Capillary electrophoresis–mass spectrometry as a characterization tool for therapeutic proteins

Abstract With the increasing use of capillary electrophoresis (CE) in the biotechnology industry, there is a demand for analytical tools and methodology that can be used to characterize CE profiles. This article describes the implementation and optimization of a robust online CE–mass spectrometry (CE–MS) system used for the characterization of several CE assays developed at Genentech Inc. These assays include CE as a complement to reverse-phase peptide mapping for the identification of small peptides eluting in the void volume, profiling N-linked glycopeptide heterogeneity, and determining O-linked site occupancy. In addition, CE–MS was used to confirm major 8-aminopyrene-1,3,6-trisulfonate (APTS)-labeled glycans released from recombinant antibodies that are routinely profiled by CE–laser-induced fluorescence (CE–LIF). For each study, CE–MS was able to successfully identify components seen in UV or LIF electropherograms, thereby expanding the capability of CE and CE–MS for profiling biomolecules.