Determining Antibody-Drug Conjugate Positional Isomer Distribution with Nonreduced CE-SDS and HIC

Antibody-drug conjugates are among the most structurally complex biotherapeutics in development, and their efficacy, safety, and biophysical behavior depend not only on the drug-to-antibody ratio (DAR) but on where the payload sits on the antibody. The techniques used routinely in ADC characterization each capture only part of that picture: hydrophobic interaction chromatography (HIC) resolves DAR0 through DAR8 species but carries no positional information, while nonreduced CE-SDS quantifies the fragments generated when payloads displace interchain disulfides, revealing insights into drug location, but not the DAR. This webinar shows how combining both measurements, acquired on the Agilent ProteoAnalyzer system and the Agilent 1290 Infinity III Bio LC, resolves individual positional isomers through a system-of-equations approach.

Attendees will learn:

• How conjugation chemistry drives the fragment patterns observed under nonreduced denaturing CE-SDS
• Practical method considerations for robust HIC DAR profiling, including salt selection, organic modifiers, and column care
• How to combine CE-SDS and HIC data to estimate positional isomer distribution without additional analytical techniques
• How automated parallel CE-SDS and bio-compatible LC streamline ADC characterization workflows 

Presenter: Whitney Pike, M.A. (Application Scientist, Agilent Technologies, Inc.)

Whitney joined Agilent in 2018 with the acquisition of Advanced Analytical (AATI) and is an application scientist with a focus on the Agilent Automated Electrophoresis instruments. During her years at AATI, she played a pivotal role in the release of the Femto Pulse system and kits for the Fragment Analyzer as a scientist on the application development team.

Presenter: Dr. Lisa Engelbart (Application Scientist, Agilent Technologies)

Lisa is an application scientist at Agilent Technologies in Waldbronn, Germany. In this role, she develops application notes for LC and LC-MS, with a special focus on the Agilent Infinity III LC system. Lisa studied food chemistry and holds a PhD in analytical chemistry from the University of Tübingen.