Qualification and robustness study of a bio-layer interferometry-based C1q binding assay for therapeutic antibodies

Bio-layer interferometry (BLI) assays have been widely used to study the interaction between therapeutic antibodies and complement component 1q protein (C1q), assessing the efficiency of antibody-induced complement-dependent cytotoxicity (CDC). In this study, a strategy was developed to comprehensively qualify BLI-based C1q binding assay using commercial therapeutic antibody Trastuzumab as an example, in accordance with the ICH Q2(R2) Guideline. The assay's specificity, linearity, precision (repeatability and intermediate precision), accuracy, and robustness were rigorously evaluated. Specificity tests showed no C1q binding for Trastuzumab's formulation buffer and IgG4 samples. The assay showed good accuracy, with relative bias ranged from -0.4-13.2 % in relative potency (%) levels ranging from 50 % to 150 %. A linear regression analysis conducted between the measured levels and theoretical levels resulted in an R² value of 0.9925. Experiments conducted with six replicates by each analyst resulted in relative standard deviations (RSD) of 3.1 % and 2.1 %, respectively. The combined analysis of twelve replicates by two analysts yielded an RSD of 2.9 %, demonstrating exceptional precision. The assay robustness was assessed using a design-of-experiment (DOE) approach that considered three major factors: protein L biosensor lot, C1q protein lot, and analyst. The assessment found no significant impact on assay results from these factors or their interactions. Overall, the method demonstrated excellent specificity, accuracy, linearity, and precision, with good robustness for detecting the binding of antibodies against C1q. This report presents the first published strategy by implementing the DOE approach to qualify BLI-based C1q binding assays, adhering to the ICH Q2(R2) and ICH Q14 guidelines to comply with regulatory standards. This framework may serve as a reference for optimizing and quantifying BLI/SPR-based binding assays for other targets.