(546d) Preservation of Protein Stability and Activity By Confinement in Michael-Type Addition Polyethylene Glycol Hydrogels

A robust formulation for preserving the structure and stability of protein therapeutics against chemical and physical instabilities is a challenge towards their biopharmaceutical development. Structure and stability are highly influenced by the local environment of the protein; highly concentrated solutions are considered to “crowd” a protein, whereas physical entrapment within a small volume is considered to “confine” a protein. While protein stability can be promoted in a crowded environment, protein structure under confinement, such as in hydrogels and the intracellular space, is much less understood. For example, could confinement in a hydrogel be optimized to preserve protein structure and function? To begin to answer this question, lysozyme and alcohol dehydrogenase were utilized as model proteins that were confined in polyethylene glycol (PEG) hydrogels crosslinked by an acrylate-thiol Michael-Type addition reaction. The conformational stability of proteins was studied by intrinsic fluorescent spectroscopy and enzymatic activity measurements. By assuming a two-state unfolding mechanism, the unfolding free energy (ΔG_unfolding) and the change in the accessible surface area of the protein — i.e., the m-value or the difference in the accessible surface area between the unfolded and the native state of the protein — were calculated from solvent denaturation curves using a linear extrapolation method.

We hypothesized that confinement would stabilize protein structure, and thus be correlated with higher ΔG_unfolding values; interestingly, we found that the m-values instead correlated with confinement. We did not observe any significant difference in ΔG_unfolding of proteins in free buffer, crowded (PEG-OH), and confined spaces (PEG-hydrogel). However, proteins confined in hydrogel had a lower m-value magnitude, suggesting that proteins were less unfolded in confinement. We suggest that the conformational stabilization arose from a change in the entropy of unfolding. The unfolded protein structure is less favorable in confined space vs free buffer solution due to excluded volume and spatial barriers limiting the number of possible unfolded conformations. Also, we observed that under mild stressed conditions such as incubation at 50°C, lysozyme activity was equal to or greater in confinement compared with free and crowded spaces.

Therefore, when comparing crowded vs confined environments, we found that the m-value is more affected when the protein is confined compared with the free protein in a buffer or crowded solution due to the physical characteristics of each environment. Ongoing studies are investigating additional proteins that are unstable and have therapeutic significance. Ultimately, a deeper understanding of protein conformational stability in these different microenvironments can provide for rationally-designed technologies for the improved formulation, storage and delivery of unstable protein therapeutics.