Heterogeneity in Desiccated Solutions: Implications for Biostabilization

Abstract

Biopreservation processes such as freezing and drying inherently introduce heterogeneity. We focused on exploring the mechanisms responsible for heterogeneity in isothermal, diffusively dried biopreservation solutions that contain a model protein. The biopreservation solutions used contained trehalose (a sugar known for its stabilization effect) and salts (LiCl, NaCl, MgCl2, and CaCl2). Performing Fourier transform infrared spectroscopy analysis on the desiccated droplets, spatial distributions of the components within the dried droplet, as well as their specific interactions, were investigated. It was established that the formation of multiple thermodynamic states was induced by the spatial variations in the cosolute concentration gradients, directly affecting the final structure of the preserved protein. The spatial distribution gradients were formed by two competing flows that formed within the drying droplet: a dominant peripheral flow, induced by contact line pinning, and the Marangoni flow, induced by surface tension gradients. It was found that the changes in cosolute concentrations and drying conditions affected the spatial heterogeneity and stability of the product. It was also found that trehalose and salts had a synergistic stabilizing effect on the protein structure, which originated from destructuring of the vicinal water, which in turn mediated the interactions of trehalose with the protein. This interaction was observed by the change in the glycosidic CO, and the CH stretch vibrations of the trehalose molecule.

Are the Soft, Liquid-Like Structures Detected around Bacteria by Ambient Dynamic Atomic Force Microscopy Capsules?

“…In any case, it was always observed that a clear peripheral ring was formed, indicating a higher concentration of mass in that area, together with a clearer inner zone. Evaporating droplets carrying hydrophobic inorganic or organic particles, including microorganisms, have been shown to give rise to rings (26). This phenomenon is usually referred as a "coffee ring" effect and was deeply studied by Deegan et al (6).…”

Effect of trehalose on protein structure

“…86,87 Simultaneous use of trehalose and ionic salts has been shown to have a synergistic effect on the freeze-drying stability of hen egg white lysozyme. 88 This stabilization results from the interaction of trehalose with the protein, as confirmed by changes in the CO stretching vibration, resulting from the destructuring of the surrounding water molecules. This study also serves as a reminder that, during freeze-drying, the mixture does not remain homogeneous and a concentration gradient of components of the lyophilization mixture is set up.…”

“…This study also serves as a reminder that, during freeze-drying, the mixture does not remain homogeneous and a concentration gradient of components of the lyophilization mixture is set up. 88 Proposing any hypothesis needs to take into account this often-ignored fact. Recent studies on structural relaxation times with human growth hormone have shown only a weak correlation between the secondary structure and aggregation propensity of the protein.…”

Effects of Heat Shock Treatment on Enzymatic Proteolysis for LC-MS/MS Quantitative Proteome Analysis

“…The heat shock inactivate proteins by virtue of heat to break the intermolecular hydrogen bonds, polar, and van der Waals interactions to make proteins lose their secondary and tertiary structure. 27 This results in protein denaturation in a reproducible and irreversible manner, facilitates trypsin cleavage, and prevents protein degradation, thereby leading to increased identification of protein and peptide IDs in plasma, tissue, neutrophils, and yeasts. 13,14,28 This is an interesting additional use of the instrument, which otherwise is mainly used to denature degradation enzymes irreversibly along with other proteins to preserve the proteome intact.…”