Mechanistic Study of the Adsorption and Desorption of Proteins on Silica

Flora Felsovalyi

Mechanistic Study of the Adsorption and Desorption of Proteins on Silica
Felsovalyi, Flora
Thesis Advisor(s):
Banta, Scott A.
Ph.D., Columbia University
Chemical Engineering
Persistent URL:
Proteins are complex biomacromolecules that are intrinsically surface-active. Through the process of protein adsorption, the surface can act as a catalyst to facilitate dramatic structural alteration and destabilization of the protein. Understanding the mechanisms governing protein adsorption to a solid/liquid interface is pertinent in the wide range of applications in which this ubiquitous phenomenon plays a key role. One particular application, in which the consequences of adsorption must be especially well-characterized, is prefilled drug container systems for protein-based therapeutics. Here, high-value biologics are exposed to solid/liquid interfaces often for extended periods of time, and any surface-induced change in concentration or conformation of the protein is strictly regulated. The goal of this research is to further our understanding about the factors affecting protein adsorption and desorption and the global interplay between various adsorption-related subprocesses. Our strategy is to expand the traditional design space of protein adsorption studies to target a wider range of surface coverages, longer desorption time scales and proteins with unique characteristics. We begin our investigation with a critical assessment of the impact of surface-induced structural perturbations on protein desorption, where irreversible conformational changes might lead to various forms of protein destabilization. We study the adsorption of lysozyme on silica and find that not only is adsorption reversible, but also that desorption is predictable in a coverage-dependent manner. Because we see evidence of coverage-independent structural perturbation on the surface, we speculate that more local descriptors, such as the number of amino acids per chain that are physically adsorbed on the surface, likely control the desorption process.To evaluate the effects of protein stability on interfacial behavior, we employ two naturally occurring stability variants from the aldo-keto reductase superfamily. We compare their adsorption, structural transitions and desorbability in the presence of silica nanoparticles. We find little correlation between a protein's thermostability, surface-affinity and susceptibility to surface-induced unfolding. Our results question the idea that thermal stability is an accurate predictor of adsorption behavior. In a similar effort to evaluate the effects of electrostatic interactions, we use supercharged GFP variants that have dramatically different surface charge distributions. Here, we find that protein/surface charge differences correlate more strongly with surface affinity and desorption kinetics. These results highlight the more dominant role of electrostatics, compared to intrinsic structural stability, in determining protein interfacial behavior on hydrophilic surface. Finally, we question the widely accepted notion that due to the complex, multi-segment binding of proteins and surface-induced unfolding, protein adsorption is a thermodynamically irreversible process. We study the desorption of several proteins and find that all proteins exhibit reversible binding and structural refolding, albeit at very different time scales. To interpret our protein desorption data, we take an interdisciplinary approach in applying models from polymer theory. In this way, we uncover new similarities between the two fields and gain interesting insight into the heterogeneity of the adsorbed protein layer. By showing reversibility of adsorption, we also analyze the role of the Langmuirian parameters, and combined with intrinsic protein parameters, we develop a framework for predicting protein desorption behavior.
Chemical engineering
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Suggested Citation:
Flora Felsovalyi, , Mechanistic Study of the Adsorption and Desorption of Proteins on Silica, Columbia University Academic Commons, .

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