The formation and structure of precipitated protein phases
Date
2016
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
From 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.