The Suitability of Latex Particles to Evaluate Critical Process Parameters in Steric Exclusion Chromatography.

The steric exclusion chromatography (SXC) is a rather new method for the purification of large biomolecules and biological nanoparticles based on the principles of precipitation. The mutual steric exclusion of a nonionic organic polymer, i.e., polyethylene glycol (PEG), induces target precipitation and leads to their retention on the chromatographic stationary phase. In this work, we investigated the application of latex particles in the SXC by altering the particle's surface charge as well as the PEG concentration and correlated both with their aggregation behavior. The parameters of interest were offline precipitation kinetics, the product recovery and yield, and the chromatographic column blockage. Sulfated and hydroxylated polystyrene particles were first characterized concerning their aggregation behavior and charge in the presence of PEG and different pH conditions. Subsequently, the SXC performance was evaluated based on the preliminary tests. The studies showed (1) that the SXC process with latex particles was limited by aggregation and pore blockage, while (2) not the aggregate size itself, but rather the aggregation kinetics dominated the recoveries, and (3) functionalized polystyrene particles were only suitable to a limited extent to represent biological nanoparticles of comparable size and charge.