Resolution limits of size exclusion chromatography columns identified from flow reversal and overcome by recycling liquid chromatography to improve the characterization of manufactured monoclonal antibodies.

This study focuses on resolution limits of size exclusion chromatography columns identified from flow reversal and overcome by recycling liquid chromatography to improve the characterization of manufactured monoclonal antibodies.. The research employs high-performance liquid chromatography (HPLC) techniques to address analytical challenges in the biopharmaceutical field. The flow reversal (FR) technique consists of reversing the flow direction along a chromatographic column. It is used to reveal the origin (such as poor column packing, active sites, or slow absorption/escape kinetics) for the resolution limit of 4.6 mm × 150 mm long columns packed with 1.7 μm 200... Research Background and Significance Size exclusion chromatography (SEC) is widely used in the biopharmaceutical industry for the characterization of large biomolecules, including monoclonal antibodies (mAbs). Despite its broad utility, SEC is often limited by resolution constraints that hinder the effective separation of closely related molecular species such as aggregates, fragments, and monomers. These resolution limits are critical because accurate characterization of mAbs influences drug safety, efficacy, and regulatory compliance. The study by Gritti (2023) addresses these limitations by investigating the underlying causes of resolution loss in SEC columns, specifically using flow reversal (FR) techniques. By exploring the chromatographic behavior under reversed flow conditions, the research aims to identify column packing imperfections, active sites, and kinetic factors that contribute to peak broadening and poor separation. The significance of this work lies in its potential to enhance analytical capabilities and improve quality control in biopharmaceutical manufacturing through refined chromatograph