Adsorption of Poly(ethylene glycol)-Modified Lysozyme to Silica Susan M. Daly, Todd M. Przybycien, and Robert D. Tilton* Departments of Chemical Engineering and Biomedical Engineering, Center for Complex Fluids Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 Received July 6, 2004. In Final Form: November 8, 2004 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. Introduction Covalent attachment of poly(ethylene glycol) (PEG) chains to therapeutic proteins, “PEGylation”, is growing in application due to increased renal clearance time, decreased proteolytic degradation, and decreased immune response relative to the unmodified forms. 1,2 The PEGylated forms of adenosine deaminase and asparagi- nase are now approved for human use by the U.S. Food and Drug Administration, 2,3 and other PEG-modified proteins are being developed for possible therapeutic use, including PEG epidermal growth factor 4 and PEGylated single-chain Fv proteins. 5 The numerous scenarios in which PEGylated proteins encounter solid/liquid interfaces during their manufac- ture, storage, and end use motivate this study of the effect of PEGylation on protein adsorption behavior. When PEGylated proteins are prepared for therapeutic applica- tions, it is necessary that the distribution of modified forms be reproducible. Over-PEGylation can decrease bioactivity, and under-PEGylation may allow a molecule to be cleared from the circulatory system too rapidly. A reproducible population of modified forms could be achieved by controlling the PEGylation reaction and/or by fractionation of the PEGylated product. Chromatography is a common technique for therapeutic protein fractionation, and most chromatographic methods depend on adsorption behavior. Furthermore, PEG-modified proteins may also encoun- ter solid/liquid interfaces during drug delivery. Protein loss to deposition and surface-induced conformational changes at these interfaces is manifested as lost bioavail- ability. Tzannis and co-workers observed ∼90% loss of the biological activity of interleukin 2 after 24 h of continuous delivery via commercial silicone rubber cath- eter tubing. 6 Additionally, insulin activity is reduced upon delivery from micropumps, due to surface-induced ag- gregation. 7 In delivery technologies, where dosage is critical, unpredictable protein-surface interactions can have a significant impact on the amount of biofunctional drug administered to a patient. The extent of adsorption, the adsorption kinetics, and the layer structural evolution upon the adsorption of protein will dictate the yield of biochemically functional protein and will all likely be affected by PEGylation. Interest in the adsorption of PEGylated proteins is piqued by the so-called “stealthy” characteristics of PEG. It is well-known that surfaces coated with PEG tend to be resistant to protein adsorption 8-10 and the protective effect of PEG modification on circulating proteins (as well as PEGylated liposomes) in vivo is attributed to strong steric and hydration repulsions between the PEGylated species and other proteins or cell surfaces. 11 Nevertheless, PEG itself is surface active on a variety of surfaces, and * Corresponding author. E-mail: tilton@andrew.cmu.edu. (1) Harris, J. M.; Martin, N. E.; Modi, M. Clin. Pharmacokinet. 2001, 40, 539. (2) Michaelis, M.; Cinatl, J.; Pouckova, P.; Langer, K.; Kreuter, J.; Matousek, J. Anti-Cancer Drugs 2002, 13, 149. (3) Hershfield, M. S. Biochemistry and Immunology of Poly(ethylene glycol)-Modified Adenosine Deaminase (PEG-ADA); ACS Books: Wash- ington, DC, 1997. (4) Lee, H.; Park, T. Pharm. Res. 2002, 19, 845. (5) Lee, L.; Conover, C.; Shi, C.; Whitlow, M.; Filpula, D. Bioconjugate Chem. 1999, 10, 973. (6) Tzannis, S. T.; Hrushesky, W. J. M.; Wood, P. 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