Mechanisms of activity loss for a multi-PEGylated protein by experiment and simulation

Abstract

Over the past five decades, one of the most investigated strategies to increase the circulatory half-life and to reduce the immunogenicity of non-human therapeutic proteins has been to cover their surface with multiple copies of methoxy poly(ethylene glycol) (mPEG). This multi-‘PEGylation’ strategy, however, is often associated with loss of protein activity, which is most often rationalized as direct modification of the protein's active site or to steric hindrance near its surface. While these mechanisms are generally accepted and likely valid, very few studies investigate other possible mechanisms of activity loss. To answer this question, this study investigates the mechanisms of activity loss for glutamate dehydrogenase (GDH) bearing up to 25 chains of mPEG per protein. Through a combination of experimental design and simulation, the most common proposed causes of protein inactivation (i.e. active-site PEGylation and steric hindrance near the surface of the protein) are circumvented to shed light on less characterized mechanisms such as altered protein structure, altered microenvironment at the surface of the protein, and altered protein dynamics. Experiments suggest that the secondary and tertiary structure of GDH was (sometimes significantly) affected by PEGylation, though these changes do not necessarily result in a loss of activity. Structure-activity correlations could not reconcile all trends within the library of bioconjugates tested. Moreover, by measuring catalytic activity in the presence of five different allosteric modulators, loss of activity could not be ascribed to an altered local microenvironment at the protein's surface. As such, coarse-grained simulations were exploited to reveal the effect of PEGylation on protein dynamics, which are long-ranged, cooperative, and essential for GDH catalytic activity. Normal mode analysis of native bovine GDH (i.e. not PEGylated) revealed that the lowest ranking mode displayed several features that bore similarity to movements known for the native protein. These included rotation/distortion of the ‘antenna’ regions, closure of the ‘catalytic cleft’, and constriction of the internal cavity. A reduced magnitude of this mode was observed as a function of degree of PEGylation and mPEG molecular weight and correlated well with trends observed for the library of bioconjugates tested experimentally. Overall, this study provides new insight into fundamental considerations associated with loss of activity, which will contribute to the design of future bioconjugates by providing a better understanding of possible mechanisms of loss of activity.