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 freeze-drying. Fourier transform infrared spectroscopy was used to monitor the structural integrity of recombinant hemoglobin frozen and lyophilized in the separated phases of a polyethylene glycol (PEG)-dextran system. Protein in each phase of an equilibrated biphasic PEG-dextran system experiences similar levels of structural protection against freezing stresses despite large differences in polymer concentration. This result further demonstrates previous suggestions that proteins are protected during freezing by the preferential exclusion mechanism. There are, however, distinct differences in the level of structural protection that polymers in equilibrium phases provide to proteins during lyophilization, emphasizing that the mechanisms of protein protection during freezing and drying are fundamentally different. In addition, we provide evidence that phase separation per se occurring during the course of the lyophilization cycle can be detrimental to the structural stability of a protein.