Self-association, crystallization, and phase separation: Understanding intermolecular interactions for a monoclonal antibody

Protein intermolecular interactions govern the physical stability of liquid formulations of therapeutic proteins. Intermolecular repulsion typically yields a formulation with long term physical stability. When there is intermolecular attraction, the result can be the formation of oligomers or a phase separation in which crystals, amorphous solids, or phase-separated liquids are generated, depending on the strength, range, and type of the interaction. Understanding the nature of the interactions responsible for the different physical behaviors of rhuMAb VEGF provides specific information regarding the factors that should be considered for stabilizing this antibody as well as contributes more general knowledge regarding protein:protein interactions and their roles in stability of therapeutic protein formulations.;A comparison of the rhuMAb VEGF structures under different pH conditions as well as an exploration of the conformational stability was performed and concluded that native state interactions are responsible for the self-association of rhuMAb VEGF, with the specific amino acids in the CDR playing a critical role in the dimerization reaction. The thermodynamics of the self-association reaction were explored by monitoring the equilibrium constant for dissociation as a function of pH, temperature, pressure, and surface tension. The pH and temperature dependence of the equilibrium constant suggest that protonation of a single histidine residue may be responsible for the differences in K d over the narrow pH range studied.;Phase separation is observed with rhuMAb VEGF under certain solution conditions. Under some conditions, liquid-liquid phase separation (LLPS) was preceded by critical opalescence which obeyed the critical exponent, gamma. This study concluded that opalescence of protein formulations is not necessarily attributable to aggregates and provided considerations to the impact of LLPS on formulation development. Protein crystallization is the result of short range, slightly attractive protein interactions. The nucleation and growth kinetics of rhuMAb VEGF crystals were studied by using a mixed suspension, mixed product removal reactor and population balance model. Growth kinetics were limited by the incorporation of new molecules from the bulk solution into the crystalline lattice. The results demonstrate that the population and mass balance approach is suitable for determining the nucleation and growth kinetics for protein crystallization.