Preparation and characterization of agarose-encapsulated ceramic hydroxyapatite particles for flow-through chromatography

Composite adsorbents were prepared by encapsulating ceramic hydroxyapatite particles (CHT Type I) within inert agarose beads using an emulsion method as stationary phases for the removal of small protein and nucleic acid impurities from large proteins and bioparticles in a flow-through mode. The composite particles were prepared with 6% and 10% agarose and contain 35% and 25% (v/v) CHT, respectively, with effective pore radii of 15.1 and 8.8 nm. The functional properties were studied using yeast RNA, bovine serum albumin (BSA), thyroglobulin (Tg), IgM, and 30 and 50 nm silica nanoparticles (NP) as models. The RNA and BSA adsorption capacities are smaller than for unencapsulated CHT on a bead volume basis but comparable when normalized by the volume of CHT particles in the composite beads attaining values between 30 and 40 mg/mL. Confocal laser scanning microscopy shows that, while RNA and BSA have complete access to the encapsulated CHT particles, Tg, IgM, and the NPs are largely excluded. RNA and BSA breakthrough experiments in laboratory-scale columns at a 5-min residence time show RNA and BSA binding capacities comparable to those measured in a batch mode with either single component feeds or with feed mixtures containing 30 nm NPs.