A multifactorial screening strategy to identify anti-idiotypic reagents for bioanalytical support of antibody therapeutics.

Abstract Antibodiesare critical tools for protein bioanalysis; their quality and performance dictate the caliberand robustness of ligand binding assays. After immunization, polyclonal B cells generate a diverse antibodyrepertoire against constant and variable regions of the therapeutic antibodyimmunogen. Herein we describe a comprehensive and multifactorial screening strategy to eliminate undesirable constant region-specific antibodiesand select for anti- idiotypic antibodieswith specificity for the unique variable region. Application of this strategy is described for the therapeutic antibodyMab-A case study. Five different factors were evaluated to select a final antibodypair for the quantification of therapeutics in biological matrices: (i) matrix effect in preclinical and clinical matrices, (ii) assay sensitivitywith lower limit of quantification goal of single-digit ng/ml (low pM) at a signal-to-background ratio greater than 5, (iii) epitope distinction or nonbridging antibodypair, (iv) competition with target and inhibitory capacity enabling measurement of free drug, and (v) neutralizing bioactivity using bioassay. The selected antibodypair demonstrated superior assay sensitivitywith no or minimal matrix effect in common biological samples, recognized two distinct binding epitopes on the therapeutic antibodyvariable region, and featured inhibitory and neutralizing effects with respect to quantification of free drug levels.