Native SEC/MS Analysis of mAbs on an Entirely Biocompatible Flow Path

Size exclusion chromatography (SEC) of recombinant therapeutic proteins, such as monoclonal antibodies (mAbs), is an essential analytical tool to detect the presence of size variants, which may have adverse effects on safety and efficacy. SEC is commonly carried out using nonvolatile phosphate buffers, which makes the separation incompatible with mass spectrometry (MS). For this reason, a desalting step (online or offline) is required for peak identification.The use of volatile MS-compatible buffers, such as ammonium acetate, has been demonstrated to be significantly less effective in comparison with phosphate buffers when applied on classic stainless-steel instrument/column configurations due to nonspecific interactions. The latter can be counteracted by using biologically inert or biocompatible flow paths. This application note demonstrates the potential of the combination of a polyether ether ketone (PEEK)-lined Agilent AdvanceBio SEC column with the Agilent 1290 Infinity II Bio LC System for native SEC/MS analysis of mAbs.