Assessing and Improving Protein Stability and Activity in iFNP Encapsulation

Abstract

Biologics are a promising variety of pharmaceutical therapy and present many advantages over traditional small molecule compounds, particularly in their specificity and lack of off-target effects. However, they suffer from greatly reduced stability and permeability, necessitating the design of novel delivery systems to ensure their clinical efficacy. An inverse Flash NanoPrecipitation (iFNP) process has been developed by the Prud’homme group by which proteins can be encapsulated into nanoparticles, enabling higher circulation times and protection from external conditions. However, although the final particles are quite stable, iFNP requires the use of organic solvents which pose a significant risk to protein structure and activity. In this thesis, we aim to develop means by which the model proteins, lysozyme and β-galactosidase, might be stably encapsulated using the iFNP process without suffering loss of activity. We develop quantitative activity assays, which enable a determination of protein activity, an indicator of overall stability, for both model enzymes. We then quantify the effects of organic solvent exposures on the proteins. Finally, we successfully encapsulate and recover lysozyme while retaining 99% activity. Additional work is required to accurately assess the activity of β-galctosidase following various processing steps. Specifically, it is necessary as a future direction to investigate alternative total protein quantification assays for this model enzyme.