Exploiting the potential of preparative multimodal chromatography resins for increasing selectivity in an orthogonal...

Multimodal or mixed-mode chromatography (MMC) resins are promising for pharmaceutical recombinant proteins due to the potential for developing orthogonal purification steps with unique selectivity. However, the complex behavior of multimodal ligands makes them difficult to use. Generally, several factors, including ligand design (functional groups, spacer/ligand length and flexibility, ligand orientation), ligand density and distribution, and using optimal operational conditions (elution strategy and composition), define the final selectivity of the purification process. Adjusting multimodal interactions results in modified binding strength and adsorption. In some cases, optimizing the interaction potential of each modality in the structure of the multimodal ligand could achieve the desired property, while others show contrasting results. The ideal linker length between various moieties improves retention in some cases. A high adsorption rate and selectivity have been achieved respectively, with a uniform and heterogeneous distribution of ligand surface. However, the result of functional group variation on resin behavior varied based on protein characteristics. Moreover, control over operational conditions like pH, salt concentration, temperature, additives, strategies for protein purification, and displacers is essential for producing selective purification steps using these resins. Nevertheless, a deep understanding of the molecular interactions between ligands and the target biomolecules during binding and elution processes provides superior assistance for exploiting the selectivity of these resins by selecting an optimal purification strategy, ligand type, and ligand design. For this purpose, various experimental and modeling tools (mechanistic or empirical) have been used for the successful study of the chromatographic process. Although all of these methods have their potential and limitations, using them provides a beneficial understanding of the purification process. An inclusive overview of the mechanisms and methods for exploiting the selectivity and orthogonality of MMC resins is a gap that is being addressed in this review for further successful purification studies.