SURFACE DENATURATION AT SOLID-VOID INTERFACE - A POSSIBLE PATHWAY BY WHICH OPALESCENT PARTICULATES FORM DURING THE STORAGE OF LYOPHILIZED TISSUE-TYPE PLASMINOGEN-ACTIVATOR AT HIGH-TEMPERATURES
SURFACE DENATURATION AT SOLID-VOID INTERFACE - A POSSIBLE PATHWAY BY WHICH OPALESCENT PARTICULATES FORM DURING THE STORAGE OF LYOPHILIZED TISSUE-TYPE PLASMINOGEN-ACTIVATOR AT HIGH-TEMPERATURES
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DOI：
10.1023/a:1016270103863
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发表时间：
1995-01-01
影响因子：
3.7
通讯作者：
PEARLMAN, R
中科院分区：
文献类型：
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作者：
HSU, CC;NGUYEN, HM;PEARLMAN, R
在蛋白质冷冻干燥过程中，为了避免蛋白质变性，以及获得质量均一的最终产品，通常需要尽快完成冷冻步骤。我们报告了在精氨酸配制的重组组织型纤溶酶原激活剂(t-PA)的冷冻干燥过程中所做的一个相互矛盾的观察。冷冻干燥过程中的快速冷却导致冻干产品在150 mTorr氮气密封气体环境下，在50摄氏度的长期储存下产生更多乳白色颗粒。快速冷却还会导致内表面积较大的冻干蛋糕。对含有1%(w/w)残余水分和不同蛋糕表面积(0.22-1.78m(2)/g)的冷冻干燥产品的研究表明，所有冷冻干燥蛋糕都处于无定形状态，玻璃化转变温度相似(103-105摄氏度)。然而，在储存期间，冷冻干燥产品中乳白色颗粒的形成速度(通过在360至340 nm范围内对重组溶液进行紫外光密度测量来确定)与蛋糕表面积成正比。我们认为这是一种表面相关的现象，在这种现象中，冷冻干燥蛋糕的固-空界面上的蛋白质在高温下储存期间变性。在冷冻干燥过程中，冰液界面的不可逆变性不太可能发生，因为反复冷冻/解冻对蛋白质没有任何不利影响。红外光谱分析不能确定蛋白质在冷冻干燥后，在固-空界面上是否仍然是天然的。
During protein lyophilization, it is common practice to complete the freezing step as fast as possible in order to avoid protein denaturation, as well as to obtain a final product of uniform quality. We report a contradictory observation made during lyophilization of recombinant tissue-type plasminogen activator (t-PA) formulated in arginine. Fast cooling during lyophilization resulted in a lyophilized product that yielded more opalescent particulates upon long term storage at 50 degrees C, under a 150 mTorr nitrogen seal gas environment. Fast cooling also resulted in a lyophilized cake with a large internal surface area. Studies on lyophilized products containing 1% (w/w) residual moisture and varying cake surface areas (0.22 - 1.78 m(2)/gm) revealed that all lyophilized cakes were in an amorphous state with similar glass transition temperatures (103 - 105 degrees C). However, during storage the rate of opalescent particulate formation in the lyophilized product (as determined by UV optical density measurement in the 360 to 340 nm range for the reconstituted solution) was proportional to the cake surface area. We suggest that this is a surface-related phenomenon in which the protein at the solid-void interface of the lyophilized cake denatures during storage at elevated temperatures. Irreversible denaturation at the ice-liquid interface during freezing in lyophilization is unlikely to occur, since repeated freezing/thawing did not show any adverse effect on the protein. Infrared spectroscopic analysis could not determine whether protein, upon lyophilization, at the solid-void interface would still be in a native form.