Resin structure impacts two-component protein adsorption and separation in anion exchange chromatography

The influence of the resin structure, on the competitive binding and separation of a two-component pro-tein mixture with anion exchange resins is evaluated using conalbumin and green fluorescent protein as a model system. Two macroporous resins, one with large open pores and one with smaller pores, are compared to a resin with grafted polymers. Investigations include measurements of single and two-component isotherms, batch uptake kinetics and two-component column breakthrough. On both macro-porous resins, the weaker binding protein, conalbumin, is displaced by the stronger binding green fluo-rescent protein. For the large pore resin, this results in a pronounced overshoot and efficient separation by frontal chromatography. The polymer-grafted resin exhibits superior capacity and kinetics for one-component adsorption, but is unable to achieve separation due to strongly hindered counter-diffusion. Intermediate separation efficiency is obtained with the smaller pore resin. Confocal laser scanning mi-croscopy provides a mechanistic explanation of the underlying intra-particle diffusional phenomena re-vealing whether unhindered counter-diffusion of the displaced protein can occur or not. This study demonstrates that the resin's intra-particle structure and its effects on diffusional transport are crucial for an efficient separation process. The novelty of this work lies in its comprehensive nature which in-cludes examples of the three most commonly used resin structures: a small pore agarose matrix, a large -pore polymeric matrix, and a polymer grafted resin. Comparison of the protein adsorption properties of these materials provides valuable clues about advantages and disadvantages of each for anion exchange chromatography applications.& COPY; 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )