Application of a thermodynamic model to the prediction of phase separations in freeze-concentrated formulations for protein lyophilization

Many of the compounds considered for use in pharmaceutical formulations dem onstrate incompatibilities with other components at high enough concentrati ons, including pairs of polymers, polymers and salts, or even proteins in c ombination with polymers, salts, or other proteins. Freeze concentration ca n force solutions into a region where incompatibilities between solutes wil l manifest as the formation of multiple phases. Such phase separation compl icates questions of the stability of the formulation as well as labile comp onents, such as proteins. Yet, phase separation events are difficult to ide ntify by common formulation screening methods. In this report, we use the o smotic virial expansion model of Edmond and Ogston (1) to describe phase-se parating behavior of ternary aqueous polymer solutions. Second osmotic viri al coefficients of polyethylene glycol 3350 (PEG) and dextran T500 were mea sured by light scattering. Assuming an equilibrium between ice and water in the freeze-concentrated solution, a degree of freeze concentration can be estimated, which, when combined with the phase separation spinodal, describ es a "phase separation envelope" in which phase separation tendencies can b e expected in the frozen solution. The phase separation envelope is bounded at low temperatures by the glass transition temperature of the freeze-conc entrated solution. Scanning electron microscopic images and infrared spectr oscopy of protein structure are provided as experimental evidence of the ph ase separation envelope in a freeze dried system of PEG, dextran, and hemog lobin. (C) 1999 Academic Press.