We have recently reported that poly(ethylenimine) (PEI)-modified Sepharose resins show unique behavior as protein anion exchangers (J. Chromatogr. A, 2013, 1305, 76-84 & 85-93). Herein, we have extended the use of PEI resins by further modifying the PEI brushes with benzoic acid, leading to the creation of a new mixed-mode ligand, benzoyl poly(ethylenimine) (B-PEI). Various B-PEI-Sepharose resins were fabricated by modifying PEI-Sepharose of different ionic capacities (ICs, 330, 520 & 700mmol/L) at different benzyol group densities. We investigated the chromatographic behavior of bovine serum albumin and human ?-immunoglobulin G on the mixed-mode chromatography (MMC) as well as protein adsorption equilibria and kinetics as a function of ionic strength (IS). It was found that PEI-Sepharose at an ionic capacity of 330 mmol/L modified with a benzoyl group density of 160 mmol/L (the MMC medium denoted as B160-PEI330) exhibited favorable protein adsorption and pH-dependent elution performance. By increasing IS, the adsorption capacity of B160-PEI330 decreased significantly till reaching a plateau (60 mg/mL) at IS=0.3 mol/L. However, the effective pore diffusivity increased rapidly at IS<0.1 mol/L and then decreased slowly till reaching a plateau at 0.5 mol/L. As a result, the MMC medium presented much higher interior protein transport rate as compared to its counterpart, PEI-based anion exchanger, in addition to its substantial salt-tolerant performance. The research demonstrated that the fabrication strategy was promising for creating useful MMC media.

Protein chromatography on a new mixed-mode adsorbent fabricated from benzoyl-modified poly(ethylenimine)-grafted Sepharose

247th National Meeting (2014)

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