A Nanoparticle-Based Assay To Evaluate Surface-Induced Antibody Instability

Protein stability against aggregation represents a major quality attribute for the successful development of biopharmaceuticals. Increasing evidence indicates that the formation of protein aggregates in aqueous solutions is often triggered by interactions between proteins and interfaces. Yet, in contrast to a large number of methods available to test protein bulk properties, high-throughput assays to investigate protein instability at interfaces remain much less developed. Major challenges include the control of the amount and type of surfaces, as well as the presence of synergistic effects between interfaces and hydrodynamic flows. Here, we describe and develop a highly controlled surface-mediated stress assay of protein instability based on polymeric nanoparticles. We show that hydrophobic nanoparticles are remarkably powerful in destabilizing large proteins such as antibodies. We further show that this approach can be implemented on a high-throughput microfluidic platform by compartmentalizing the protein solution in picoliter droplets surrounded by an oil phase. Our method allows the evaluation of protein instability at hydrophobic interfaces in a time scale of minutes and requires amounts of the sample in the order of a few hundred micrograms. We demonstrate that our assay represents a good mimic of air-water interfaces and finds application as a screening tool to optimize protein stability toward surface-induced aggregation. We provide a concrete example by identifying the optimal concentration range of Tween 80 that prevents antibody instability in the presence of interfaces. Overall, our hydrophobic nanoparticle surface-mediated stress assay (HNSSA) represents an attractive tool for accelerated tests of protein instability at interfaces under both stagnant and flow conditions, with implications for the optimization of buffer composition and the selection of stable biotherapeutic candidate molecules during early stage development.