Stabilization of biologicals during freezing and drying is of great commercial importance, particularly to the pharmaceutical, medical, and food industries. Due to the complex nature of damage that occurs during processing, much work has been done to develop protective compounds for biomolecules and cells. Notable among these compounds are the saccharides. They have been found to protect proteins during freezing and drying as well as prevent membrane damage during cooling of cells. Trehalose (a-D-glucopyranosyl-a-D-glucopyranoside), a naturally occurring disaccharide of glucose, has been found to be a highly effective cryoprotectant. We have been conducting research into the thermophysical properties of trehalose and its interaction in solution with ionic species, which are ubiquitous in biological systems. Our findings have been applied to cryo- and lyo-protection of proteins and cells. We have studied the electrical conductivity of ambient and supercooled aqueous solutions of trehalose containing non-complexing ions because ionic transport has important consequences for preservation of biological systems. We have observed a remarkable increase in the Walden product (the product of viscosity and conductivity) for trehalose solutions in the presence of sodium chloride. No such effect was observed with aqueous glycerol/sodium chloride solutions. The results of molecular dynamics simulations of the diffusion of Na+ and Cl- ions in trehalose/water solutions [1] confirm that the product of viscosity and diffusion coefficient increases with the concentration of trehalose (that is, the Stokes-Einstein relation does not hold). The molecular explanation for this behavior is found in microheterogeinities close to the ions. The local environment of the ions contains more water than the bulk. One particularly interesting protectant system is that of trehalose and the complexing borate ions (added in the form of sodium tetraborate). This novel formulation forms a cross-linked network resulting in dramatic increases in glass transition temperature. Such behavior was not observed for solutions of trehalose and NaCl, KCl, or MgCl2. The combination of trehalose with borate has been the subject of several recent studies of enzyme and bacterial cell stabilization in the frozen and dried states [2,3]. In all cases the systems preserved with trehalose and borate performed significantly better than more common protectants, including trehalose alone. This improved protection was seen for vacuum dried, freeze-dried as well as frozen samples. Long-term storage of dried proteins and cells at elevated temperatures further emphasized the improvements.

[1] Miller, D.P, Conrad, P.B., Fucito, S., de Pablo, J.J, Corti, H.R., J.Phys.Chem. B., paper submitted.

[2] Miller, D. P., Anderson, R. E., de Pablo, J. J., Pharm. Res., 15, 1215-1221, 1198.

[3] Conrad, P. B., Miller, D. P., Cielenski, P. R., de Pablo, J. J., Pharm. Res., paper submitted.