Unveiling the effects of light exposure Bevacizumab stability in clinical settings

Bevacizumab was the first available anti-angiogenic therapy with a monoclonal antibody, initially approved against colorectal cancer. It is now used intravenously in the treatment of several other malignancies and in combination with chemotherapy. It binds to all circulating, soluble VEGF-A isoforms, inhibiting angiogenesis, and thereby reducing tumor vascularization. During storage, transport, dilution and administration, Bevacizumab is exposed to different stressors, including ambient light, which can compromise its efficacy and safety. Here, the chemico-physical instability induced by real-life doses of light on Bevacizumab-bvzr is reported in relation to the observed target recognition, anti-angiogenic activity and in vitro immunogenicity. By irradiating the diluted formulation with real-life light doses, mimicking the IV bag infusions, no conformational changes were found by UV and CD spectroscopy. However, light-exposed Bevacizumab-bvzr exhibited a marked reduction in VEGF-A binding, along with decreased anti-angiogenic efficacy in HUVEC-based assays. This loss of activity can be explained by the formation of a small amount of high molecular weight aggregates, detected by SEC, SDS-PAGE and TEM analyses, in all irradiated samples and in a concentration-dependent manner. The immunogenic properties of Bevacizumab aggregates were assessed on human monocytes-derived dendritic cells, which revealed no evidence of dendritic cell activation in vitro. At very high light doses, highlighting the amino acid modifications, mono/dioxidations and deamidation were detected by LC-MS fingerprinting analysis, involving Trp and Met, and Asn and Gln, respectively. Serine formylation and tyrosine oxidation mostly in 5 different peptides were also found. Notably, none of the identified modifications appeared within the complementarity-determining regions (CDRs). Although Bevacizumab is typically exposed to low levels of artificial and indoor light under clinical conditions, our findings indicate that it should be always protected from light. This is especially true during long IV administration periods, to avoid aggregate formation with potential reduction of the mAb therapeutic activity.