mD-UPLC-MS/MS: Next Generation of mAb Characterization by Multidimensional Ultraperformance Liquid Chromatography-Mass Spectrometry and Parallel On-Column LysC and Trypsin Digestion

Abstract

For the past few years, multidimensional liquid chromatography-mass spectrometry (LC-MS) systems have been commonly used to characterize post-translational modifications (PTMs) of therapeutic antibodies (mAbs). In most cases, this is performed by fractionation of charge variants by ion-exchange chromatography and subsequent online LC-MS peptide mapping analysis. In this study, we developed a multidimensional ultra-performance-liquid-chromatography-mass spectrometry system (mD-UPLC-MS/MS) for PTM characterization and quantification, allowing both rapid analysis and decreased risk of artificial modifications during sample preparation. We implemented UPLC columns for peptide mapping analysis, facilitating the linkage between mD-LC and routine LC-MS workflows. Furthermore, the introduced system incorporates a novel in-parallel trypsin and LysC on-column digestion setup, followed by a combined peptide mapping analysis. This parallel digestion with different enzymes enhances characterization by generating two distinct peptides. Using this approach, a low retentive ethylene oxide adduct of a bispecific antibody was successfully characterized within this study. In summary, our approach allows versatile and rapid analysis of PTMs, enabling efficient characterization of therapeutic molecules.

Comprehensive Multidimensional Liquid Chromatography–Mass Spectrometry for the Characterization of Charge Variants of a Bispecific Antibody

“…Another report on workflow optimization for peptide mapping describes the improved retention of hydrophilic peptides . More recently, the implementation of a precolumn reducing the back pressure and thus enabling the application of any offline analytical C18 method was reported and will improve further 4D-LC-MS investigations …”

Novel Multidimensional Liquid Chromatography Workflow with In-Loop Enzymatic Digests of Multiple Heart-Cuts for Fast and Flexible Characterization of Biotherapeutic Protein Variants

“…However, by relying on only the most commonly used protease (trypsin), PTMs may be missed due to lower sequence coverage or incomplete proteolytic digestion caused by steric hindrance of the modification preventing the protease access to the substrate . Sequence coverage is thus more complete with parallel digestions using multiple endoproteases, each generating a different set of peptide fragments. − While immobilization likely lowers the risk of overdigestion, available HPLC-immobilized enzymes are expensive and limited in both the choice of enzymes and their lifetime, depending on the buffer conditions.…”

Enhanced Identification of Ginsenosides Simultaneously from Seven Panax Herbal Extracts by Data-Dependent Acquisition Including a Preferred Precursor Ions List Derived from an In-House Programmed Virtual Library

“…It is crucial to establish precise and fit-for-purpose analytical techniques that can implement the characterization and quantification of metabolites for a number of research areas (e.g., natural product research and food science). , At the same time, it is still confronted with challenges for the detection and analysis of the metabolites owing to the severe endogenous interference and, in most cases, the low content in a complex matrix. In recent years, liquid chromatography/mass spectrometry (LC/MS) has provided a powerful and practical method enabling the systematic characterization of known compounds, as well as the discovery of potentially new components from natural products. − Nowadays, the utilization of multidimensional chromatography (MDC) greatly improves the selectivity and expands the peak capacity, , while various MS strategies are available to maximize the qualitative MS information supporting the identification of metabolites. , The utilization of various scan methods has been demonstrated as potent in the in-depth characterization and multicomponent quantitation of various metabolites from herbal extracts. , …”

“…In recent years, liquid chromatography/mass spectrometry (LC/MS) has provided a powerful and practical method enabling the systematic characterization of known compounds, as well as the discovery of potentially new components from natural products. 3−6 Nowadays, the utilization of multidimensional chromatography (MDC) greatly improves the selectivity and expands the peak capacity, 7,8 while various MS strategies are available to maximize the qualitative MS information supporting the identification of metabolites. 9,10 The utilization of various scan methods has been demonstrated as potent in the in-depth characterization and multicomponent quantitation of various metabolites from herbal extracts.…”