Fluorescence Correlation Spectroscopy for Particle Sizing in Highly Concentrated Protein Solutions

Highly concentrated solutions of biomolecules play an increasingly important role in biopharmaceutical drug development. In these systems, the formation of reversible aggregates by self-association creates a significant analytical challenge, since dilution is often required for techniques such as HPLC/liquid chromatography and analytical ultracentrifugation. There is a growing demand for methods capable of analyzing these assemblies, ideally under formulation conditions (i.e., in the presence of excipients). One approach that addresses this need is based on fluorescence correlation spectroscopy (FCS), which is a flexible and powerful technique to measure the diffusion of fluorescently labeled particles. It is particularly suited to measuring the size distribution of reversible aggregates of proteins or peptides in highly concentrated formulations, since it overcomes some of the challenges associated with other methods. In this protocol, we describe state-of-the-art measurement and analysis of protein self-assembly by determination of particle size distributions in highly concentrated protein solutions using FCS.