Yoshioka Sumie, Tajima Shinsuke, Aso Yukio, Kojima Shigeo
National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158, Japan.
Pharm Res. 2003 Oct;20(10):1655-60. doi: 10.1023/a:1026151721212.
To examine whether the empirical Kohlrausch-Williams-Watts (KWW) equation is applicable not only to protein aggregation but also to protein denaturation in lyophilized formulations. Lyophilized beta-galactosidase (beta-GA) formulations containing polyvinylalcohol and methylcellulose were used as model formulations. The possibility of predicting storage stability based on the temperature dependence of the estimated parameters of inactivation/aggregation--time constant (tau) and its distribution (beta) is discussed.
Protein aggregation in lyophilized beta-GA formulations at 10-70 degrees C and 6-43% relative humidity was determined as a function of time by size exclusion chromatography. Enzyme activity was also determined using 2-nitrophenyl-beta-D-galactopyranoside as a substrate.
Inactivation and aggregation of beta-GA were describable with the empirical KWW equation, regardless of whether the temperature was above or below the NMR relaxation-based critical mobility temperature (Tmc) or whether protein molecules with different degrees of deformation resulting from stresses during lyophilization exist in formulation. The estimated beta parameter for protein aggregation creased rapidly as temperature increased beyond Tmc becausethe mobility of polymer molecules increased in the initial stages of glass transition. The time required for 10% enzyme to aggregate (t90) calculated from the tau and beta parameters exhibited a change in temperature dependence gradient near Tmc. In contrast, t90 for protein inactivation exhibited temperature dependence patterns varying withthe excipients.
The t90 calculated from the estimated tau and beta parameters was found to be a useful parameter for evaluating the stability of lyophilized beta-GA formulations. The prediction of t90 by extrapolation was possible in the temperature range in which beta did not rapidly vary with temperature.
研究经验性的科尔劳施 - 威廉姆斯 - 瓦特（KWW）方程是否不仅适用于蛋白质聚集，还适用于冻干制剂中的蛋白质变性。含有聚乙烯醇和甲基纤维素的冻干β - 半乳糖苷酶（β - GA）制剂用作模型制剂。讨论了基于失活/聚集估计参数的温度依赖性——时间常数（τ）及其分布（β）预测储存稳定性的可能性。
通过尺寸排阻色谱法测定冻干β - GA制剂在10 - 70℃和6 - 43%相对湿度下蛋白质聚集随时间的变化。还使用2 - 硝基苯基 - β - D - 吡喃半乳糖苷作为底物测定酶活性。
无论温度是高于还是低于基于核磁共振弛豫的临界迁移温度（Tmc），或者冻干过程中因应力导致不同变形程度的蛋白质分子是否存在于制剂中，β - GA的失活和聚集都可用经验性KWW方程描述。随着温度超过Tmc，蛋白质聚集的估计β参数迅速增加，因为在玻璃化转变的初始阶段聚合物分子的迁移率增加。根据τ和β参数计算的10%酶聚集所需时间（t90）在Tmc附近呈现温度依赖性梯度变化。相比之下，蛋白质失活的t90表现出随辅料变化的温度依赖性模式。
发现根据估计的τ和β参数计算的t90是评估冻干β - GA制剂稳定性的有用参数。在β不随温度快速变化的温度范围内，可以通过外推法预测t90。
