IS STABILITY PREDICTION POSSIBLE FOR PROTEIN DRUGS - DENATURATION KINETICS OF BETA-GALACTOSIDASE IN SOLUTION

Denaturation and aggregation kinetics of Aspergillus oryzae beta-galac tosidase in solution were studied in order to determine whether the st ability of protein drugs can be predicted. Denaturation of beta-galact osidase, monitored by measuring enzyme activity, conformed to first-or der kinetics, whereas aggregation of the denatured form, monitored by high performance size exclusion chromatography, showed a reaction orde r higher than 1. Denaturation of beta-galactosidase was irreversible a nd exhibited a biphasic kinetic pattern which could be explained by as suming that two isoenzymes denatured irreversibly at different rates. Linear Arrhenius plots were obtained for the estimated rate constants, and Delta H double dagger and Delta S double dagger were estimated ac cording to the Eyring equation. The estimated Delta H double dagger wa s much larger than Delta H double dagger observed in usual chemical re actions. The present study suggests that the denaturation of protein d rugs can be analyzed by the Eyring equation in the same manner as chem ical degradation, contradicting the general consensus that accelerated testing can not be used to predict the stability of protein formulati ons.