Denatured state aggregation parameters derived from concentration dependence of protein stability

Abstract

Protein aggregation is a major issue affecting the long-term stability of protein preparations. Proteins exist in equilibrium between the native and denatured or partially denatured conformations. Often denatured or partially denatured conformations are prone to aggregate because they expose to solvent the hydrophobic core of the protein. The aggregation of denatured protein gradually shifts the protein equilibrium toward increasing amounts of denatured and ultimately aggregated protein. Recognizing and quantitating the presence of denatured protein and its aggregation at the earliest possible time will bring enormous benefits to the identification and selection of optimal solvent conditions or the engineering of proteins with the best stability/aggregation profile. In this article, a new approach that allows simultaneous determination of structural stability and the amount of denatured and aggregated protein is presented. This approach is based on the analysis of the concentration dependence of the Gibbs energy (ΔG) of protein stability. It is shown that three important quantities can be evaluated simultaneously: (i) the population of denatured protein, (ii) the population of aggregated protein, and (iii) the fraction of denatured protein that is aggregated.

Conformational stability and self-association equilibrium in biologics

“…This behavior has been reported for cetuximab in urea denaturation experiments [22]. Δ G decreased from approximately 9 kcal/mol at 24 Mg/ml to 7 kcal/mol at 180 Mg/ml, consistent with denatured state aggregation.…”

“…In this case, the equilibrium is shifted to the left, Δ G of the denatured and partially denatured states increases and, consequently, the stability of the native state increases. If this situation occurs, the measured ΔG, usually referred to as apparent Δ G , Δ G app , is given by Equation 2 [22]: $$\mathrm{normal\Delta }{G}_{\mathit{\text{app}}}=\mathrm{normal\Delta }{G}_{\mathit{0}}^{\mathit{0}}+RT\text{italicln}false(1+j{K}_{N,i}{false[Nfalse]}^{j-1}false),$$ where $\mathrm{normal\Delta }{G}_{\mathit{0}}^{\mathit{0}}$ is equal to the value of the Gibbs energy extrapolated to zero protein concentration (in practice, a concentration low enough at which aggregation is not present), j is the average stoichiometry of the self-associated state, and K N,j the self-association constant. Equation 2 indicates that native state self-association will cause the measured Gibbs energy to increase as a function of protein concentration and that the magnitude of the increase is proportional to the self-association constant and the average size of the aggregates.…”

“…In this case, the equilibrium will be shifted to the right, Δ G will decrease and the stability of the native state will decrease. If this situation occurs, the measured Δ G is given by Equation 2 [22]. …”

Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry

“…For the system of coupled reactions, the apparent or measured conformational equilibrium constant can be defined as: …”

“…Under irreversible conditions most proteins, especially high molecular weight proteins, undergo unfolding followed by aggregation and precipitation . Protein unfolding is an endothermic process whereas aggregation and precipitation are exothermic processes.…”