Adams G D, Ramsay J R
Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire, U.K.
J Pharm Sci. 1996 Dec;85(12):1301-5. doi: 10.1021/js960146p.
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) 70000 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 (Tc) measured using a freeze-drying microscope or glass transition temperatures (Tg') 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 Tc temperatures than monosaccharides, or drying below Tc. 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.
抗白血病酶欧文氏菌L - 天冬酰胺酶以四聚体形式存在，可因冻干应力解离为四个亚基（亚基分子量为34000道尔顿）。通过在制剂中添加保护剂来稳定生物聚合物，可减少解离，同时产品干燥后应形成药学上美观、货架稳定且易溶的块状物。我们利用分析超速离心、高效液相色谱和圆二色性技术，将冻干过程中酶的结构解离与生物活性联系起来。甘露醇等添加剂可防止小瓶内容物损失并干燥形成外观美观的块状物，但几乎没有生物保护作用，因为在冷冻过程中它们会结晶并从制剂中析出。在整个循环过程中以无定形状态存在的辅料可提供更好的生物保护，尽管高分子量化合物如葡聚糖（分子量70000道尔顿）仅在产品冷冻或储存期间最有效。低分子量糖类在整个循环过程中都具有保护作用，尽管含有单糖的制剂通常表现出较低的塌陷温度（通过冻干显微镜测量）或玻璃化转变温度（通过热分析测量），但随着干燥过程的进行，这些制剂会变形形成塌陷的、外观不可接受的块状物，活性降低、稳定性差、水分含量高且溶解度降低。通过使用二糖进行配方可避免塌陷，二糖的塌陷温度高于单糖，或者在塌陷温度以下干燥。当干燥样品在高于其固态塌陷温度储存时，如果它们的衰变速度比阿累尼乌斯动力学预测的速度快，也会塌陷。水分从瓶塞扩散到干燥产品中会导致样品含水量增加，从而显著降低固态塌陷温度。通过分析塌陷特性可以减少冻干循环时间，从而控制产品温度与腔室压力之间的关系，以便在确保产品在升华过程中不会熔化或塌陷的同时优化干燥速率。
