have developed a silica-based MM resin capable of weak anion-exchange and reversed-phase interactions for the simultaneous separation of acidic, basic, and neutral pharmaceutical compounds ( 9). have developed a library of MM ligands that can be employed for the capture of charged proteins under high salt conditions ( 4, 5). Recent advances in the design of multimodal (MM) chromatographic systems have produced previously undescribed classes of chromatographic materials that can provide alternative and improved selectivities as compared to traditional single mode chromatographic materials ( 3– 9). The use of NMR titration experiments in concert with chromatographic data obtained with protein libraries represents a previously undescribed approach for elucidating the structural basis of protein binding affinity in MM chromatographic systems. Finally, coarse-grained ligand docking simulations were employed to study the modes of interaction between the MM ligand and ubiquitin. The results with NMR confirmed that the protein possessed a distinct preferred binding region for the MM ligand in agreement with the chromatographic results. Chemical shift mapping and determination of dissociation constants from NMR titration experiments with the MM ligand and isotopically enriched ubiquitin were used to determine and rank the relative binding affinities of interaction sites on the protein surface. Further, the chromatographic results with the protein library indicated that mutations in a defined region induced greater changes in protein affinity to the solid support. The elution order of the mutants on the MM resin was significantly different from that obtained by ion-exchange chromatography. All rights reserved.NMR titration experiments with labeled human ubiquitin were employed in concert with chromatographic data obtained with a library of ubiquitin mutants to study the nature of protein adsorption in multimodal (MM) chromatography. Downstream processing of the Capto MMC pools showed that it is feasible to produce material with comparable purity to a process with affinity capture after two chromatography steps.Ĭapto MMC Mammalian cell culture fluid Monoclonal antibody Multimodal chromatography Selective capture.Ĭopyright © 2014 Elsevier B.V. Four separate elution strategies were identified to selectively recover the mAb and could be applied with minimal optimization to other mAb feedstocks. Analysis of the DBC versus molecular properties showed that the mAb-ligand binding interaction was predominantly charge based. A range of dynamic binding capacities was observed for 10 feedstocks (24-53g/L) and two feedstocks had poor binding properties (<10g/L) despite load conditioning towards a more favorable pH. Twelve mAb HCCF feedstocks were examined for dynamic binding capacity (DBC) and then two representative feedstocks were selected to develop a systematic approach for elution buffer development. Capto MMC is a multimodal resin that contains a ligand with the potential to participate in ionic, hydrophobic, and hydrogen boding interactions with proteins and is coupled to a highly cross-linked agarose bead matrix. This proof-of-concept study examines the applicability of using multimodal chromatography to selectively capture recombinantly produced monoclonal antibodies (mAb) directly from harvested mammalian cell culture fluid (HCCF) with minimal optimization.
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