Mechanisms underlying protein sorption and transport within polysaccharide-based stationary phases for ion-exchange chromatography

Author(s)Angelo, James Michael
Date Accessioned2016-11-21T13:41:14Z
Date Available2016-11-21T13:41:14Z
Publication Date2016
AbstractIon-exchange chromatography is a powerful tool that is widely utilized in the downstream purification of biomolecules. In the biotechnology industry, resins that exhibit enhanced chromatographic performance are widely desired to streamline purification processes and meet production requirements. Polysaccharide-based ion-exchange adsorbents meet many performance standards by possessing high binding capacities and enhanced uptake rates of protein, while being highly compatible with biologics and remaining relatively cost-efficient. The characterization of these materials is crucial for informed decisions during stationary-phase selection and process design. The structural characteristics of agarose-based, dextran-modified agarose-based and cellulosic ion-exchange materials were determined using methods to gauge the pore dimensions and the effect of ionic strength on intraparticle architecture. Inverse size exclusion chromatography (ISEC) analysis and electron microscopy techniques were used to quantitatively assess and visualize the pore structure, respectively. Adsorption behavior in these materials was characterized using methods to evaluate the dynamics of protein uptake as a function of ionic strength and protein concentration using several model proteins. Fast uptake rates were observed in both batch kinetics experiments and time-series confocal laser scanning microscopy (CLSM) for the cellulosic media, suggesting low intraparticle transport resistances relative to external film resistance, even at higher bulk protein concentrations where the opposite is typically observed. While polysaccharide-based materials possess easily accessible microstructures, elution has sometimes been observed to be undesirably slow. In order to determine which physicochemical phenomena control elution behavior, the materials were characterized by their uptake and elution profiles at various conditions. In general, effective elution rates decreased with the reduction of accessible pore volume. The use of protein stabilizing agents was explored in systems presenting atypical elution behavior. Incorporation of excipients into eluent buffer gave rise to faster elution and significantly lower pool volumes in elution from polymer-modified agarose adsorbents. Finally, thermodynamic modeling based on molecular theory was used in conjunction with single-molecule total internal reflection fluorescence microscopy (TIRFM) to further develop the understanding of fundamental mechanisms of protein adsorption within polymer-modified surface layers. These molecular methods revealed trends and characteristics that were in agreement with protein transport in stationary phase analogues to these surfaces.en_US
AdvisorLenhoff, Abraham M.
DegreePh.D.
DepartmentUniversity of Delaware, Department of Chemical and Biomolecular Engineering
Unique Identifier963836910
URLhttp://udspace.udel.edu/handle/19716/19854
PublisherUniversity of Delawareen_US
URIhttp://search.proquest.com/docview/1818564327?accountid=10457
dc.subject.lcshProteins -- Absorption and adsorption.
dc.subject.lcshProteins -- Physiological transport.
dc.subject.lcshPolysaccharides.
dc.subject.lcshStationary phase (Chromatography)
dc.subject.lcshIon exchange chromatography.
TitleMechanisms underlying protein sorption and transport within polysaccharide-based stationary phases for ion-exchange chromatographyen_US
TypeThesisen_US
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