Near UV Photo-Degradation of IgG1-Fc Promoted by His and Fe(III): Probing the Role of the Metal-Binding Pocket by Site-Directed Mutagenesis

Light exposure of therapeutic proteins can affect chemical and physical stability and compromise safety and efficacy. In order to develop stable pharmaceutical formulations without undesired side effects, it is essential to understand both product formation and reaction mechanisms which lead to light-induced protein degradation. We recently reported that visible and near UV light induce site-specific fragmentation and photo-conversion of a heavy chain Thr residue in IgG1-Fc, full length IgG1 and IgG1-based fusion proteins formulated in Fe(III)-containing histidine buffer. These post-translational modifications manifest differently in each modality, with the photo-conversion of Thr to Ala being more prominent in IgG1-Fc. Mechanistically, these photo-degradation pathways occur within a Fe(III)-binding pocket present in the CH2 domain. Here, we investigate the role of individual metal-binding residues located in the Fe(III)-binding pocket in the photo-degradation of IgG1-Fc. The single mutants of Asp25 to Ala, Thr32 to Ala, and Glu34 to Gln in IgG1-Fc completely prevent the site-specific photo-degradation of Thr32, while the mutation of Pro33 to Ala had only a relatively small effect. A comparison of crystal structures reveals that the replacement of Pro33 by Ala does not significantly affect the structure of the Fe(III)-binding pocket. Our results indicate that a single amino acid mutation can prevent the Fe(III)-catalyzed photo-degradation, suggesting that single point mutations may be a future pathway towards photo-stable pharmaceutical formulations.