Structure, rheology, and phase behavior of protein formulations under high pressure
Date
2025
Authors
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Publisher
University of Delaware
Abstract
High hydrostatic pressure (HP), defined as pressures between 100 MPa - 800 MPa, is an increasingly prevalent component of protein formulation processing for foods and pharmaceuticals. HP is found across the process development scheme, including extraction and purification of drug substances via cell disruption, freeze-thaw cycling of liquid therapeutic drug products during storage, pressure-inactivation of cell-based and virus-based assays for vaccine manufacturing, and non-thermal sterilization. HP can have significant effects on the stability, rheological properties, and phase behavior of protein formulations, yet despite the wide usage of HP in industrial processing, little is known about the mechanistic effects of HP on protein intermolecular behavior, formulation properties, or phase behavior. The major barrier to developing predictive models for HP protein behavior is a lack of available in situ HP protein data, which itself stems from the limited availability of in situ characterization approaches for structure, rheology, and phase behavior under HP. Therefore, the research in this dissertation is motivation primarily by two goals: an expanded understanding of the mechanistic effects of HP on protein behavior by building structure-property relationships across multiple length scales, and an expansion upon current HP analytical capabilities to provide better tools for formulation screening under HP. ☐ First, we apply HP small-angle X-ray scattering (HP-SAXS) to investigate the simultaneous effects of HP and dissolved salt on ovalbumin intermolecular interactions. Interaction trends are quantified by the reduced second virial coefficient and a synergistic effect is observed, with enhanced net attraction with either increasing ionic strength or applied pressure. A significant contribution of the work in this dissertation, inspired by the apparent pressure-salt synergism, is a new semi-empirical model which captures the effects of both pressure and ionic strength with a single effective pressure parameter. Applying the model results in smooth alignment of interaction data to a master curve across a wide range of applied pressures and ionic strengths. The model is then applied to protein data in the literature, and the correlation is shown to apply broadly but not universally to other protein-salt systems. Importantly, the synergism with ion effects strongly supports a hydration-driven mechanism of pressure-induced protein-protein attraction and provides critical insight into the mechanistic effects of pressure on protein stability. ☐ We also investigate the effects of pressure holding time on the semi-empirical master curve, using HP small-angle neutron scattering (HP-SANS) as a non-destructive method for time-resolved studies. Performing a similar screening of ovalbumin intermolecular interactions across applied pressures and ionic strengths reveals that net attractive interactions are significantly enhanced with increased pressure holding time. Pressure-induced effects are also shown not to be fully reversible in the presence of salt, and slow aggregation processes emerge more than 24 h after depressurization. Interaction data from long pressure incubations are then used to predict the protein relative viscosity. We use a combination of conventional viscometry and HP diffusing wave spectroscopy (HP-DWS) microrheology to demonstrate agreement between predicted and experimental viscosity data, providing a route to building predictive models by correlating multiple data sets together from distinct characterization approaches. ☐ Another novel contribution of the research in this dissertation is the first demonstration of in situ HP rheology for studying protein sol-gel behavior. HP-DWS is performed with in situ temperature control to measure the effects of HP on a thermoreversible protein sol-gel transition. One major contribution of this work is clear evidence that pressure and temperature induce orthogonal gelation mechanisms, and the rheological behavior of a pressure-induced gel varies significantly from that of a preset thermal gel under HP. We present a pressure-temperature phase diagram, from which the structural and mechanical properties of a gel can be potentially tuned using stepwise variations in pressure and temperature. The work presented here significantly advances current HP characterization capabilities by developing a new analytical tool for HP rheology of protein-based complex fluids. We additionally present a proposed design for simultaneous HP-SANS-DWS, which would enable optimized screening of protein formulation structure and rheology with in situ pressure and temperature control. ☐ We further demonstrate the irreversible effects of HP on salted-out ovalbumin dense phases using ex situ static light scattering (SLS) and SAXS. Gel microparticles formed at moderate salt concentrations and dense core-shell protein particles formed beyond a high-salt phase transition boundary both dissociate during pressure treatment, allowing for repacking into more tightly arranged structures that persist after depressurization. In contrast, gel microparticles near the phase transition line exhibit pressure-induced phase separation. Significantly, the gel particle network is replaced by dense core-shell structures, while the constituent gel clusters persist. This result implies a gradually reduced favorability of the gel phase with pressurization, allowing crystallization to dominate with a sufficiently high applied pressure, and confirms the hypothesis that competition between gelation and crystallization dictates the dominant phase behavior. ☐ The research presented in this dissertation makes significant contributions to the mechanistic understanding of protein behavior under high pressure and begins to correlate data across multiple characterization approaches into structure-property relationships. By probing HP behavior simultaneously with other formulation and process parameters, the mechanisms through which each parameter influences structure, rheology, and phase behavior can be determined and decoupled. The results of the research in this dissertation have significant implications for property control during protein formulation processing, as material properties can be tuned through a carefully designed series of process steps.
Description
Keywords
High-pressure, Interactions, Method development, Proteins, Rheology, Small-angle scattering