EFFECTS OF PHASE-SEPARATING SYSTEMS ON LYOPHILIZED HEMOGLOBIN

Polymer liquid-liquid two-phase systems offer a unique opportunity to study the mechanisms of protein stabilization during freezing and free ze-drying. Fourier transform infrared spectroscopy was used to monitor the structural integrity of recombinant hemoglobin frozen and lyophil ized in the separated phases of a polyethylene glycol (PEG)-dextran sy stem. Protein in each phase of an equilibrated biphasic PEG-dextran sy stem experiences similar levels of structural protection against freez ing stresses despite large differences in polymer concentration. This result further demonstrates previous suggestions that proteins are pro tected during freezing by the preferential exclusion mechanism. There are, however, distinct differences in the level of structural protecti on that polymers in equilibrium phases provide to proteins during lyop hilization, emphasizing that the mechanisms of protein protection duri ng freezing and drying are fundamentally different. In addition, we pr ovide evidence that phase separation per se occurring during the cours e of the lyophilization cycle can be detrimental to the structural sta bility of a protein.