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.
摘要：
用聚乙二醇共价标记治疗药物和蛋白质（聚乙二醇化）是提高稳定性的重要修饰，溶解度和半衰期。聚乙二醇化通过增加排除体积对热力学非理想性的影响来改变蛋白质溶液的行为，以及通过增加摩擦阻力来实现流体动力非理想性。要了解聚乙二醇化的影响，我们使用分析超速离心（AUC）和动态光散射（DLS）研究了由聚乙二醇化人血清白蛋白（PEG-HSA）衍生物组成的模型系统的热力学和流体动力学特性。我们构建了单,线性5K，10K,20K,40KPEG链和40K(2X20K)的单支链PEG。使用SEDANAL直接边界拟合分析了沉降速度（SV）实验，以提取理想的沉降系数，因此，水动力非理想性ks，和热力学非理想性2BM1SV项。这些量允许确定斯托克斯半径Rs，摩擦比f/fo，和肿胀或夹带的体积Vs/v，测量尺寸，形状,和溶剂相互作用。我们进行了沉降平衡实验，以获得热力学非理想性2BM1SE的独立测量值。从DLS测量中，我们确定了相互作用参数，KD,表观扩散系数的浓度依赖性，D,从D到c=0的外推得出R的第二个估计值。然后使用从SV和DLS测量和集合模型计算得到的Rs值(参见补充研究)来显示ks+kD=理论2B22M1。相比之下,来自SV和SE数据的实验BM1值共同允许对蛋白质-PEG缀合物进行类似分析，并显示kskD=1.02-1.07*BM1，而不是为硬球开发的广泛使用的kskD=2BM1。PEG的无规卷曲行为主导了PEG-蛋白质缀合物的胶体性质，并且由于夹带过多的水而超过了无规卷曲和硬球体积的总和。
