ELECTROPHORETIC MOBILITY MODELING OF PROTEINS IN FREE ZONE CAPILLARY ELECTROPHORESIS AND ITS APPLICATION TO MONOCLONAL-ANTIBODY MICROHETEROGENEITY ANALYSIS

A semi-empirical model for describing the electrophoretic mobility of proteins in free solution is derived. Protein mobility is found to be influenced, as dictated by the Debye-Huckel-Henry theory, by protein valence, size and shape, and by solution ionic strength, pH, viscosity and temperature. Protein valence, the most important mobility determining parameter intrinsic to the protein, is calculated for a given pH from its amino acid content using the Henderson-Hesselbalch equation. Electrostatic charge suppression causes actual valence to be less than that calculated. To equate the two an experimentally determined proportionality constant (F(Z)) is introduced. Consequently, F(Z) can be applied to the calculated valence and mobility-pH titration curve for a protein, resulting in the actual mobility of the protein at any given pH. The model further predicts that the molecular weight (M) dependency of mobility should be a continuous function of M-1/3 to M-2/3, depending on the magnitude of the protein molecular weight and buffer ionic strength under investigation. Many aspects of the model are demonstrated by its application to the resolution of immunoglobulin G isoelectrotypes, normally only resolved using isoelectric focusing.