Inactivation and aggregation of beta-galactosidase in lyophilized formulation described by Kohlrausch-Williams-Watts stretched exponential function.

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 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.

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 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.

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.