RECEIVED DATE: TITLE RUNNING HEAD: Size and SEC of PEGylated Proteins

Size exclusion chromatography (SEC) was used to determine the viscosity radii of equivalent spheres for proteins covalently grafted with poly(ethylene glycol) (PEG). The viscosity radius of such PEGylated proteins was found to depend on the molecular weight of the native protein and the total weight of grafted PEG but not on PEG molecular weight, or PEG-to-protein molar grafting ratio. Results suggest grafted PEG’s form a dynamic layer over the surface of proteins. The geometry of this layer results in a surface area to volume ratio approximately equal to that of a randomly coiled PEG molecule of equivalent total molecular weight. Two simple methods are given to predict the viscosity radius of PEGylated proteins. Both methods accurately predicted (3 % absolute error) the viscosity radii of various PEG-proteins produced using three native proteins, α-lactalbumin (14.2 kDa MW), β−lactoglobulin dimer (37.4 kDa MW) and bovine serum albumin (66.7 kDa MW), three PEG reagents (2400, 5600, and 22500 MW), and molar grafting ratios of 0 to 8. Accurate viscosity radius prediction allows calculation of the distribution coefficient, Kav, for PEG-proteins in SEC. The suitability of a given SEC step for the analytical or preparative fractionation of different PEGylated protein mixtures may therefore be assessed mathematically. The methods and results offer insight to several factors related to the production, purification, and uses of PEGylated proteins