Interaction of recombinant factor VIII and the nonionic surfactant Tween 80 at interfaces Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/nc580q95x

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  • The role of the nonionic surfactant Tween 80 on the behavior of the therapeutic recombinant protein Factor VIII (rFVIII) was investigated at solid/liquid and air/water interfaces. In order to provide a model system to compare results obtained for the complicated rFVIII-Tween system, a well-characterized globular protein lysozyme was used. The experimental scheme involved the introduction of the protein and Tween to the adsorption substrate in different manners, either lysozyme Tween together or in sequence as lysozyme followed by Tween or vice versa. It was observed that the addition of Tween together with lysozyme reduced the amounts adsorbed at hydrophobic surfaces, while no such reduction was observed on hydrophilic surfaces. A high Tween concentration was required to effect the removal of the lysozyme molecules from the hydrophobic surface and Tween was not effective in removing lysozyme from the hydrophilic surface at any concentration. These results suggest that the Tween-surface interaction is important in determining lysozyme adsorption. Similar observations were made for the rFVIII-Tween system at hydrophobic and hydrophilic silica interfaces. In this case, the presence of interfacial and solution Tween together resulted in complete prevention of rFVIII adsorption. Electrostatic forces were observed to be play an important role in rFVIII adsorption. The rFVIII-Tween interactions at solid interfaces were also evaluated using intrinsic fluorescence and biological activity measurements. Results obtained with respect rFVIII adsorbed mass, and structure or biological activity change upon adsorption, were evaluated in parallel. This parallel evaluation suggested that rFVIII adsorption on hydrophilic, negatively charged surfaces is likely to be highly ordered and oriented in a manner that retains the solvent accessibility of the active sites in rFVIII. On the other hand, rFVIII may adsorb to hydrophobic surfaces in different orientations, with a likelihood of surface induced unfolding. rFVIII-Tween interaction at the air/water interface was investigated separately. Surface tension data recorded for rFVIII-Tween mixtures suggested that Tween dominated the air/water interface as the Tween concentration was increased. Reduced interface-induced unfolding was observed at high Tween concentrations. These results were also thought to contribute to the reduction in rFVIII aggregation typically observed as a result of exposure to the air/water interface.
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