From proof of concept to the routine use of an automated and robust multi-dimensional liquid chromatography mass spectrometry workflow applied for the charge variant characterization of therapeutic antibodies.

•Thorough optimization of a multi-dimensional LC/MS setup to identify charge variants.•Similar levels of most protein PTMs reported by off-line and on-line approaches.•Faster turnaround time achieved by on-line vs. off-line peptide mapping.•Ultra-fast on-column tryptic digestion can limit artificial deamidation.