Purpose High hydrostatic pressures (1-4kbar) reverse or foster protein aggregation. Recombinant human interleukin-1 receptor antagonist (IL-1ra) was used as a pharmaceutically relevant, recombinant protein to study the effects of pressure on protein aggregation. The purpose of this research was to investigate the thermodynamics of pressure-modulated structural changes on native and aggregated IL-1ra, with application to pressure-modulated aggregate refolding and pressure-accelerated formulation studies.

Methods Purified monomer was provided by Amgen Corporation and used as received. Pressure-induced, disulfide crosslinked aggregates of IL-1ra were formed in oxidizing buffer as a function of pressure by unfolding IL1-ra and and exposing interior free cysteines. Aggregates were also formed by temperature treatment by incubating for 40 minutes at 55oC. For refolding, the aggregates were suspended in reducing buffer and pressure treated at 0.001, 1.5 and 2.5 kbar bar at 31oC for 15 hours. Refolding yields were determined by size exclusion (HPLC-SEC) chromatography, activity assays, and extinction coefficients. Density and infrared spectroscopic studies were also conducted.

Results Temperature-induced aggregates of IL-1ra refolded at 59% yields after pressure treatment at 1.5 kbar. In contrast, if pressure treatment of 2.5 kbar was used, refolding from theramally-induced aggregates did not occur. Temperature-induced aggregates of IL-1ra are less dense than native IL-1ra, with specific volumes of 0.761 ml/g vs. 0.752 ml/g respectively. Partial specific adiabatic compressibility measurements suggest differences in the number of solvent-free cavities within aggregated and native forms of IL-1ra. Pressurization of native, monomeric IL1-ra to 1.5 kbar results in a minor loss of native secondary structure. However, pressures of 2.5 kbar denature IL-1ra, and result in formation of aggregates.

Conclusions Pressure is a practical tool for modulating aggregate structure. Moderate pressure treatment (1-2kbar) can be used to dissociate and refold protein aggregates in conditions that maintain native protein structure. In comparison, traditional chaotrope refolding requires denaturation prior to refolding. We conclude that pressure thermodynamically drives refolding since aggregates are typically less dense than native proteins, due to packing defects. Sufficiently high pressures (2-4kbar) can be used to partially denature native proteins. In these conditions, pressure treatment can foster and accelerate protein aggregation. Elevated temperatures are currently used to accelerate protein aggregation to predict protein formulation conditions. We propose that pressure can be used as an orthogonal tool to conduct accelerated formulation studies.