Li Jinghan, Lin Xinhao, Zhen Zixuan
Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, United States.
Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269, United States.
Eur J Pharm Biopharm. 2025 Sep;214:114764. doi: 10.1016/j.ejpb.2025.114764. Epub 2025 Jun 7.
Freezing is a common unit operation during the processing and storage of therapeutic proteins. Upon freezing, protein molecules and other solutes are excluded from the ice crystals, resulting in the formation of a freeze concentrated solution (FCS). Protein destabilization can be prevented due to the low temperature and the formation of a viscous FCS. However, the changes in temperature, pH, salt concentration, and the formation of ice-solution interfaces can induce protein destabilization. The current paper reviews the stresses associated with freezing and thawing and discusses the stabilization strategies. At subambient conditions, protein unfolding is thermodynamically favored when the solution is cooled below the cold denaturation temperature. The formation of a viscous FCS, specifically at a temperature below the glass transition temperature (T'), immobilizes and stabilizes the proteins. Protein can adsorb at the ice/solution interface, followed by unfolding and aggregation. Therefore, freezing and thawing rates need to be carefully controlled to minimize the ice surface area (due to the formation of small ice crystals) and to avoid ice recrystallization. Besides, stabilizers, including sugars, surfactants, and amino acids, are added as stabilizers (cryoprotectants) to prevent protein destabilization upon freezing and frozen storage. In situ spectroscopic (infrared, Raman, and solid-state NMR) and scattering (X-ray and neutron) techniques provide useful insights into protein stability and phase transitions of excipients in frozen solutions. Finally, we discuss the importance of the freezing process and excipient selection in the successful lyophilization of protein formulations. The review paper comprehensively discusses the challenges during the cryopreservation of therapeutic proteins and provides valuable insights into the critical stabilizers in frozen protein solutions.
冷冻是治疗性蛋白质加工和储存过程中常见的单元操作。冷冻时，蛋白质分子和其他溶质被排除在冰晶之外，导致形成冷冻浓缩溶液（FCS）。由于低温和粘性FCS的形成，可以防止蛋白质不稳定。然而，温度、pH值、盐浓度的变化以及冰-溶液界面的形成会导致蛋白质不稳定。本文综述了与冷冻和解冻相关的应力，并讨论了稳定化策略。在低于环境温度的条件下，当溶液冷却至低于冷变性温度时，蛋白质解折叠在热力学上是有利的。粘性FCS的形成，特别是在低于玻璃化转变温度（T'）的温度下，会固定并稳定蛋白质。蛋白质可吸附在冰/溶液界面，随后发生解折叠和聚集。因此，需要仔细控制冷冻和解冻速率，以最小化冰表面积（由于形成小冰晶）并避免冰的重结晶。此外，添加包括糖、表面活性剂和氨基酸在内的稳定剂作为保护剂（冷冻保护剂），以防止蛋白质在冷冻和冷冻储存时不稳定。原位光谱（红外、拉曼和固态核磁共振）和散射（X射线和中子）技术为冷冻溶液中蛋白质稳定性和辅料的相变提供了有用的见解。最后，我们讨论了冷冻过程和辅料选择在蛋白质制剂成功冻干中的重要性。这篇综述文章全面讨论了治疗性蛋白质冷冻保存过程中的挑战，并为冷冻蛋白质溶液中的关键稳定剂提供了有价值的见解。
