Investigations of Mixed-Mode Cation Exchange Chromatography for Control of Aggregates in Bispecific Antibodies

Bispecific antibodies are a class of engineered antibodies with the ability to bind to two distinct antigens. Compared to monoclonal antibodies, single chain variable fragment-IgG bispecific antibodies are prone to high levels of aggregate (10-30%), which can impact product yields and must be removed during downstream polishing steps. Mixed mode chromatography (MMC) provides a potential method of aggregate control due to its multiple modes of protein-ligand interactions, resulting in enhanced selectivity compared to traditional ion exchange and hydrophobic interaction chromatography resins. Ceramic hydroxyapatite (CHT) is an MMC resin often used as a polishing step for many high aggregate forming species; however, its scalability issues raise the need for an alternative aggregate removal step. Initial studies showed that mixed-mode cation exchange (CEX) resins clear aggregates with high selectivity and minimal breakthrough while operating in flow-through mode. To further investigate potential alternatives to CHT, we evaluated prototype POROS™ mixed-mode CEX resins from Thermo Fisher Scientific to study the impact of ligand density, operating pH and arginine concentration on aggregate removal ability.