Hemoglobin has been previously shown to unfold during freeze drying when lyophilized from formulations that undergo freeze-concentration induced phase separation (Heller et al. 1997. Biotechnol Frog 13:590-596). In this report, we show that such damage may be avoided using kinetic strategies to arrest the phase separation. By rapidly cooling samples during liquid nitrogen spray-freeze drying, the time that the formulation spends in temperature regimes (ca. -3 to -23 degrees C) in which phase separation is both thermodynamically favorable and kinetically realizable is minimized. Increased protein damage with decreasing cooling rates and/or longer annealing periods at -7 degrees C is observed by FTIR spectroscopy. Phase separation and concomitant protein damage may also be avoided by addition of mannitol at concentrations sufficient to cause crystallization. Mannitol crystals segregate the freeze concentrated solution into microscopic domains that block propagation and nucleation of phase separating events. Addition of noncrystallizing sugars, such as sucrose and trehalose, or nonionic surfactants, such as Tween 80 and Triton X-100, has little protective effect against phase separation induced damage during freezing drying.