Effects of rotational speed on the hydrodynamic properties of pharmaceutical antibodies measured by analytical ultracentrifugation sedimentation velocity

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.