Confident Sequence Analysis Of A Trispecific Antibody Using An Electron-Activated Dissociation-Based Middle-Down Workflow

By Haichuan Liu, Andrew Mahan, Hirsh Nanda, and Zoe Zhang, SCIEX, USA; Janssen, USA

This study demonstrates an EAD-based middle-down workflow using the ZenoTOF 7600 system and Biologics Explorer software from SCIEX was leveraged to confirm the sequences of major subunits of a tsAb treated with GlySERIAS and IdeS proteases. The results were used to determine the enzymatic cleavage sites on the linkers and to localize the glycosylation.

Key features of the EAD-based middle-down workflow for antibody characterization

• Single-injection platform method: The EAD-based middle-down workflow provides high sequence coverage in a single injection with 1 fragmentation technique
• Confident sequence confirmation: Zeno EAD leads to fragment-rich spectra of tsAb subunits for confident sequence confirmation and determination of enzymatic cleavage sites
• Accurate localization of PTMs: High sequence coverage and preservation of labile modifications by EAD allows for comprehensive PTM analysis
• Powerful software tools: Biologics Explorer software provides powerful tools for data visualization, results review and comparison
• Streamlined: The workflow consists of simple sample preparation, efficient data acquisition and automatic data analysis, with limited method development required and is easy to adopt by various levels of LC-MS users