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High‐pressure studies of aggregation of recombinant human interleukin‐1 receptor antagonist: Thermodynamics, kinetics, and application to accelerated formulation studies
Author(s) -
Seefeldt Matthew B.,
Kim YongSung,
Tolley Kevin P.,
Seely Jim,
Carpenter John F.,
Randolph Theodore W.
Publication year - 2005
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.051490205
Subject(s) - chemistry , denaturation (fissile materials) , kinetics , thermodynamics , activation energy , nucleation , intermolecular force , covalent bond , crystallography , organic chemistry , molecule , nuclear chemistry , physics , quantum mechanics
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 (Δ G ∓ aggregation ) was found to be 37 ± 3 kJ/mol, whereas the activation volume (Δ V ∓ aggregation ) was −120 ± 20 mL/mol. These values compare closely with equilibrium values for denaturation: The free energy for denaturation, Δ G denaturation , was 20 ± 5 kJ/mol, whereas the partial specific volume change for denaturation, Δ V 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, Δ G ∓ nuc , was 42 ± 4 kJ/mol, and the activation volume for nucleation, Δ V ∓ 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 cm 2 /dyn, suggests that a significant component of the Δ V 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.