Abstract:
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.
