Application of high hydrostatic pressure to dissociate aggregates and refold proteins.

Non-denaturing pressures of around 2000 bar are effective for eliminating and refolding protein aggregates and may be applicable in various phases of protein manufacturing to decrease aggregate levels in products and improve process yields. Lower aggregate levels can result in reduced immunogenicity of proteins and enable the correct refolding of proteins that might not be recovered with traditional techniques. High pressure treatment can also be used to conduct selective PEGylation and protease cleavage reactions while minimizing protein aggregation. High pressure processes have been used in the food industry for over 50 years and large scale (300 L) systems are commercially available, enabling production of proteins on the kilogram scale. This review summarizes the utility of high pressure refolding to remove and refold protein aggregates, enhance therapeutic proteins, and facilitate manufacturing improvements at industrial scales.

Confronting high-throughput protein refolding using high pressure and solution screens.

Over-expression of heterologous proteins in Escherichia coli is commonly hindered by the formation of inclusion bodies. Nevertheless, refolding of proteins in vitro has become an essential requirement in the development of structural genomics (proteomics) and as a means of recovering functional proteins from inclusion bodies. Many distinct methods for protein refolding are now in use. However, regardless of method used, developing a reliable protein refolding protocol still requires significant optimization through trial and error. Many proteins fall into the category of "Challenging" or "Difficult to Express" and are problematic to refold using traditional chaotrope-based refolding techniques. This review discusses new methods for improving protein refolding, such as implementing high hydrostatic pressure, using small molecule additives to enhance traditional protein refolding strategies, as well as developing practical methods for performing refolding studies to maximize their reliability and utility. The strategies examined here focus on high-throughput, automated refolding screens, which can be applied to structural genomic projects.

Specific volume and adiabatic compressibility measurements of native and aggregated recombinant human interleukin-1 receptor antagonist: density differences enable pressure-modulated refolding.

High hydrostatic pressures have been used to dissociate non-native protein aggregates and foster refolding to the native conformation. In this study, partial specific volume and adiabatic compressibility measurements were used to examine the volumetric contributions to pressure-modulated refolding. The thermodynamics of pressure-modulated refolding from non-native aggregates of recombinant human interleukin-1 receptor antagonist (IL-1ra) were determined by partial specific volume and adiabatic compressibility measurements. Aggregates of IL-1ra formed at elevated temperatures (55 degrees C) were found to be less dense than native IL-1ra and refolded at 31 degrees C under 1,500 bar pressure with a yield of 57%. Partial specific adiabatic compressibility measurements suggest that the formation of solvent-free cavities within the interior of IL-1ra aggregates cause the apparent increase in specific volume. Dense, pressure-stable aggregates could be formed at 2,000 bar which could not be refolded with additional high pressure treatment, demonstrating that aggregate formation conditions and structure dictate pressure-modulated refolding yields.

High-pressure studies on protein aggregates and amyloid fibrils.

High hydrostatic pressure (HHP) modulates protein-protein and protein-solvent interactions through volume changes and thereby affects the equilibrium of protein conformational species between native and denatured forms as well as monomeric, oligomeric, and aggregated forms without the addition of chemicals or use of high temperature. Because of this unique property, HHP has provided deep insights into the thermodynamics and kinetics of protein folding and aggregation, including amyloid fibril formation. In particular, HHP is a useful tool to stabilize and populate specific folding intermediates, the characterization of which provides thorough understanding of protein folding and aggregation pathways. Furthermore, recent application of HHP for dissociation of protein aggregates, such as inclusion bodies (IBs), into native proteins in a single step facilitates protein preparation for structural and functional studies. This chapter overviews recent HHP studies on the population and characterization of folding intermediates associated with protein aggregation and protein refolding from protein aggregates of amyloid fibrils and IBs. Finally, we describe overall experimental procedures of HHP-mediated protein refolding and provide a detailed discussion of each operating parameter to optimize the refolding.

High-pressure studies of aggregation of recombinant human interleukin-1 receptor antagonist: thermodynamics, kinetics, and application to accelerated formulation studies.

Recombinant human interleukin-1 receptor antagonist (IL-1ra) in aqueous solutions unfolds and aggregates when subjected to hydrostatic pressures greater than about 180 MPa. This study examined the mechanism and thermodynamics of pressure-induced unfolding and aggregation of IL-1ra. The activation free energy for growth of aggregates (DeltaG-/+(aggregation)) was found to be 37 +/- 3 kJ/mol, whereas the activation volume (DeltaV-/+(aggregation)) was -120 +/- 20 mL/mol. These values compare closely with equilibrium values for denaturation: The free energy for denaturation, DeltaG(denaturation), was 20 +/- 5 kJ/mol, whereas the partial specific volume change for denaturation, DeltaV(denaturation), was -110 +/- 30 mL/mol. When IL-1ra begins to denature at pressures near 140 MPa, cysteines that are normally buried in the native state become exposed. Under oxidizing conditions, this results in the formation of covalently cross-linked aggregates containing nonnative, intermolecular disulfide bonds. The apparent activation free energy for nucleation of aggregates, DeltaG-/+(nuc), was 42 +/- 4 kJ/mol, and the activation volume for nucleation, DeltaV-/+(nuc),was -175 +/- 37 mL/mol, suggesting that a highly solvent-exposed conformation is needed for nucleation. We hypothesize that the large specific volume of IL-1ra, 0.752 +/- 0.004 mL/g, coupled with its relatively low conformational stability, leads to its susceptibility to denaturation at relatively low pressures. The positive partial specific adiabatic compressibility of IL-1ra, 4.5 +/- 0.7 +/- 10(-12) cm2/dyn, suggests that a significant component of the DeltaV(denaturation) is attributable to the elimination of solvent-free cavities. Lastly, we propose that hydrostatic pressure is a useful variable to conduct accelerated formulation studies of therapeutic proteins.

High-pressure refolding of bikunin: efficacy and thermodynamics.

Bikunin is a glycosylated protein that aggregates extensively during mammalian cell culture, resulting in loss of activity, loss of native secondary structure, and the formation of nonnative disulfide bonds. We investigated the use of high hydrostatic pressure (1000-3000 bar) for the refolding of bikunin aggregates. The refolding yield obtained with pressure-modulated refolding at 2000 bar was 70 (+/-5%) by reverse-phase chromatography (RP-HPLC), significantly higher than the value of 55 (+/-6%) (RP-HPLC) obtained with traditional guanidine HCl "dilution-refolding." In addition, we determined the thermodynamics of pressure-modulated refolding. The change in volume for the transition of aggregate to monomer DeltaV(refolding) was calculated to be -28 (+/-5) mL/mole. Refolding was accompanied by a loss of hydrophobic exposure, resulting in a positive contribution to the DeltaV(refolding). These findings suggest that the disruption of electro-static interactions or the differences in size of solvent-free cavities between the aggregate and the monomer are the prevailing contributions to the negative DeltaV(refolding).