Increasing attention has been paid to the purity of therapeutic proteins imposing extensive costs and challenges to the downstream processing of biopharmaceuticals. One of the efforts, that has been exerted to overcome such limitations, was developing multimodal or mixed-mode chromatography (MMC) resins for launching selective, orthogonal, non-affinity purification platforms. Despite relatively extensive usage of MMC resins, their real potential and fulfillment have not been extensively reviewed yet. In this work, the explanation of practical and key aspects of downstream processing of recombinant proteins with or without MMC resins was debated, as being useful for further purification process development. This review has been written as a step-by-step guide to deconvolute both inherent protein purification and MMC complexities. Here, after complete elucidation of the potential of MMC resins, the effects of frequently used additives (mobile phase modifiers) and their possible interactions during the purification process, the critical characteristics of common product-related impurities (e.g., aggregates, charge variants, fragments), host-related impurities (e.g., host cell protein and DNA) and process related impurities (e.g., endotoxin, and viruses) with solved or unsolved challenges of traditional and MMC resins have been discussed. Such collective experiences which are reported in this study could be considered as an applied guide for developing successful downstream processing in challenging conditions by providing a clear insight into complex MMC resins and impurities.
