B-155 Analysis of Monoclonal Antibodies Using a Quantification LC-MS/MS Kit - Spotlight on Infliximab
Authors: Calton L , Lebert D , Foley D 
Published: October 2024

Abstract

Background
The use of immunoassays 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. Using LC-MS/MS associated with the ready-to-use commercial mAbXmise Kit is a simple way to implement mAbs measurement for infliximab, providing high analytical performance, ease of use, and flexibility.

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. Briefly, 20µL samples 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 allow washing of the samples. Samples are eluted into a collection plate, evaporated to dryness, 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 Column, eluted using a water/acetonitrile/formic acid gradient profile and quantified with both a Waters Xevo™ TQ-XS and Xevo TQ-S micro Mass Spectrometer.

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.

Conclusions
We have demonstrated a clinical research method for quantifying Infliximab in plasma using a commercially available kit for sample preparation followed by LC-MS/MS analysis. The Promise Proteomics mAbXmise 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. 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. Analytical performance depends on the instrument characteristics and its settings. The end user is responsible for completion of the method development and validation. Proteomics mAbXmise Kits are not available for sale in all countries. For Research Use Only. Not for use in diagnostic procedures.