Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan.
Eur J Pharm Sci. 2012 Sep 29;47(2):367-74. doi: 10.1016/j.ejps.2012.06.005. Epub 2012 Jun 21.
Analytical ultracentrifugation sedimentation velocity (AUC-SV) has recently become one of the most important tools for the measurement of hydrodynamic properties of proteins. Although a number of studies using AUC-SV as applied to pharmaceutical antibodies have been conducted, the effect of rotational speed on molecular properties has not been systematically examined. The present study aimed to elucidate the influence of rotational speed on the hydrodynamic parameters of pharmaceutical antibodies. A monoclonal and a polyclonal antibody were studied by using AUC-SV at 5 different rotor speeds, and the acquired data were analyzed either by using the computer programs SEDFIT or UltraScan. The frictional ratio of the studied antibodies decreased at high rotor speeds, resulting in underestimation of molecular weight. The frictional ratio value of the monoclonal antibody measured at the low rotor speed was consistent with that of human immunoglobulin G1 computed from its three-dimensional structure. The best agreement between the measured molecular weight and the value calculated from the antibody sequence was achieved at the lower rotor speed. Similar to the results obtained using antibodies, AUC-SV analysis of human serum albumin revealed that the frictional ratio and apparent molecular weight behave in a speed-dependent manner. We deduced that the findings were mainly attributable to the hydrostatic pressure in the analytical ultracentrifuge. The current study implies that rotor speed should be carefully considered in antibody studies using AUC-SV.
分析超速离心沉降速度（AUC-SV）最近已成为测量蛋白质流体力学性质的最重要工具之一。尽管已经进行了许多使用 AUC-SV 应用于药物抗体的研究，但旋转速度对分子性质的影响尚未系统地检查。本研究旨在阐明旋转速度对药物抗体流体力学参数的影响。使用 AUC-SV 在 5 种不同的转子速度下研究了一种单克隆抗体和一种多克隆抗体，并使用 SEDFIT 或 UltraScan 计算机程序分析获得的数据。在高转子速度下，研究抗体的摩擦比降低，导致分子量低估。在低转子速度下测量的单克隆抗体的摩擦比与从其三维结构计算得出的人免疫球蛋白 G1 的摩擦比一致。在较低的转子速度下，测量的分子量与从抗体序列计算得出的值之间的最佳一致性。与使用抗体获得的结果类似，AUC-SV 对人血清白蛋白的分析表明，摩擦比和表观分子量表现出与速度相关的行为。我们推断，这些发现主要归因于分析超速离心机中的静水压力。本研究表明，在使用 AUC-SV 进行抗体研究时，应仔细考虑转子速度。
