Adsorption of Poly(ethylene glycol)-Modified Lysozyme to Silica

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Abstract
Covalent grafting of poly(ethylene glycol) (PEG) to pharmaceutical proteins, “PEGylation”, is becoming more commonplace due to improved therapeutic efficacy. As these conjugates encounter interfaces in manufacture, purification, and end use and adsorption to these interfaces may alter achievable production yields and in vivo efficacies, it is important to understand how PEGylation affects protein adsorption mechanisms. To this end, we have studied the adsorption of unmodified and PEGylated chicken egg lysozyme to silica, using optical reflectometry, total internal reflection fluorescence (TIRF) spectroscopy, and atomic force microscopy (AFM) under varying conditions of ionic strength and extent of PEG modification. PEGylation of lysozyme changes the shape of the adsorption isotherm and alters the preferred orientation of lysozyme on the surface. There is an abrupt transition in the isotherm from low to high surface excess concentrations that correlates with a change in orientation of mono-PEGylated conjugates lying with the long axis parallel to the silica surface to an orientation with the long axis oriented perpendicular to the surface. No sharp transition is observed in the adsorption isotherm for di-PEGylated lysozyme within the range of concentrations examined. The net effect of PEGylation is to decrease the number of protein molecules per unit area relative to the adsorption of unmodified lysozyme, even under conditions where the surface is densely packed with conjugates. This is due to the area sterically excluded by the PEG grafts. The other major effect of PEGylation is to make conjugate adsorption significantly less irreversible than unmodified lysozyme adsorption.