Abstract
Covalent labeling of therapeutic drugs and proteins with polyethylene glycol (PEGylation) is an important modification for improving stability, solubility, and half-life. PEGylation alters protein solution behavior through its impact on thermodynamic nonideality by increasing the excluded volume, and on hydrodynamic nonideality by increasing the frictional drag. To understand PEGylation's impact, we investigated the thermodynamic and hydrodynamic properties of a model system consisting of PEGylated human serum albumin derivatives using analytical ultracentrifugation (AUC) and dynamic light scattering (DLS). We constructed PEGylated human serum albumin derivatives of single, linear 5K, 10K, 20K, and 40K PEG chains and a single branched-chain PEG of 40K (2 × 20K). Sedimentation velocity (SV) experiments were analyzed using SEDANAL direct boundary fitting to extract ideal sedimentation coefficients s(o), hydrodynamic nonideality k(s), and thermodynamic nonideality 2BM(1)(SV) terms. These quantities allow the determination of the Stokes radius R(s), the frictional ratio f/f(o), and the swollen or entrained volume V(s)/v, which measure size, shape, and solvent interaction. We performed sedimentation equilibrium experiments to obtain independent measurements of thermodynamic nonideality 2BM(1)(SE). From DLS measurements, we determined the interaction parameter, k(D), the concentration dependence of the apparent diffusion coefficient, D, and from extrapolation of D to c = 0 a second estimate of R(s). R(s) values derived from SV and DLS measurements and ensemble model calculations (see complementary study) are then used to show that k(s) + k(D) = theoretical 2B(22)M(1). In contrast, experimental BM(1) values from SV and sedimentation equilibrium data collectively allow for similar analysis for protein-PEG conjugates and show that k(s) + k(D) = 1.02-1.07(∗)BM(1), rather than the widely used k(s) + k(D) = 2BM(1) developed for hard spheres. The random coil behavior of PEG dominates the colloidal properties of PEG-protein conjugates and exceeds the sum of a random coil and hard-sphere volume due to excess entrained water.