Thermodynamic Basis of Sugar-Dependent Polymer Stabilization: Informing Biologic Formulation Design

TL;DR

This study develops a thermodynamic framework to understand how four sugars ($\alpha$-glucose, $\beta$-fructose, trehalose, and sucrose) modulate the stability of simplified protein-like polymers (HP and CP). Using replica-exchange umbrella sampling, PMF decomposition, preferential interaction coefficients, and local mixing entropy, it reveals a concentration-dependent trend: sugars stabilize folded states at low concentrations but can promote unfolding at higher concentrations, with disaccharides generally exerting stronger effects than monosaccharides and mixtures sometimes outperforming pure sugars due to entropic contributions. The work shows that polymer–sugar interactions, polymer–water interactions, and entropy (notably polymer–solvent interaction entropy) jointly govern stability, and that local mixing entropy can serve as a practical proxy for entropic stabilization in formulation design. The findings provide mechanistic insight for rationalizing excipient selection in biologics and highlight the nuanced role of sugar structure, concentration, and solvent entropy in protein formulation stability, with potential relevance to cellular crowding as well.

Abstract

The stabilization of macromolecules is fundamental to developing biological formulations, such as vaccines and protein therapeutics. In this study, we employ coarse grained polymer models to investigate the impact of four sugars: $α$-glucose, $β$-fructose, trehalose, and sucrose on macromolecule stability. Free energy decomposition and preferential interaction analysis indicate that polymer-sugar interactions favor folding at low concentrations while driving unfolding at higher concentrations. In contrast, the polymer-solvent soft interaction entropy consistently favors unfolding across all sugar concentrations under study. At low sugar concentrations, polymer-solvent interactions predominantly govern stabilization, whereas at higher concentrations, entropic penalties dictate polymer stability. Local mixing entropy demonstrates that binary sugar mixtures introduce entropic contributions that preferentially stabilize the folded state. These findings contribute to a more nuanced understanding of sugar-based excipient stabilization mechanisms, offering guidance for the rational design of stable biological formulations.

Figures (10)

• Figure 1: (a) The polymer models considered in the current study. The colors of the beads correspond to their partial charges: purple $\rightarrow$ 0, silver $\rightarrow$ +0.5, yellow $\rightarrow$ -0.5, (b) 2D structures of the sugars considered in this study, (c) Representative polymer conformations: Top -- fully collapsed (I), hairpin (II), and unfolded state (III) for HP; Bottom -- hairpin (I) and unfolded state (II) for CP.
• Figure 2: Hydrophobic polymer PMFs and free energy of hydrophobic polymer unfolding in different excipient solutions. Red -- $\alpha$-glucose, cyan -- $\beta$-fructose, blue -- trehalose, green -- sucrose. Increasing shading corresponds to increasing concentration of sugars. Increasing additive concentration is denoted by increased shading (light to dark). Error bars were estimated from bootstrapping (100 samples).
• Figure 3: Charged polymer PMFs and free energy of charged polymer unfolding in different excipient solutions. Red -- $\alpha$-glucose, cyan -- $\beta$-fructose, blue -- trehalose, green -- sucrose. Increasing shading corresponds to increasing concentration of sugars. Increasing additive concentration is denoted by increased shading (light to dark). Error bars were estimated from bootstrapping (100 samples). Hatching patterns are used to differentiate CP results from HP.
• Figure 4: The preferential interaction coefficients for the folded and unfolded state of (a) HP and (b) CP in different sugar solutions. The cut-off separating the local and bulk domains is set to 1.2 nm. Refer to SI Figs. S2 and S3 for the variation of $\Gamma$ with the choice of this cut-off. Mean $\Gamma_{ps}$ of the last four 20 ns simulation trajectory blocks and the standard error of those means are reported.
• Figure 5: Decomposition of HP unfolding free energy contributions in (a) $\alpha$-glucose, (b) $\beta$-fructose, (c) trehalose, and (d) sucrose solutions. Positive values make the unfolding in the presence of sugar molecules less favorable than in pure water. Increasing additive concentration is denoted by increased shading (light to dark).