Optimizing the lyophilization cycle and the consequences of collapse on the pharmaceutical acceptability of Erwinia L-asparaginase
Optimizing the lyophilization cycle and the consequences of collapse on the pharmaceutical acceptability of Erwinia L-asparaginase
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DOI：
10.1021/js960146p
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发表时间：
1996-12-01
影响因子：
3.8
通讯作者：
Ramsay, JR
中科院分区：
文献类型：
--
作者：
Adams, GDJ;Ramsay, JR
抗白血病酶--欧文氏L天冬酰胺酶以四聚体形式存在，在冻干胁迫下可解离为四个亚基(M(R)34000Da)。可以通过在配方中添加保护剂来稳定生物聚合物来减少离解，而产品应该干燥以形成药学上优雅的、货架稳定的易于溶解的蛋糕。利用分析超速离心法、高效液相色谱和圆二色谱，我们将冻干过程中酶的结构解离与生物活性联系起来。像甘露醇这样的添加剂可以防止小瓶内容物的烧蚀和干燥，以形成美观的优雅蛋糕，但提供的生物保护很少，因为在冷冻过程中，它们会结晶并从制剂中移除。赋形剂在整个循环中以非晶态持续存在，可提供更好的生物保护，尽管高分子量化合物，如葡聚糖(M(R)70000 Da)仅在产品冷冻或储存期间最有效。虽然含有单糖的配方通常表现出较低的崩塌温度(T-c)或通过热分析测量的玻璃化转变温度(T-g‘)，但这些配方在干燥过程中会扭曲，形成折叠的、在化妆品上不可接受的蛋糕，活性降低，稳定性差，水分含量高，溶解性降低。双糖的T-C温度高于单糖，或在T-C以下干燥，可避免崩解。保持无定形状态的干燥样品在高于固态崩塌温度时也会崩塌，当它们以比Arrhenius动力学预测的速度更快的速度衰减时。通过将水分从塞子扩散到干燥产品中，可以显著降低固态崩塌温度，从而增加样品的水分含量。通过分析崩解特性，可以缩短冻干循环时间，从而控制产品温度和腔体压力之间的关系，从而优化干燥速度，同时确保产品在升华过程中不会熔化或崩塌。
The antileukemia enzyme, Erwinia L-asparaginase, occurs as a tetramer which can be dissociated by the stresses of lyophilization into four subunits (subunit M(r) 34 000 Da). Dissociation can be reduced by adding protectants to the formulation to stabilize the biopolymer, while the product should dry to form a pharmaceutically elegant, shelf-stable cake which is readily soluble. Using analytical ultracentrifugation, HPLC, and circular dichroism we have related structural dissociation of the enzyme during lyophilization to biological activity. Additives such as mannitol prevent ablation loss of vial contents and dry to form cosmetically elegant cakes but provide little biological protection, since during freezing they crystallize and are removed from the preparation. Excipients persisting throughout the cycle in the amorphous state provide improved biological protection, although high molecular weight compounds such as Dextran (M(r) 70 000 Da) are most effective only during product freezing or storage. Low molecular weight sugars are protective throughout the cycle although formulations containing monosaccharides often exhibit low collapse temperatures (T-c) measured using a freeze-drying microscope or glass transition temperatures (T-g') measured by thermal analysis, but these formulations distort as drying progresses to form a collapsed, cosmetically unacceptable cake, with reduced activity, poor stability, a high moisture content, and reduced solubility. Collapse can be avoided by formulating with disaccharides, which display higher T-c temperatures than monosaccharides, or drying below T-c. Dried samples which persist in the amorphous state can also collapse when stored above their solid-state collapse temperatures when they decay at a faster rate than predicted by Arrhenius kinetics. The solid-state collapse temperature can be significantly decreased by the diffusion of moisture from the stopper into the dry product resulting in an increase in sample water content. Lyophilization cycle times can be reduced by analyzing collapse characteristics so that the relationship between product temperature and chamber pressure can be controlled so that drying rates can be optimized while ensuring that the product does not melt or collapse during sublimation.