Impact of process impurities on developability assessments of complex antibody formats: the striking role of leachable copper

The assessment of developability and physical stability of antibody therapeutics is a cornerstone of modern biopharmaceutical discovery, increasingly relying on high-throughput automation to screen candidates to identify the most suitable lead candidate. In this study, we report a severe, anomalous fragmentation event observed in therapeutic antibodies during routine automated buffer exchange into phosphate-buffered saline (PBS) at physiological pH. Through a systematic investigation, we demonstrate that this instability is not intrinsic to the proteins, but is driven by the specific physicochemical environment of the purification process. We identify that at pH 7.4, the dominant phosphate species may act as a ligand capable of mobilizing trace catalytic copper from disposable chromatography columns. Our data suggests the leached copper coordinates to the antibody hinge region, driving a highly localized, Fenton-like oxidative cleavage. Comparative analysis reveals that this vulnerability is influenced by molecular architecture. Complex formats, such as 2 + 1 CrossMabs, exhibited significantly higher susceptibility than standard IgGs, suggesting that steric crowding may enhance the accessibility of the metal-binding site. Finally, we present a robust mitigation strategy using an ethylenediaminetetraacetic acid-based column conditioning protocol that effectively eliminates the leachable catalyst. These findings highlight a critical, often-overlooked source of chemical instability in automated workflows and underscore the necessity of controlling process-related impurities, particularly when developing complex antibody modalities.