β-Relaxation governs protein stability in sugar-glass matrices

Abstract

The stabilizing effect of sugar-glass matrix materials for freeze-drying proteins or nucleic acids has been variously ascribed to the thermodynamic effect of ‘water replacement’ by sugar molecules or to the kinetic effect of slowed α relaxation associated with sugar matrix vitrification. While evidence for each of these hypotheses exists, we show that neither can adequately account for the observed stabilization of proteins embedded in sugar-glasses. Instead, we find firm evidence that protein stability in these glasses is directly linked to high frequency β relaxation processes of the sugar matrix. Specifically, we observe that when the β relaxation time, τ_β, of sugar-glasses is increased with antiplasticizing additives, protein stability increases in linear proportion to the increase in τ_β, even though these same additives simultaneously decrease the glass transition temperature, T_g, and the α relaxation time, τ_α, of the sugar matrix materials. Moreover, we find that while sugars ‘replace’ water by stabilizing protein native-like conformation in the dry state, the resulting enhanced protein conformational stability does not have a significant impact on the degradation rate of the proteins in sugar-glasses. We discuss implications of these findings for the fundamental physics of glass formation and for effective engineering of protein stabilizing glasses through the modification of τ_β.