Characterization of lysozyme adsorption in cellulosic chromatographic particles using small-angle neutron scattering

Koshari, Stijn
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University of Delaware
Polymer-derivatized chromatographic media (PDM) for protein separation have been shown to display clear benefits in key measures of performance compared to conventional media. However, mechanistic understanding of protein mobility in these media is limited, and more experimental research is necessary to provide the insight necessary to develop and validate models to design these media and the processes in which they are used. Small-angle neutron scattering (SANS) is an entirely novel way to study and characterize these systems. As a first, preliminary feasibility study, the thesis discusses the difference between the SANS spectra from chromatographic media with and without adsorbed protein. In particular, we look at the adsorption of lysozyme on cellulosic S HyperCel (Pall Corporation) particles. Contrast matching techniques are not viable for studying these systems, as the scattering length densities of the materials are too similar. Instead, we offer a framework that allows quantitative analysis of the protein adsorption by direct comparison of the scattering spectra before and after adsorption. To support this framework, reduction techniques like background removal and scaling are provided to allow quantitative comparison of the data, in addition to a theoretically derived model for the scattering from cellulosic gel-like particles. The scattering spectrum from the particles with adsorbed protein has three contributions: (1) the pure particle without adsorbed protein, as captured by the theoretically derived model; (2) the form factor of protein monomers, which can be seen at high values of the momentum transfer vector Q; and (3) the change in the fractal-like structure of the media evident at low Q upon protein adsorption. The intermediate- Q region of the scattering spectrum is not influenced by the adsorption of protein. These contributions are investigated for different protein loadings of the resin particles and successfully linked to the sample composition. The sample incoherent background can be predicted from the sample composition, and the total concentration of protein in the sample can be accurately acquired from the SANS spectrum. The findings support the idea that protein adsorption is uniform and leads to a virtual densification of the cellulosic gel-like particle structure. To conclude, it is clear that SANS is capable of probing these structures, and that it can provide additional information on protein adsorption when analyzed within the context of the model framework developed in the thesis.