(160g) Engineering New Salt-Tolerant and Mixed-Mode Chromatography Resins for Orthogonal Separations

The design of downstream processes for non-mAb therapeutics such as enzymes, viral vectors, and non-Fc containing mAb fragments are often challenging due the lack of existing or robust affinity purifications steps. Purification strategies for these non-mAb processes often involves the pairing of orthogonal chromatography steps such as ion exchange (IEX) and hydrophobic interaction chromatography (HIC). The use of these single modality resins is limited by the need for low ionic strength conditions or high concentrations of kosmotropic additives, which limits downstream capacity and increases costs and complexity. The use of salt-tolerant and mixed-mode resins for these processes can address these challenges, provide unique selectivities between products and impurities, and enable direct capture of product from post-harvest material. Developing these steps, however, can be challenging since chromatographic behaviors in mixed-mode and salt-tolerant systems are often less intuitive than for their single-mode counterparts. Further, our previous work has shown that developing orthogonal mixed-mode or salt-tolerant chromatography steps is difficult and existing resins are not explicitly designed in the context of the other resins used in a process. Designing sets of mixed-mode and salt-tolerant resins engineered for orthogonal selectivities can greatly facilitate their incorporation into downstream processes and allow for pre-packaged resin set solutions for purifying a wide range of non-mAb products. In this work we designed an extensive library of mixed-mode and salt-tolerant resins encompassing a wide range of physiochemical properties to understand the impact of ligand chemistry on orthogonal selectivities. Protein-resin interactions and binding strengths were characterized on a set of non-mAb proteins using a high-throughput multi-well plate-based assay. A newly developed framework to quantify the degree of orthogonality in mixed-mode and salt-tolerant resin systems was implemented to directly leverage binding strength data to predict orthogonal resin sets from our resin library. With this approach, we identified new sets of resins that when operated together provide highly orthogonal separations for non-mAb proteins. Further, the characterization of this resin library allowed us to investigate the impact of ligand density and different chemistries of the charged and hydrophobic groups on creating orthogonal selectivities. We believe that this work represents a paradigm shift in designing next-generation chromatographic materials where resins are designed to operate in the context of one another to facilitate process development for non-mAb products.