Freeze-concentration separates proteins and polymer excipients into different amorphous phases

Purpose. To study the miscibility of proteins and polymer excipients in frozen solutions and freeze-dried solids as protein formulation models. Methods. Thermal profiles of frozen solutions and freeze-dried solids containing various proteins (Iysozyme, ovalbumin, BSA), nonionic polymers (Ficoll, polyvinylpyrrolidone [PVP]), and salts were analyzed by differential scanning calorimetry (DSC). The polymer miscibility was determined from the glass transition temperature of maximally freeze-concentrated solute (T-g') and the glass transition temperature of freeze-dried solid (T-g). Results. Frozen Ficoll or PVP 40k solutions showed T-g' at -22 degreesC, while protein solutions did not show an apparent T-g'. AII the protein and nonionic polymer combinations (5% w/w, each) were miscible in frozen solutions and presented single T-g's that rose with increases in the protein ratio. Various salts concentration-dependently lowered the single T-g's of the proteins and Ficoll combinations maintaining the mixed amorphous phase. In contrast, some salts induced the separation of the proteins and PVP combinations into protein-rich and PVP-rich phases among ice crystals. The T-g's of these polymer combinations were jump-shifted to PVP's intrinsic T-g' at certain salt concentrations. Freeze-dried solids showed varied polymer miscibilities identical to those in frozen solutions. Conclusions. Freeze-concentration separates same combinations of proteins and nonionic polymers into different amorphous phases in a frozen solution. Controlling the polymer miscibility is important in designing protein formulations.