MANIPULATION OF LYOPHILIZATION-INDUCED PHASE-SEPARATION - IMPLICATIONS FOR PHARMACEUTICAL PROTEINS

Lyophilization, or freeze-drying, of pharmaceutical proteins ia often the only processing method that provides requisite long-term product s tability. Freezing and drying, however, can cause acute damage to prot eins. To alleviate damage, formulations frequently include protein sta bilizers (often polymers and/or sugars), as well as buffering salts an d ''inert'' bulking agents. While great efforts are placed on developi ng a formulation and suitable lyophilization cycle, incompatibilities among components through freezing and drying have been almost complete ly ignored. We demonstrate that solutions of poly(ethylene glycol) (PE G) and dextran, initially below critical concentrations for phase sepa ration, do indeed experience a liquid-liquid phase separation induced by freeze concentration during the lyophilization cycle. The separatio n is shown to evolve with annealing at -7 degrees C and can be effecti vely inhibited simply by replacing NaCl with KCI in the formulation bu ffer. In addition, we show that phase separation causes unfolding of a model protein, recombinant hemoglobin, when freeze-dried in the PEG/d extran system. When the phase separation is averted by switching to KC l, the protein structural damage is also avoided. Measurements of pH i n the frozen solutions show that the structural damage is not a result of pH changes. We suggest that KCl forms a glass with rapid cooling w hich kinetically prevents the phase separation and thus the protein st ructural damage.