Gabor\nTransform-Based Antibody Quantitation\nin Serum: An Interlaboratory Liquid Chromatography/High-Resolution\nMass Spectrometry Investigation

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