Development of a 3D–LC/MS workflow for fast, automated and effective characterization of glycosylation patterns of biotherapeutic products

Glycosylation is a common post-translational modification of therapeutic monoclonal antibodies produced in mammalian cells and is considered an important critical quality attribute (CQA), as it is known to impact efficacy, stability, half-life and immunogenicity. For these reasons, glycosylation requires characterization and close monitoring during the manufacturing process. Due to the complexity of the glycosylation patterns, sophisticated analytical tools with high resolving power are required for the characterization of the glycoforms. This study describes, for the first time, the development and use of an online three-dimensional high-performance liquid chromatography/mass spectrometry (3D-HPLC/MS) approach for the monitoring of glycosylation patterns at the middle-up level. An immobilized IdeS-enzyme column was used in the first dimension for the digestion of mAbs in 10 minutes. Then, following an online reversed phase liquid chromatography (RPLC) column reduction, the ≈25 kDa proteolytic fragments w...