Liquid phase separation techniques for the characterization of monoclonal antibodies and bioconjugates

The characterization of biopharmaceuticals is a challenging task due to the structural complexity of such products. During both manufacturing and storage, biopharmaceuticals are exposed to a high risk of undergoing several desired and undesired chemical and enzymatic modifications. Final products are therefore heterogeneous mixtures of quite diverse chemical entities, rather than titrated blends of molecules. For this reason, the development of advanced separation techniques is paramount to allow the characterization of the different constituents of biopharmaceuticals. Several separation methods have been reported so far, each one based on specific physicochemical mechanism and uniquely suited for the characterization of distinctive product features, called Critical Quality Attributes (CQAs). If such diversity offers on one hand a wide array of possibilities, on the other hand it raises some issues concerning regulation, validation and harmonization of the results obtained in different laboratories or by different methods. Moreover, a comprehensive characterization of biopharmaceuticals can nowadays be achieved only by multiple analyzes with orthogonal methods or by the combination of orthogonal techniques in one method. Herein, we reviewed the main variants of Liquid Chromatography (LC), Capillary Electrophoresis (CE), and other less popular liquid phase separation techniques that are used for the characterization of monoclonal Antibodies (mAbs) and Antibody Drug Conjugates (ADCs).