A framework for calculating orthogonal selectivities in multimodal systems directly from cell culture fluid

This paper presents a straightforward approach for measuring and quantifying orthogonality directly in complex cell culture fluids (CCFs) without the requirement for tracking the retention behaviors of large sets of proteins. Null‐producing CCFs were fractionated using linear salt gradients at constant pH on a set of multimodal resins. Fractions were then analyzed by ultraperformance‐reversed phase liquid chromatography and the resulting chromatograms provided host cell protein (HCP) “fingerprints.” Using these fingerprints, an inner product vector‐based approach was employed to quantify the degree of orthogonality between pairs of resins and operating conditions for these large HCP protein sets. To compare resin orthogonality behavior in different expression systems, the Chinese hamster ovary and Pichia pastoris null‐producing CCFs were examined. Orthogonality in multimodal systems was found to strongly depend on the expression system and the HCPs being screened. We also identified several unexpected pairs of multimodal resins within the same family that exhibited significant orthogonality. Furthermore, “self‐orthogonality” was evaluated between resins operated at different pHs, and important operating regimes were identified for maximizing orthogonal selectivities. The framework developed in this paper for calculating orthogonality without the need for labor‐intensive HCP tracking has important implications for efficient process development and resin/operating condition selection for both monoclonal antibody (mAb) polishing steps and non‐mAb processes. In addition, this study provides a tool to unlock the untapped potential of multimodal resins by aiding in their rational selection and incorporation. Finally, the orthogonality framework here can facilitate the development of sets of next‐generation multimodal resins specifically designed to provide highly orthogonal and efficient separations tailored for different expression systems. The authors present a straightforward approach for measuring and quantifying orthogonality directly in complex cell culture fluids without the requirement for tracking the retention behaviors of large numbers of proteins.