Heller M C, Carpenter J F, Randolph T W
Department of Chemical Engineering, University of Colorado at Boulder, Boulder, Colorado 80309-0424, USA.
Biotechnol Bioeng. 1999 Apr 20;63(2):166-74. doi: 10.1002/(sici)1097-0290(19990420)63:2<166::aid-bit5>3.0.co;2-h.
Hemoglobin has been previously shown to unfold during freeze drying when lyophilized from formulations that undergo freeze-concentration induced phase separation (Heller et al. 1997. Biotechnol Prog 13:590-596). In this report, we show that such damage may be avoided using kinetic strategies to arrest the phase separation. By rapidly cooling samples during liquid nitrogen spray-freeze drying, the time that the formulation spends in temperature regimes (ca. -3 to -23 degrees C) in which phase separation is both thermodynamically favorable and kinetically realizable is minimized. Increased protein damage with decreasing cooling rates and/or longer annealing periods at -7 degrees C is observed by FTIR spectroscopy. Phase separation and concomitant protein damage may also be avoided by addition of mannitol at concentrations sufficient to cause crystallization. Mannitol crystals segregate the freeze concentrated solution into microscopic domains that block propagation and nucleation of phase separating events. Addition of noncrystallizing sugars, such as sucrose and trehalose, or nonionic surfactants, such as Tween 80 and Triton X-100, has little protective effect against phase separation induced damage during freezing drying.
先前的研究表明，当从经历冷冻浓缩诱导相分离的制剂中冻干时，血红蛋白会发生解折叠（Heller等人，1997年。《生物技术进展》13:590 - 596）。在本报告中，我们表明可以使用动力学策略来阻止相分离，从而避免这种损伤。通过在液氮喷雾冷冻干燥过程中快速冷却样品，制剂在相分离在热力学上有利且动力学上可实现的温度范围（约 - 3至 - 23摄氏度）内停留的时间被最小化。通过傅里叶变换红外光谱法观察到，随着冷却速率降低和/或在 - 7摄氏度下退火时间延长，蛋白质损伤增加。通过添加足以引起结晶的浓度的甘露醇，也可以避免相分离和伴随的蛋白质损伤。甘露醇晶体将冷冻浓缩溶液分隔成微观区域，阻止相分离事件的传播和成核。添加非结晶糖，如蔗糖和海藻糖，或非离子表面活性剂，如吐温80和曲拉通X - 100，对冷冻干燥过程中相分离诱导的损伤几乎没有保护作用。
