Inactivation and Aggregation of beta-Galactosidase in Lyophilized Formulation Described by Kohlrausch-Williams-Watts Stretched Exponential Function

Inactivation and Aggregation of beta-Galactosidase in Lyophilized Formulation Described by Kohlrausch-Williams-Watts Stretched Exponential Function
October 2003
Sumie Yoshioka1, Shinsuke Tajima1, Yukio Aso1 and Shigeo Kojima1
(1) National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158, Japan.
Pharmaceutical Research Volume 20, Number 10
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Abstract
Purpose. 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.
Methods. Protein aggregation in lyophilized beta-GA formulations at 10-70?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.
Results. 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 the formulation. The estimated beta parameter for protein aggregation decreased rapidly as temperature increased beyond Tmc because the 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 with the excipients.
Conclusions. 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.