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 process. During freezing, the physical environment of a protein changes dramatically leading to the development of stresses that impact protein stability. Low temperature, freeze-concentration, and ice formation are the three chief stresses resulting during cooling and freezing. Because of the increase in solute concentrations, freeze-concentration could also facilitate second order reactions, crystallization of buffer or non-buffer components, phase separation, and redistribution 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 thermodynamic versus kinetic mechanisms of stabilization in frozen environments and the current state of knowledge concerning those mechanisms are also reviewed in this publication. An understanding of the relative contributions of freezing stresses coupled with the knowledge of cryoprotection mechanisms is central to the development of more rational formulation and process design of stable lyophilized proteins.

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