A Facile Method for Gel Electrophoresis with Intrinsic Fluorescence Imaging for Self-Aggregation and Stability Assay of Monoclonal Antibody

Abstract

Monoclonal antibodies (mAbs) are widely used in tumor and autoimmune disease therapy and clinical diagnosis, but they suffer from self-aggregation, particularly dimer formation, impacting their stability, efficacy, and potentially causing severe allergies. Traditional methods for detecting mAb dimers, such as TEM, AUC, DLS, SEC, and CE, are limited by low throughput, high costs, and qualitative data, making them unsuitable for large-scale sample assays in biopharmaceuticals industry as well as antibody research. To address these issues, we developed a facile method of protein cross-linking gel electrophoresis with online intrinsic fluorescence imaging (PC-GE-IFI) for self-aggregation and stability assays of mAbs. This method enables the real-time quantification of monoclonal antibody (mAb) monomers and dimers with exceptional sensitivity, characterized by low detection limits (monomer: 0.9 nM; dimer: 0.45 nM) and a broad dynamic range (monomer: 2.50–2500 nM; dimer: 1.25–1250 nM). Furthermore, the sample wells can be used as windows for the assay of precipitates formed by mAb aggregation. The method supports accurate assessment of mAb dimerization and monomer purity under various stress conditions, including thermal stress, mechanical agitation, and freeze–thaw cycles. Moreover, the method allows concurrent analysis of dimers and precipitates across multiple samples at different concentrations, while nonlinear fitting provides the dissociation constant (K_d) for monomer–dimer interactions, a critical parameter that aids in assessing aggregation propensity, and informs the design and development of mAb products The PC-GE-IFI method has great potential for development, quality control, and safety assessment of mAbs, bispecific antibodies, ADCs, and protein drugs.