Protein Stability During Freezing: Separation of Stresses and Mechanisms of Protein Stabilization

Abstract

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.

Functional properties of missense variants of human tryptophan hydroxylase 2

“…The difference is explained by the use of a more effective cell lysis procedure and avoidance of repeated freeze/thaw cycles of the cells. Freezing and thawing is a wellstudied stressor of protein stability that promotes denaturation and aggregation [Bhatnagar et al, 2007;Cao et al, 2003].…”

Effect of freezing methods on the properties of lyophilized porous silk fibroin membranes

“…The denaturation during freezing is greater when proteins are frozen under conditions that generate a relatively large ice surface area. The observed results indicate that the freezing rate is an important factor that determines the integrity of lyophilized porous SF membranes 11,23 .…”

“…Low temperature and ice formation are some of the stresses resulting during freezing 23 . The differences observed in the integrity of the porous SF membranes may be explained by the influence of the freezing rate.…”

Identification of Clinical Biomarkers for Pre-Analytical Quality Control of Blood Samples

“…Repeated freeze-thawing cycling is known to affect the stability of biological samples and, notably, to induce conformational changes in proteins that may ultimately lead to aggregation or degradation. [26][27][28] However, previous studies have shown that some molecules are stable in serum and plasma under conditions of repeated freezing and thawing, even up to 10 cycles. 29,30 The present study showed that even one freeze-thaw cycle led to changes in the concentrations of four analytes (AST, GGT, LDH, and BUN); the change in plasma LDH concentration was similar to that seen FIG.…”