Polymer-Grafted and Agarose-Encapsulated Adsorbents for Protein and Bioparticle Purification
Fuks, Preston, Chemical Engineering - School of Engineering and Applied Science, University of Virginia
Carta, Giorgio, EN-Chem Engr Dept, University of Virginia
Understanding the interplay between the stationary phase architecture and the mechanism of protein adsorption and transport is vital for the optimal design of chromatographic processes. This work studies the properties of stationary phases obtained by modifying a base matrix by grafting dextran polymers or by the incorporation in agarose gels.
Anion exchangers with low graft content (Nuvia HR Q and Nuvia HP Q) and high graft content (Nuvia Q) are considered. The accessible pore volume decreases with increasing polymer-graft content. Conversely, the binding capacity for bovine serum albumin (BSA) and thyroglobulin (Tg) increases with polymer-graft content. Dynamic binding capacities significantly increase at moderate NaCl concentrations for all polymer-grafted adsorbents. Breakthrough of mixtures of BSA and Tg demonstrates a higher selectivity for resins with intermediate graft density as well as a reversal in the order of breakthrough with the addition of NaCl. Pore diffusion best describes the adsorption kinetics of BSA and Tg on ungrafted resins while single file diffusion best describes two-component adsorption for highly grafted resins indicating that counter diffusion of displaced species hinders transport.
Novel composite-particles, obtained by encapsulating ceramic hydroxyapatite type-I particles (CHT), in inert agarose-beads were prepared via an emulsion method. The composite beads combine two chromatographic principles: separation by molecular size and separation by selective adsorption occurring within the encapsulated CHT particles. Adsorption capacities of BSA and RNA are lower on a bead volume basis relative to unencapsulated CHT; however, on a CHT volume basis, adsorption is similar to unencapsulated CHT suggesting that the agarose does not inhibit adsorption. Confocal laser scanning microscopy images show that large proteins such as Tg and IgM, and 30 nm and 50 nm nanoparticles are largely excluded from encapsulated CHT particles. Two-component breakthrough of mixtures containing 30 nm nanoparticles and either BSA or RNA demonstrate that the applicability of these novel materials to the flow-through purification of large proteins and bioparticles.
PHD (Doctor of Philosophy)
Protein Chromatography, Polymer-grafted anion exchange, Agarose-encapsulated hydroxyapatite, Chromatography modelling
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