance, lack of standardization, a high cost when processing a limited number of samples, limited dynamic range, and the potential for cross-reactivity. Moreover, commercial kits are only available for a limited number of mAbs. Mass spectrometry, a technology widely used in clinical laboratories for monitoring small molecules, is an interesting alternative to overcome these limitations. Here we demonstrate Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) associated with the ready-to-use commercial mAbXmise™ kit is a simple way to implement mAbs measurement for the anti-TNFα mAb, infliximab, providing high analytical performance, ease of use, and high flexibility for laboratory personnel.

Methods: The Promise Proteomics Inflammation mAbXmise Kit contains all the calibrators, QCs, internal standard and sample preparation consumables to run the LC-MS/MS method for infliximab. The QC material was used to evaluate method precision. Anonymized plasma samples (Synnovis Analytics, UK) were extracted using the mAbXmise Kit and analyzed using an in-house developed LC-MS/MS method.
Briefly, 20µL plasma samples, including calibrator, and quality controls, are dispensed into the mAbXmise plates containing lyophilized stable labelled infliximab. Samples are transferred to the PuriXmise plate to perform the immunocapture of infliximab and allows washing of the sample to reduce matrix interferences. Samples are eluted into a collection plate and evaporated to dryness. Samples are resuspended, and the protease (CutXmise) is added to the sample to digest infliximab overnight. The digestion is quenched, with the samples immediately ready for analysis. Using an ACQUITY™ UPLC™ I-Class PLUS FL System, samples were injected onto a Waters XSelect™ Premier HSS T3, 2.5µm, 2.1 x 50 mm Column using a water/acetonitrile/formic acid gradient elution profile and the infliximab signature peptides SINSATHYAESVK (SIN), ASQFVGSSIHWYQQR (ASQ), GLEWVAEIR (GLE), SAVYLQMTDLR (SAV) and DILLTQSPAILSVSPGER (DIL) were quantified with both a Waters Xevo™ TQ-XS Mass Spectrometer (5µL injection) and Xevo TQ-S micro Mass Spectrometer (15µL injection), with a run time of 4.5 minutes.

Results: The method was shown to be linear from 2 - 100 µg/mL. Analytical sensitivity at lowest calibrator of 2µg/mL provided S/N (PtP) ≥15:1 for all tryptic peptides on both mass spectrometers. Coefficients of variation (CV) at 4 and 25µg/mL QC concentrations were all ≤ 10.0% (n = 5). The relative concentrations of the tryptic peptides were compared across 29 anonymized samples and good agreement was observed for GLE, SAV, DIL and ASA peptides. However, the SIN peptide demonstrated significant negative method bias, which could be attributed to its susceptibility to deamidation.

Conclusions: We have demonstrated a method for quantifying Infliximab in plasma using a commercially available kit for sample preparation followed by LC-MS/MS analysis. The use of the Promise Proteomics mAbXmise Kit makes the analysis accessible and easy to implement, while also being amenable to automation. The extended measuring range, between 2 µg/mL and 100 µg/mL, avoids the requirement for dilution sometimes observed with immunoassays, improving turnaround times for higher concentration samples. In addition, reduction in dilution and re-analysis improves utilization of the kit, which aids cost management. The Kit aids in the day-to-day reproducibility of the immunocapture and tryptic digestion of IFX for targeted LC-MS/MS analysis with the correct peptide selection. Compared to a direct digest surrogate peptide workflow, the process used in the kit reduces matrix interference, improves analytical sensitivity, and enables the use of lower sample volumes.

It has been demonstrated that the method can be run on both the Xevo TQ-XS and Xevo TQ-S micro Mass Spectrometers which provide the dynamic range to quantify IFX across the expected range, and the selectivity and analytical sensitivity to obtain low-level quantification of the IFX surrogate peptide in plasma samples.

The data presented combines the use of a kit dedicated to the preparation of samples and the use of liquid chromatography and mass spectrometry instrumentation to perform the quantitative analyses. The mAbXmise Kit described has not been cleared by any regulatory entity for diagnostic purposes outside of Europe. Proteomics mAbXmise Kits are not available for sale in all countries.