Roy Ipsita, Gupta Munishwar Nath
Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India.
Biotechnol Appl Biochem. 2004 Apr;39(Pt 2):165-77. doi: 10.1042/BA20030133.
Freeze-drying (lyophilization) removes water from a frozen sample by sublimation and desorption. It can be viewed as a three-step process consisting of freezing, primary drying and secondary drying. While cryoprotectants can protect the protein from denaturation during early stages, lyoprotectants are needed to prevent protein inactivation during drying. The structural changes as a result of freeze-drying have been investigated, especially by FTIR (Fourier-transform IR) spectroscopy. In general, drying results in a decrease of alpha-helix and random structure and an increase in beta-sheet structure. In the case of basic fibroblast growth factor and gamma-interferon, enhanced FTIR showed large conformational changes and aggregation during freeze-drying, which could be prevented by using sucrose as a lyoprotectant. It is now well established that structural changes during freeze-drying are responsible for low activity of freeze-dried powders in nearly anhydrous media. Strategies such as salt activation can give 'activated' enzyme powders, e.g. salt-activated thermolysin-catalysed regioselective acylation of taxol to give a more soluble derivative for therapeutic use. In the presence of moisture, freeze-dried proteins can undergo disulphide interchange and other reactions which lead to inactivation. Such molecular changes during storage have been described for human insulin, tetanus toxoid and interleukin-2. Some successful preventive strategies in these cases have also been mentioned as illustrations. Finally, it is emphasized that freeze-drying is not an innocuous process and needs to be understood and used carefully.
冷冻干燥（冻干）通过升华和解吸从冷冻样品中去除水分。它可被视为一个由冷冻、一次干燥和二次干燥组成的三步过程。虽然冷冻保护剂可在早期阶段保护蛋白质免受热变性，但需要冻干保护剂来防止蛋白质在干燥过程中失活。人们已经对冷冻干燥导致的结构变化进行了研究，尤其是通过傅里叶变换红外光谱（FTIR）。一般来说，干燥会导致α-螺旋和无规结构减少，β-折叠结构增加。就碱性成纤维细胞生长因子和γ-干扰素而言，增强傅里叶变换红外光谱显示在冷冻干燥过程中发生了大的构象变化和聚集，而使用蔗糖作为冻干保护剂可以防止这种情况。现在已经明确，冷冻干燥过程中的结构变化是导致冻干粉末在几乎无水介质中活性较低的原因。盐活化等策略可以得到“活化”的酶粉末，例如盐活化的嗜热菌蛋白酶催化紫杉醇的区域选择性酰化反应，以得到一种更易溶的衍生物用于治疗。在有水分存在的情况下，冷冻干燥的蛋白质会发生二硫键交换和其他导致失活的反应。已经描述了人胰岛素、破伤风类毒素和白细胞介素-2在储存过程中的这种分子变化。在这些情况下还提到了一些成功的预防策略作为示例。最后，强调冷冻干燥并非无害过程，需要谨慎理解和使用。
