The formation and structure of precipitated protein phases

Lenhoff, AbrahamWagner, NormanSandler, StanleyFrom downstream processing to crystallization screens, protein dense phases, such as gels, aggregates and precipitates, arise during protein solution processing. Several phase separation mechanisms exist that lead to precipitation, including liquidliquid phase separation and crystallization. Competition between these mechanisms and non-equilibrium aggregation mechanisms, such as percolation and gelation, results in complex kinetic phenomena that lead to distinctive microstructures. While the aggregation boundaries have been mapped out for several proteins as a function of various additives, less information is known about the specific microstructures that form within these non-crystalline protein dense phases. Structural biologists have reported qualitative evidence that these dense phases can contain small, highly ordered regions of protein and these data are supported by simulations of colloidal spheres. However, direct experimental measurement of these microstructures is lacking. ☐ First, we utilize small-angle scattering techniques and electron microscopy to measure the microstructure of salted-out ovalbumin. Ovalbumin aggregates into gel beads on the addition of sufficient quantities of ammonium sulfate. Within these gel beads, ovalbumin molecules hierarchically pack into nanocrystallites, approximately 12 nm in size, that assemble into a porous network. Importantly, the results strongly support the hypothesis that non-crystalline protein dense phases can and do contain small, highly ordered regions. ☐ While salted-out ovalbumin exhibits nanocrystals, it is unclear if this structural motif also arises within the dense phases of other proteins or if it is unique to ovalbumin. To address this, the microstructures that form on salting out several other protein species, including lysozyme, ribonuclease A, catalase, and a monoclonal antibody, are then quantified. On shallow quenches nanocrystals ranging in size from 10 to 100 nm develop within these dense phases, further supporting the hypothesis that these macroscopically non-crystalline dense phases do contain highly ordered regions. With recent advances in X-ray sources, we expect that these nanocrystals will be useful for crystallographic measurements. ☐ For deep quenches, a structural motif arises that is consistent with the structure of an arrested spinodal decomposition. The scattering spectra of these deeply quenched samples exhibit a broad peak that is associated with a correlation length scale. The length scale has a power-law dependence on the quench depth and a single power-law index is sufficient to describe the behavior of all the proteins studied, indicating that the behavior may be universal. ☐ The structures discussed above are the result of specific kinetic pathways that arise due to a competition between different growth mechanisms. Therefore, we utilize time-resolved small-angle neutron scattering (TR-SANS) to better understand and characterize these complex pathways. While we are unable to conclusively determine a growth mechanism, TR-SANS reveals that the nanocrystal growth is rapid and can occur in less than an hour. ☐ In many situations, for example during protein purification, precipitation occurs in the presence of multiple protein species. Therefore, we quantify the structure and growth mechanism of a precipitate that forms during the purification of an industrially relevant therapeutic protein when the pH is increased from 3.5 to 5. The precipitate has a mass-fractal microstructure with fractal dimension 2. This structure arises from diffusion-limited aggregation. The process is driven by attractive electrostatic interactions that are modulated by salt and there appears to be a specific ion effect that alters the pH at which the precipitation occurs. ☐ Taken together, these results highlight that precipitation can follow a variety of pathways that lead to very distinct microstructures. A better understanding of these pathways is critical in designing next-generation precipitation processes and the nanocrystals that develop on salting-out may be useful for structural biology. These results establish the first steps in understanding this complex phenomenon on a molecular level.University of Delaware, Department of Chemical and Biomolecular EngineeringPh.D