{Reference Type}: Journal Article
{Title}: Hydrodynamic and thermodynamic analysis of PEGylated human serum albumin.
{Author}: Correia JJ;Stafford WF;Erlandsen H;Cole JL;Premathilaka SH;Isailovic D;Dignam JD;
{Journal}: Biophys J
{Volume}: 0
{Issue}: 0
{Year}: 2024 Jun 18
{Factor}: 3.699
{DOI}: 10.1016/j.bpj.2024.06.015
{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 so, hydrodynamic nonideality ks, and thermodynamic nonideality 2BM1SV terms. These quantities allow the determination of the Stokes radius Rs, the frictional ratio f/fo, and the swollen or entrained volume Vs/v, which measure size, shape, and solvent interaction. We performed sedimentation equilibrium experiments to obtain independent measurements of thermodynamic nonideality 2BM1SE. From DLS measurements, we determined the interaction parameter, kD, the concentration dependence of the apparent diffusion coefficient, D, and from extrapolation of D to c = 0 a second estimate of Rs. Rs values derived from SV and DLS measurements and ensemble model calculations (see complementary study) are then used to show that ks + kD = theoretical 2B22M1. In contrast, experimental BM1 values from SV and sedimentation equilibrium data collectively allow for similar analysis for protein-PEG conjugates and show that ks + kD = 1.02-1.07∗BM1, rather than the widely used ks + kD = 2BM1 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.