Protein formulation and lyophilization cycle design: Prevention of damage due to freeze-concentration induced phase separation

Hemoglobin has been previously shown to unfold during freeze drying when ly ophilized from formulations that undergo freeze-concentration induced phase separation (Heller et al. 1997. Biotechnol Frog 13:590-596). In this repor t, we show that such damage may be avoided using kinetic strategies to arre st the phase separation. By rapidly cooling samples during liquid nitrogen spray-freeze drying, the time that the formulation spends in temperature re gimes (ca. -3 to -23 degrees C) in which phase separation is both thermodyn amically favorable and kinetically realizable is minimized. Increased prote in damage with decreasing cooling rates and/or longer annealing periods at -7 degrees C is observed by FTIR spectroscopy. Phase separation and concomi tant protein damage may also be avoided by addition of mannitol at concentr ations sufficient to cause crystallization. Mannitol crystals segregate the freeze concentrated solution into microscopic domains that block propagati on and nucleation of phase separating events. Addition of noncrystallizing sugars, such as sucrose and trehalose, or nonionic surfactants, such as Twe en 80 and Triton X-100, has little protective effect against phase separati on induced damage during freezing drying. (C) 1999 John Wiley & Sons, Inc.