Gábor Transform-Based Antibody Quantitation in Serum: An Interlaboratory Liquid Chromatography/High-Resolution Mass Spectrometry Investigation

Therapeutic monoclonal antibodies (t-mAbs) are crucial for treating various conditions, including cancers and autoimmune disorders. Accurate quantitation and pharmacokinetic monitoring of t-mAbs in serum are essential, but current methods like ligand binding assays (LBAs) and bottom-up peptide liquid chromatography-tandem mass spectrometry (LC-MS/MS) can lack the sensitivity and specificity needed to meet clinical demands. Emerging techniques using high-resolution mass spectrometry (HRMS) in top-down and middle-up approaches offer improved ability to accurately quantify mAb proteoforms apart from degradation products by keeping the sample proteins intact or minimizing digestion. This study describes the first use of Gábor transform (GT)-based iFAMS Quant+ software to quantify a t-mAb (vedolizumab) from ∼400 samples using an Agilent 6545XT AdvanceBio Q-TOF at the University of Oregon. These results are compared to a previously validated laboratory-developed test (LDT) from Mayo Clinic utilizing a Thermo Q Exactive Plus Orbitrap. The Mayo method used conventional extracted ion chromatograms (XICs) of select charge states for quantitation, while the iFAMS Quant+ method utilized GT-based charge state deconvolution, background subtraction, and signal integration. Calibration and quality control (QC) analyses and Passing-Bablok regression of 351 subject samples demonstrated excellent agreement between the two methods. The iFAMS Quant+ workflow exhibited unique advantages for characterizing interferents and analyte signal anomalies due to its deconvolution-based approach.