Bhatnagar Bakul S, Bogner Robin H, Pikal Michael J
School of Pharmacy, U-3092, University of Connecticut, Storrs, CT 06269, USA.
Pharm Dev Technol. 2007;12(5):505-23. doi: 10.1080/10837450701481157.
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.
尽管蛋白质在加工过程中常被冷冻或在制剂后进行冻干以提高其稳定性，但它们在这个过程中仍可能发生降解，导致生物活性丧失。在冷冻过程中，蛋白质的物理环境会发生巨大变化，从而产生影响蛋白质稳定性的应力。低温、冷冻浓缩和冰晶形成是冷却和冷冻过程中产生的三个主要应力。由于溶质浓度增加，冷冻浓缩还可能促进二级反应、缓冲液或非缓冲成分的结晶、相分离以及溶质的重新分布。了解这些应力对于确定蛋白质在冷冻过程中何时发生降解至关重要，因此在稳定剂系统的设计中也很重要。除了少数研究外，冻干文献中尚未探讨各种应力对冷冻蛋白质不稳定性的相对贡献。本综述的目的是描述冷冻的各个阶段，研究冷冻过程中产生的各种应力对蛋白质稳定性的影响，并评估它们对去稳定化过程的相对贡献。本出版物还综述了关于冷冻环境中稳定化的热力学与动力学机制的 ongoing debate 以及有关这些机制的当前知识状态。了解冷冻应力的相对贡献以及冷冻保护机制的知识对于开发更合理的稳定冻干蛋白质的制剂和工艺设计至关重要。
