From Enrichment to Insight: Leveraging Preparative cIEF Enrichments and Characterization to Support Data‐Driven Integration Practices for cIEF‐based Analytics
Ramya Rao, Dominik Herrmann, Seamus Kelley, Victoria Cotham, Logan Figular, Youmi Moon, Deanna Di Grandi, Kathir Muthusamy, Yue Fu, Nisha Palackal, Shunhai Wang, Ning Li, Jennifer B. Nguyen, Michael P. Rosconi, Erica A. PylesABSTRACT
Biologics such as therapeutic monoclonal antibodies (mAbs) require comprehensive chemistry, manufacturing, and control (CMC) strategies to ensure product safety, efficacy, and consistency. Among these strategies is the comprehensive assessment of product‐related variants arising throughout the product development lifecycle. Routine capillary isoelectric focusing (cIEF) analysis of a therapeutic IgG molecule (mAb1) revealed a novel, poorly resolved basic peak at varying abundance across several lots, prompting an investigation. A preparative cIEF‐based fractionation strategy with downstream identity characterization by both intact and site‐specific mass spectrometry was performed, revealing several basic posttranslational modifications (PTMs), including incomplete N‐terminal pyroglutamate conversion and Fc methionine oxidation. These PTMs were ultimately deemed low risk due to their overall low abundance in the unfractionated control and lack of impact on target binding, informing data‐driven decision‐making for cIEF integration at product release.