M-Protein semi-quantification in MM serum patients based on Immuno-Capture and Liquid Chromatography coupled to High Resolution Mass Spectrometry

In order to determine the disease status, and response to treatment in Multiple Myeloma (MM) patients, the amount of M-Protein (most often IgG or IgA with kappa or lambda light chains) in the serum is measured. According to International Myeloma Working Group (IMWG) criteria, detection and quantification of M-Protein, by Serum Protein Electrophoresis (SPEP) and Immuno-Fixation Electrophoresis (IFE) are essential for patient response evaluation in MM. Isatuximab, an IgG-kappa, anti-CD38 monoclonal antibody (currently under clinical development) has recently been shown to prolong progression-free survival and improve tumor response in combination with pomalidomide-dexamethasone in Relapsed and Refractory Multiple Myeloma (RRMM). However, isatuximab may be detected on conventional SPEP and IFE assays that are used to monitor patients with IgG kappa type M-Protein. This interference could lead to false-positive assay results and, consequently, an inaccurate determination of patient's response to the treatment according to IMWG criteria. In order to overcome the potential interference of isatuximab in clinical samples, we developed and validated a hybrid assay based on Immuno-Capture and Liquid Chromatography coupled to High Resolution Mass Spectrometry (IC-LC-HRMS). The first step of this approach is to perform an Immuno-Capture (anti-LC kappa/lambda beads) of Igs and free Light Chains (LC) in serum of MM patients. Heavy Chains (HC) and LC are then dissociated (reduction with dithiothreitol) and sorted using Liquid Chromatography. M-Protein and isatuximab LC are analyzed using HRMS and are identified according to their monoisotopic intact mass. The association of Liquid Chromatography and HRMS enables to characterize and discriminate signals of isatuximab and M Protein. As M-Proteins are specific for each MM patient, no standards are available to allow their quantification using a bioanalytical approach such as LC-HRMS. In this work, we used alemtuzumab (IgG kappa) as a standard to semi-quantify M-Protein in MM serum patients. This analytical assay was successfully validated from 10 to 200 μg/mL for M-Protein in serum, with precision and accuracy within 20% (25% at LLOQ level). This complex assay was developed in compliance with Good Clinical Laboratory Practices (GCLP) recommendations and Data Integrity policy. This new IC-LC-HRMS method was successfully applied to clinical samples evaluation and permits to abrogate potential interference of isatuximab in monitoring MM patients using SPEP and IFE. Representative clinical cases in which the MS approach assisted in response assessment will be described. In conclusion, this novel assay helps in accurate assessment of tumor response to isatuximab treatment. Moreover, this methodology may be adapted to new therapeutic antibodies to characterize a potential interference and distinguish endogenous M-protein from these antibodies.