Automated middle-up approach for the characterization of biotherapeutic products by combining on-line hinge-specific digestion with RPLC-HRMS analysis

This study describes, for the first time, the development of an automated liquid chromatography-mass spectrometry (LC-MS) workflow comprised of an immobilized IdeS-HPLC column for on-line sample digestion, followed by a reversed-phase liquid chromatography (RPLC) for protein subunit separation, and a high-resolution MS for molecular weight analysis. A proof of concept study was described here for the characterization of a therapeutic mAb and a bsAb. For the mAb, this automated workflow enabled rapid characterization of post-translational modifications (PTMs) such as N-glycosylation, glycation and N-terminal lysine. For the bsAb, the same workflow was successfully employed to identify product impurities due to chain pairing. The sample analysis using this workflow can be accomplished within less than one day. This workflow demonstrated its potential as a multi-attribute method for characterization of therapeutic proteins.Graphical abstract
Source: Journal of Pharmaceutical and Biomedical Analysis - Category: Drugs & Pharmacology Source Type: research