Therapeutic\nMonoclonal AntibodyAntidrug Antibody\nAffinity Constant Determination Using Capillary Electrophoresis–Tandem\nMass Spectrometry

Monoclonal antibody (mAbs) therapeutics cannot evade\nthe occurrence\nof adverse effects. Thus, mAbs are commonly triggering immune responses\ncorresponding to the expression of antidrug antibodies. Antidrug antibodies\ncan neutralize mAbs, leading to their inhibition and hasten clearance,\nwhich dramatically hampers their therapeutic effects. Therefore, studies\nconcerning the affinity between a mAbs and the corresponding antidrug\nantibody are demonstrating a growing interest to further understand\nthe outcome of biotherapeutics after administration. We describe a\nnovel analytical approach for the determination of the dissociation\nconstant (<i>K</i><sub>d</sub>) between a mAb and an antidrug\nantibody using capillary electrophoresis coupled to mass spectrometry\n(CE-MS/MS). The CE-MS/MS method employs a competitive assay, followed\nby the quantification of the residual amount of the active mAbs. The\nmethodology allowed for the measurement of <i>K</i><sub>d</sub> using serum samples without the implementation of immobilization\nto achieve protein–protein interaction. This characteristic\nenabled us to generate the interaction in conditions reflecting the\nphysiological environment. A mathematical treatment was developed\nto calculate <i>K</i><sub>d</sub> from MS/MS data, taking\ninto account the stoichiometry of the mAbs/antidrug antibody complex\nand the bivalent properties of the two immunoglobulins. Prior to CE-MS/MS\nanalysis, the interaction of the two proteins was studied by using\nmass photometry (MP) to determine equilibrium conditions and complexation\nstoichiometry. CE-MS/MS was used to investigate the interaction between\ninfliximab and a neutralizing anti-infliximab antibody. Results allowed\nto measure a <i>K</i><sub>d</sub> of 14.4 ± 2.9 nM.\nMS instrumentation sensitivity and specificity showed to be relevant\nto achieve accurate and robust <i>K</i><sub>d</sub> measurements\nfor strong interactions in the nanomolar range.