Here we report the results of such a structure–function study in which the backbone and ligand chemistry remain fixed but the ligand density and its distribution are varied. Additionally, ligand chemistry must be chosen and optimized. agarose, including selection of particle size, solid content, and rigidity. End users generally see only the final commercial products of resin development and are rarely privy to the results of any exploration of structural properties on separation performance, such as efforts to optimize characteristics of resin backbone materials, e.g. Ion-exchange chromatography has played a major role in the purification of proteins for decades, and a wide variety of stationary phases have been developed for preparative separations. Using these trends, this work could be useful in guiding resin selection or design. Column breakthrough data for lysozyme and lactoferrin appear to support the hypothesis, though it appears that whether a resin charge density is low or high must be considered in relation to the protein charge density. Collectively examining the lysozyme and lactoferrin data along with information from previous studies suggests that a trade-off in maximizing dynamic capacities should exist between static capacities that increase to a finite extent with increased resin charge density and uptake rates that decrease with increased charge density. For the larger protein lactoferrin, it was found that increasing dextran content led to increased protein exclusion from the dextran layer, but that increasing resin charge density helped overcome the exclusion, presumably due to the increased electrostatic attraction between the resin and protein. Isocratic retention data and confocal microscopy imaging for this protein revealed a consistent ordering of the resins linking stronger protein–resin interactions with higher static capacities but slower intraparticle uptake rates over the range of properties studied. For the small protein lysozyme, resin charge density had the greatest effect on equilibrium capacity, consistent with calculations suggesting that lysozyme capacity should be limited by the available charge on the resin. Custom-synthesized variants of the commercial Capto S resin were used to examine the effects of resin charge density and dextran content on protein adsorption and intraparticle uptake.
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