Protein concentration and analyzing charge variants in a co-formulation comprising three monoclonal antibodies: A cation-exchange chromatography approach.

In the realm of therapeutic antibodies, co-formulations comprising two or more monoclonal antibodies (mAbs) have emerged as a promising strategy, offering enhanced treatment efficacy, improved efficiency, and prolonged intellectual property protection. These advantages have sparked significant interest among both patients and pharmaceutical companies. However, the quantification and analysis of individual mAbs within such co-formulations pose a substantial challenge due to their similar physicochemical properties. To address this challenge, we introduce a pH gradient cation exchange chromatography (CEX) method designed to effectively separate three mAbs that share significant similarities in molecular weight, structure, and isoelectric points (pIs) etc. This versatile approach not only facilitates the accurate quantification of each mAb's concentration and their respective ratios within the co-formulation, but also allows for the comprehensive characterization of all charge variants present. In the case of a co-formulation containing three antibodies, the developed CEX method demonstrated superior performance compared to other techniques. The method's robustness was further underscored by its qualification parameters, including acceptable precision (RSD ≤ 3 %), accuracy (95 %-115 % recovery), and linearity (R > 0.99) across a range of 10 to 30 μg load for each mAb. Moreover, the method has been successfully applied in stability studies to quantitatively analyze individual mAb concentrations within co-formulations, marking a significant advancement in the field. Through this work, we contribute a crucial analytical insight into mAb co-formulations, especially those comprising three or more molecules, underscoring its considerable potential to propel the field of biotherapeutic co-formulations forward.