Near UV and visible light-induced site-specific fragmentation of IgG1-based modalities mediated by histidine and Fe(III): a role for intra-domain interactions?

Photo-stability represents a critical quality attribute for the development of therapeutic proteins where the exposure to near UV or visible light can lead to protein fragmentation. Here, we compare the photo-stability of three IgG1 based modalities, formulated in histidine (His) buffer containing various levels of Fe(III). We report a significant difference in the extent of photo-degradation between a high mannose Fc fragment (HM-Fc), NISTmAb and a fusion protein, Flt-3L-Ig. Our results indicate that despite preserving the Fc domain sequence, the NISTmAb and Flt-3L-Ig are more susceptible to site-specific Thr²⁵⁹ photo-fragmentation in the CH2 domain compared to the HM-Fc (amino acid numbering based on the NISTmAb sequence). Enzymatic deglycoslyation enhanced the susceptibility of both NISTmAb and HM-Fc to photo-fragmentation, while enzymatic cleavage of the Fab domain from NISTmAb decreased the extent of photo-fragmentation. Our findings suggest that differences in photo-stability may be attributed to differences in domain-domain interactions, glycan structure, and the thermal stability of these modalities. Therefore, careful consideration should be given to photostability studies during the development of such proteins into therapeutic drug products.