The Measured Electrostatic Charge on IgGs

Blood plasma is a high-concentration fluid containing ∼70 mg/ml protein. A major component of plasma (∼10 mg/ml) is a changing, heterogeneous mixture of IgGs. At plasma concentrations electrostatics (charge, dipole, induced dipoles, etc.) dominate chemical activity, hence solution properties. Protein charge can be determined accurately using a combination of electrophoretic and hydrodynamic measurements. It is essential to measure charge since calculated values (e.g., from isoelectric point determinations) may be in serious error. Monoclonal IgGs (mAbs) provide an important example where charge must be measured. Charge determinations for 11 different mAbs in 100 mM KCl at pH 6.0 show that calculated values overestimate the charge by ∼17, with the discrepancy increasing to ∼50 at pH 5.0. The mechanisms underlying charge suppression are unclear. There is nothing obvious in IgG structure (e.g., buried side chains, H-bonding, clustered charged side chains) that would account for the suppressed charge. It seems likely that weak ion binding (either site or territorial) by IgGs may occur since changing the solvent ionic strength and ion composition influence charge suppression beyond their Debye-Huckel effects. The unusual charge properties of IgGs may have both in vivo and in vitro significance. In vivo, charge suppression may provide a “buffer” that allows high plasma concentrations of IgGs with different amino acid compositions. Charge also may be important in Fc receptor binding of IgGs. Analysis of isolated Fc and Fab fragments reveals that the Fc fragment charge is less than +1 at pH 6.0, where the calculated charge is +9. In vitro, IgG charge correlates with increased solubility and reduced solution viscosity, properties that are important in drug formulation.Funding: Biomolecular Interactions Technology Center, Center to Advance Molecular Interaction Science