mD-UPLC-MS/MS: Next Generation of mAb Characterization by Multidimensional Ultraperformance Liquid...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmaceutical Science

Background:

• Multidimensional liquid chromatography-mass spectrometry (LC-MS) is standard for characterizing post-translational modifications (PTMs) in therapeutic antibodies (mAbs).
• Current methods often involve ion-exchange chromatography for charge variant fractionation followed by LC-MS peptide mapping, which can be time-consuming and risk artificial modifications.

Purpose of the Study:

• To develop a novel multidimensional ultra-performance liquid chromatography-mass spectrometry (mD-UPLC-MS/MS) system for rapid and efficient PTM characterization and quantification of therapeutic molecules.
• To integrate UPLC columns for peptide mapping, linking multidimensional LC (mD-LC) with routine LC-MS workflows.
• To introduce a parallel on-column digestion setup using trypsin and LysC for enhanced peptide mapping.

Main Methods:

• Implementation of a multidimensional ultra-performance liquid chromatography-mass spectrometry (mD-UPLC-MS/MS) system.
• Integration of UPLC columns for peptide mapping.
• Novel in-parallel trypsin and LysC on-column digestion followed by combined peptide mapping analysis.

Main Results:

• Successful characterization of a low retentive ethylene oxide adduct of a bispecific antibody.
• Demonstrated rapid analysis and reduced risk of artificial modifications during sample preparation.
• Generated distinct peptide maps through parallel digestion with different enzymes, enhancing characterization.

Conclusions:

• The developed mD-UPLC-MS/MS approach offers versatile and rapid analysis of PTMs.
• This method enables efficient characterization of therapeutic molecules, including complex adducts.
• The system facilitates integration with routine LC-MS workflows for broader applicability.