Freezing-Induced Phase Separation and Spatial Microheterogeneity in Protein Solutions

Abstract

Amid decades of research, the basic mechanisms of lyo-/cryostabilization of proteins and more complex organisms have not yet been fully established. One major bottleneck is the inability to probe into and control the molecular level interactions. The molecular interactions are responsible for the significant differences in the outcome of the preservation processes. (1) In this communication, we have utilized confocal Raman microspectroscopy to quantify the freezing-induced microheterogeneity and phase separation (solid and liquid) in a frozen solution composed of a model protein (lysozyme) and a lyo-/cryoprotectant (trehalose), which experienced different degrees of supercooling. Detailed quantitative spectral analysis was performed across the ice, the freeze-concentrated liquid (FCL) phases, and the interface region between them. It was established that the characteristics of the microstructures observed after freezing depended not only on the concentration of trehalose in the solution but also on the degree of supercooling. It was shown that, when samples were frozen after high supercooling, small amounts of lysozyme and trehalose were occluded in the ice phase. Lysozyme preserved its native-like secondary structure in the FCL region but was denatured in the ice phase. Also, it was observed that induction of freezing after a high degree of supercooling of high trehalose concentrations resulted in aggregation of the sugar and the protein.

Frozen State Storage Instability of a Monoclonal Antibody: Aggregation as a Consequence of Trehalose Crystallization and Protein Unfolding

“…Slow formation of ice allows longer time for solutes to diffuse ahead of the ice front as well as larger crystals (4). Dong et al (21) also showed that greater fraction of protein and trehalose was occluded in the ice phase when rapid freezing occurred after a high degree of supercooling. Such freezing would result in a large number of nuclei and therefore a larger number of smaller ice crystals.…”

“…It has been shown (for a number of proteins) that as freezing proceeds and ice forms, the protein at the ice surface can undergo a marked increase in flexibility due to a loosening of the native fold and loss of secondary and tertiary structure (19)(20)(21). However, once the temperature drops further (below approximately −30°C), the mobility is lost due to the increased viscosity of the matrix.…”

A New Approach to Explore the Impact of Freeze-Thaw Cycling on Protein Structure: Hydrogen/Deuterium Exchange Mass Spectrometry (HX-MS)

“…Gabellieri and coworkers measured dramatic decreases in phosphorescence lifetime at different subfreezing temperatures, suggesting significant conformational changes (4). Later, the same authors found that proteins substantially increased their binding to 1-anilino-8-naphthalene sulfonate (ANS), an extrinsic probe to measure solvent-exposed hydrophobic surface (16,17). More recently, Schwegman et al distinguished protein at the ice-solution interface vs. protein remaining in the interstitial solution space by fluorescence microscopy, and demonstrated their alteration in secondary structure by infrared microscopy (7).…”

Effects of Solute Miscibility on the Micro- and Macroscopic Structural Integrity of Freeze-Dried Solids

“…Inclusion of some solutes into small ice crystal also induces microscopic component and physical state heterogeneity in a frozen aqueous solution. 29 The purpose of this study was to elucidate the relationship between the miscibility of amorphous solutes in frozen solutions and their structural integrity during primary drying. The individual concentrated solute mixture and their unmixed phases in a frozen solution should possess different viscosities dependent on both composition and temperature.…”