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.

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.

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.

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.

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.

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.