Rational design of protective agents and processes for the stabilization of biologicals (Cryopreservation).

다국어 초록 (Multilingual Abstract)

The work compiled in this thesis consists of a range of topics related to the stabilization of biological materials. The chief goal of this work is to further the fundamental understanding of the protection of biological systems under dry or low-temperature conditions. Contributions in this area would help advance the development of superior protective formulations and processes.
Early work involved the measurement of many fundamental thermophysical properties of trehalose and its aqueous solutions. These data included: viscosity, glass transition temperature (T_g), molar volume, x-ray diffraction pattern, solubility, freezing point depression, and the heat of solution.
The ubiquity of trehalose in examples of preservation found both in nature and in the literature of biostabilization posed the question as to why trehalose appears, in many cases, to have a greater protective efficacy than other disaccharides. The prevailing hypotheses of stabilization, known as the water replacement and vitrification hypotheses, imply that the most important chemical properties are the hydrogen bonding capacity and T_g, respectively. These quantities were calorimetrically determined for trehalose and other structurally similar saccharides. Results indicated a correlation between these quantities, thereby reconciling the aforementioned hypotheses for the disaccharides. Because of their physiological importance, studies of aqueous electrolytes were a natural extension of the work with trehalose. This work involved examination of a theoretical model that predicts the T_g of dilute electrolyte solutions. Experimental results here indicate that the increase in T_g was much less than that predicted by theory. Further studies on aqueous electrolytes showed that many electrolyte/saccharide systems were found to deviate significantly from Walden's rule. The deviation was rationalized by the concept of local heterogeneities in the solvent that permitted greater ionic mobility than that predicted by Stokes' Law. This unexpected molecular mobility has implications for the long-term stabilization of biomaterials.
The synthesis of the results of the studies of trehalose and its mixtures with electrolytes resulted in the development of a superior protective formulation: trehalose/borate mixtures. The thermophysical properties of these mixtures were characterized and their feasibility was demonstrated on a labile enzyme, lactate dehydrogenase. In comparison with trehalose, dried enzyme formulations that included borate exhibited longer storage stabilities under conditions of high temperature and high relative humidity.