In-Depth Characterization of Acidic Variants Induced by Metal-Catalyzed Oxidation in a Recombinant Monoclonal Antibody

Abstract

Characterization of antibody charge heterogeneity is an important task for antibody drug development. Recently, a correlation between acidic charge heterogeneity and metalcatalyzed oxidation has been observed for antibody drugs. However, to date, the acidic variants induced by metal-catalyzed oxidation have not been elucidated. In addition, it is challenging to satisfactorily explain the induced acidic charge heterogeneity, as the existing analytical workflows, which relied on either untargeted or targeted peptide mapping analysis, could lead to incomplete identification of the acidic variants. In this work, we present a new characterization workflow by combining untargeted and targeted analyses to thoroughly identify and characterize the induced acidic variants in a highly oxidized IgG1 antibody. As a part of this workflow, a tryptic peptide mapping method was also developed for accurate determination of the relative extent of site-specific carbonylation, where a new hydrazone reduction procedure was established to minimize under-quantitation artifacts caused by incomplete reduction of hydrazones during sample preparation. In summary, we identified 28 site-specific oxidation products, which are located on 26 residues and of 11 different modification types, as the sources of the induced acidic charge heterogeneity. Many of the oxidation products were reported for the first time in antibody drugs. More importantly, this study provides new insights to understanding acidic charge heterogeneity of antibody drugs in the biotechnology industry. Additionally, the characterization workflow presented in this study can be applied as a platform approach in the biotechnology industry to better address the need for in-depth characterization of antibody charge variants.

Multiattribute Monitoring of Aggregates and Charge Variants of Monoclonal Antibody through Native 2D-SEC-MS-WCX-MS

“…In standalone WCX analysis, aggregates can indeed contribute to the charge variant profile, which can make it difficult to differentiate between modifications present in the monomer and those originating from the aggregates/fragments. Earlier studies have noted the presence of aggregate and fragment species in the acidic variants of oxidized mAb in the CEX profile . Additionally, another research group has reported the elution of aggregate species as a basic variant in the CEX method and as an acidic variant in the AEX method .…”