Higher-Order Structure and Solution Interactions of Antibodies

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Monoclonal antibodies (mAbs) are a rapidly growing class of protein therapeutics. To minimize the volume required for therapeutic administration, mAbs are typically formulated at concentrations on the order of 100 mg/ml, which is far above the range of existing particle sizing methods. While sedimentation velocity analytical ultracentrifugation has become the gold standard for measuring higher-order structures in dilute solutions, new opportunities have arisen for characterizing mAb solutions much closer to formulation conditions with the new technique of nonideal sedimentation velocity (SV), recently developed in our laboratory. In principle this allows one to study at 10fold higher protein concentrations than previously possible. Beyond the obvious high-affinity interaction of antibodies with their antigen target, there are three additional types of antibody interactions that are of interest in pharmaceutical formulations. First, short- and long-range electrostatic and hydrodynamic forces modulate protein distance distributions in solution. This may result in net repulsive or attractive interactions without formation of protein complexes. Though very subtle, such interactions are key to macroscopic solution behavior and long-term protein stability. They are commonly measured through nonideality coefficients of sedimentation, diffusion, and thermodynamic virial coefficients. A second type of antibody interactions is their propensity to self-associate through formation of short-lived, reversible protein-protein complexes. These have been associated with high solution viscosity. Both nonideality and self-association properties are concentration-dependent and cannot be measured well in dilute solution. Using a panel of monoclonal antibodies, in collaboration with AstraZeneca, we have established concentration limits of nonideal SV for IgG mAbs to be 45 mg/mL. We were able to show that with such highly concentrated samples it is possible to reliably measure nonideality coefficients of sedimentation. Simultaneously the new nonideal SV approach provides a simple and sensitive means to characterize self-association of antibodies. In theoretical work we have analyzed the impact of weak self-association on the measurement of nonideality coefficients with different biophysical methods. This has clarified the customary use of incompatible thermodynamic reference frames, for example, in static and dynamic light scattering, and explains grossly inconsistent nonideality coefficients frequently found in the literature. To overcome this problem, we have extended our global multi-method analysis (GMMA) software to allow global analysis of nonideality from different data sources. This bridges gaps in sensitivity and resolution between different techniques, and permits the use of a self-consistent reference frame that links nonideality parameters up with statistical fluid dynamics theory. Beyond the characterization of antibodies, GMMA of weak interactions and nonideality provides an experimental platform for related interests in DMAS in the field of concentrated protein solutions, including eye lens crystallins. Finally, a third category of protein interactions that are very important for antibody formulations are long-lived aggregates from misfolded protein that can form with time. These are potentially immunogenic and therefore trace quantities must be monitored and reported to FDA. In order to examine what impact the higher concentration limits of nonideal SV have on the limits of quantitation for trace protein dimers, we have previously embarked on a study of heat-denatured NISTmAb reference antibody, in collaboration with the laboratory of Dr. John Schiel (NIST). We have carried out mixing experiments of native and heat-denatured NISTmAb in parallel by nonideal SV and size exclusion chromatography. We have developed a suitable data analysis protocol for trace analysis using nonideal SV. Based on the results, we have designed control experiments to enhance comparison of techniques.