Is stability prediction possible for protein drugs? Denaturation kinetics of beta-galactosidase in solution.

Denaturation and aggregation kinetics of Aspergillus oryzae beta-galactosidase in solution were studied in order to determine whether the stability of protein drugs can be predicted. Denaturation of beta-galactosidase, monitored by measuring enzyme activity, conformed to first-order kinetics, whereas aggregation of the denatured form, monitored by high performance size exclusion chromatography, showed a reaction order higher than 1. Denaturation of beta-galactosidase was irreversible and exhibited a biphasic kinetic pattern which could be explained by assuming that two isoenzymes denatured irreversibly at different rates. Linear Arrhenius plots were obtained for the estimated rate constants, and delta H not equal to and delta S++ to were estimated according to the Eyring equation. The estimated delta H++ was much larger than delta H++ observed in usual chemical reactions. The present study suggests that the denaturation of protein drugs can be analyzed by the Eyring equation in the same manner as chemical degradation, contradicting the general consensus that accelerated testing can not be used to predict the stability of protein formulations.