Characterization and exploration of an artifact in the reducing capillary electrophoresis-sodium dodecyl sulfate analysis of the ‘me-too’ drug zuberitamab related to rituximab

For monoclonal antibody (mAb) drugs, the 'me-too' drug is a pharmacologically active compound that is structurally similar to the first-in-class drugs, acting on the same target and is used for the same therapeutic purposes, but it may differ in drug-drug interactions and adverse drug reactions. Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) has been widely used for quality evaluation of mAb drugs. The properties of the detected substances can interfere with the credibility and accuracy of the method. In the routine comparison analysis for both innovator rituximab and 'me-too' drug zuberitamab samples, an uncommon artifact related to the heavy chain (HC) of zuberitamab was observed in reducing CE-SDS and interfered with our identification of the purity of samples. In this work, the overall hydrophobicity of the HCs of rituximab, zuberitamab, and several other common mAbs was characterized and determined by reversed-phase high-performance liquid chromatography. Additionally, the local hydrophobicity and surface charge were compared using Expasy ProtScale and PyMOL software simulations. We concluded that noncovalent protein aggregation can be related to strong hydrophobicity and low electrostatic repulsion of local amino acid regions, which complicates drug quality control. These findings shed light on the relationship between protein aggregation and the local hydrophobicity region, and broaden the way to analyze the detection 'artifacts' in reducing CE-SDS studies of therapeutic proteins.