Assessment of net charge and protein-protein interactions of different monoclonal antibodies

Abstract

The purpose of this work was to compare biophysical properties of different monoclonal antibodies (mAbs). mAbs' theoretical isoelectric point (IEP) and theoretical net charge were compared with experimentally assessed values. IEP was determined by isoelectric focusing capillary electrophoresis, determination of zero electrophoretic mobility, or the minimum mutual diffusion coefficient during pH titration. Net charge was determined using electrophoretic mobility and self-diffusion coefficient. It was found that antibodies differ substantially in their biophysical properties, that is, in IEP, net charge, and zeta potential. Also, the importance of these properties was studied with respect to protein-protein interactions. This was achieved by assessing the second virial coefficient (A(2)) determined by static light scattering (SLS) and dynamic light scattering (DLS). It was found that at low ionic strength formulation conditions [20 mM histidine (His)/His-HCl buffer, pH 6.0] proteins' charge is the main driver for overall repulsive protein interactions. At high ionic strength conditions (20 mM His/His-HCl buffer, pH 6.0, + 150 mM NaCl), where counterions are shielding ionic interactions, proteins' repulsive forces were weakened, but to a different extent. Furthermore, a DLS method was developed allowing fast and easy assessment of A(2) by minimum need of material.