Protein Stability During Freezing Separation of Stresses and Mechanisms of Protein Stabilization.pdf

Pharmaceutical Development and Technology, 12:505–523, 2007 Copyright ? Informa Healthcare USA, Inc. ISSN: 1083-7450 print / 1097-9867 online DOI: 10.1080/10837450701481157 505 LPDT Protein Stability During Freezing: Separation of Stresses and Mechanisms of Protein Stabilization Protein Stability During Freezing Bakul S. Bhatnagar School of Pharmacy, U-3092, University of Connecticut, Storrs, CT, USA College of Pharmacy, University of Minnesota, Minneapolis, MN, USA Robin H. Bogner and Michael J. Pikal School of Pharmacy, U-3092, University of Connecticut, Storrs, CT, USA Institute of Materials Science, University of Connecticut, Storrs, CT, USA Although proteins are often frozen during processing or freeze- dried after formulation to improve their stability, they can undergo degradation leading to losses in biological activity during the pro- cess. During freezing, the physical environment of a protein changes dramatically leading to the development of stresses that impact pro- tein stability. Low temperature, freeze-concentration, and ice forma- tion are the three chief stresses resulting during cooling and freezing. Because of the increase in solute concentrations, freeze-concentra- tion could also facilitate second order reactions, crystallization of buffer or non-buffer components, phase separation, and redistribu- tion of solutes. An understanding of these stresses is critical to the determination of when during freezing a protein suffers degradation and therefore important in the design of stabilizer systems. With the exception of a few studies, the relative contribution of various stresses to the instability of frozen proteins has not been addressed in the freeze-drying literature. The purpose of this review is to describe the various stages of freezing and examine the consequences of the various stresses developing during freezing on protein stability and to assess their relative contribution to the destabilization process. The ongoing debate on thermody