Gel-like behavior in amorphous protein dense phases: phase behavior, neutron scattering and rheology
Date
2019
Authors
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Publisher
University of Delaware
Abstract
Protein dense phases are ubiquitous in pharmaceutical downstream processing and crystallization screens. Identifying the various dense phases that exist for different proteins and precipitants is of significant interest, with several theoretical and experimental papers published that study the various aggregation boundaries and phase behavior mechanisms that exist due to competition between various equilibrium and non-equilibrium driving forces. A protein phase diagram with dense phases such as dense liquids, gels, crystals, and precipitates can be obtained upon the addition of a precipitant or due to temperature or pH changes for a suitable set of samples. Of the dense phases discussed, the primary interest lies in gels, which are materials that are composed primarily of liquids but exhibit solid-like mechanical properties due to the individual proteins interacting and aggregating to form an interconnected structure. ☐ The goal of this project is to prepare gels of globular protein that arise from dense phases salted-out at ambient conditions (room temperature (~23ºC) and pH 7.0) and measure their structural and mechanical properties. To our knowledge, there have been studies that show gelation due to low temperature quenches in lysozyme, as well as gelation of proteins due to heating. However, there are very limited studies of the physical and structural properties of salted-out protein gel phases. Additionally, not all combinations of proteins and precipitants lead to the formation of a gel phase. To address these challenges, we conducted a screening test involving a phase behavior study to identify the protein, the precipitant and the associated concentrations that lead to an apparent gel phase. For a combination of ribonuclease A and ammonium sulfate in 5 mM phosphate buffer in D2O at pD 7.0, two distinct types of behavior are seen: (1) a clear liquid corresponding to a single-phase viscous liquid that does not show gel-like behavior; (2) an opaque gel-phase that appears near the aggregation boundary of ribonuclease A, that is attributed to spinodal decomposition and that adheres to the tube wall upon inversion. ☐ Following this, different small-amplitude oscillatory shear (SAOS) bulk-rheology experiments utilizing a cone-and-plate geometry were performed on the gel-phase: (1) an oscillation time test for 104 seconds allowing for gel formation; (2) a frequency sweep that showed a predominant storage modulus (G'(ω) > G''(ω)) that confirms the presence of a gel phase. ☐ Obtaining the structural properties of the gel is a challenge due to the opacity. Thus, a combination of small-angle neutron scattering (SANS) and ultra-small-angle neutron scattering (USANS) was used to study and characterize this system. Firstly, TR-SANS (time-resolved small-angle neutron scattering) was performed for a duration of 104 seconds corresponding to the time scale used for the oscillation time test. TR-SANS show two distinct regions of structural evolution; a low-Q region and a mid-Q region that show broad-peak evolution and monomer-monomer level interactions, respectively. SANS and USANS data for the gel formulation are fit utilizing shape independent structural models that show the presence of gel network. USANS data show the absence of any structure for the single-phase liquid indicating that the gelation behavior evidenced in rheological studies for the ‘gel phase’ are characteristic of higher-order structures that give rise to a system spanning gel. ☐ To conclude, a combination of phase behavior studies, neutron scattering, and bulk-rheology can provide an adequate framework for identifying a gel phase that exists for salted-out proteins and obtaining its structural and mechanical properties. Implications from this study could provide insight on discovering and characterizing more such protein-salt combinations that display a gel phase, for which further research is necessary.