Local Minimum in Fragility for Trehalose/Glycerol Mixtures: Implications for Biopharmaceutical Stabilization

Abstract

Approximately a decade ago it was observed that adding a small amount (5 wt %) of glycerol to trehalose could substantially improve the stability of enzymes stored in these glasses even though the final glass transition temperature (Tg) was reduced by ∼20 K. This finding inspired great interest in the fast dynamics of dehydrated trehalose/glycerol mixtures, leading to the observation that suppression of fast dynamics was optimal in the presence of ∼5 wt % of glycerol. It was also recognized that the fast dynamics should, in theory, be related to the fragility of these glass formers, but experimental confirmation of this hypothesis has been lacking for trehalose/glycerol mixtures or any other mixtures of this nature. In the present study a dynamic mechanical analyzer (DMA) was used to determine both the Tg and the kinetic fragility index (m) of trehalose/glycerol mixtures within the mass fraction range of 80-100 wt % of trehalose. It was found that the fragility index correlated with the mass fraction of trehalose in a nonmonotonic manner, with a local minimum between 87.5 and 95 wt % of trehalose, whereas the composition dependence of Tg was found to follow a Gordon-Taylor-like relationship, with no local minimum. The composition of 5-12.5 wt % glycerol in trehalose thus yielded a matrix that maximized the strong glass-forming contribution of glycerol, while minimizing its Tg lowering effect. This quantitative evidence supports speculation about the fragility characteristics of these mixtures that has been ongoing for the past decade. The DMA-based Tg and fragility determination method developed in this study represents a new approach for identifying optimal compositions for preservation of biologics.

Antiplasticization of Polymer Materials: Structural Aspects and Effects on Mechanical and Diffusion-Controlled Properties

“…Weng and Elliott [141] have used dynamic mechanical tests (DMA) on trehalose/glycerol mixtures to obtain mechanical spectra for the construction of master curves using the Williams-Landel-Ferry (WLF) shift factor a T method for the time/temperature superposition principle. In dynamic mechanical spectroscopy this can be expressed in terms of a horizontal shift for the variation of the loss modulus with a frequency over a wide range of temperatures, i.e., E"(ω, T) = E"(a T ω, Tr), where Tr is an arbitrary reference temperature.…”

Molecular Packing, Hydrogen Bonding, and Fast Dynamics in Lysozyme/Trehalose/Glycerol and Trehalose/Glycerol Glasses at Low Hydration

“…14 Yet, there are still many open questions, in particular on how water, polyols, and sugars interact with each other and/or with proteins in complex glassy mixtures. Low-molecular-weight compounds such as water, glycerol (C 3 H 8 O 3 ), or sorbitol (C 6 H 14 O 6 ), are often regarded as plasticizers of carbohydrate and protein matrices, [15][16][17][18][19][20][21][22][23][24][25][26] since the addition of these small molecules (sometimes referred to as diluents 17,20,21,23,24,[26][27][28][29] ) usually decreases their glass transition temperature, T g , as well as their elastic moduli, and increases the free volume and the water and oxygen permeabilities. However, several studies have demonstrated that they can also act as antiplasticizers, especially at low temperatures and concentrations.…”

“…28 More recently, Cicerone and Douglas confirmed such a relationship for a large series of proteins mixed with either trehalose or sucrose, and suggested that the degradation of proteins in carbohydrate glasses is governed by small amplitude, fast motions (β-relaxation processes) of the glassy matrix rather than by structural, α relaxation, 29 thus showing that the antiplasticization of fast motions at sub-T g temperatures is particularly relevant for biostabilization purposes. 26,29 The pioneering results of Cicerone and Soles 28 have triggered many studies trying to further understand the antiplasticizing effect of glycerol on trehalose, 25,[32][33][34][35][36][37] as well as the enhanced stability that such mixtures may confer to embedded proteins. 29,[38][39][40] The antiplasticization of trehalose motions by glycerol has been observed at various concentrations and temperatures in experimental and numerical studies.…”

“…%. 25 Discrepancies also emerged from studies on proteins in TG mixtures: the < u 2 > of RNase was minimum at a glycerol concentration of 12.2 wt. %.…”

Dry Preservation of Spermatozoa: Considerations for Different Species

“…23 However, motion within glasses involves a variety of molecular dynamics, ranging from atomic vibrations, motion of cages, and secondary or b-relaxation, including Johari-Goldstein ( JG) relaxation, to the fully cooperative a-relaxation associated with the transition into a glassy state. [24][25][26] For example, loss of enzymatic activity of proteins embedded in sugar glasses has been shown to not directly correlate with a-relaxation, but instead has been demonstrated to be a function of the fast highfrequency (THz) dynamics or fast b-relaxation of the solvent matrix. 25 The relationship between secondary relaxation phenomena and preservation outcome has not been well studied in more complex cellular systems.…”