Chemico-physical and biological properties of two monoclonal antibodies, Bevacizumab (Avastin®) and Durvalumab (Imfinzi®) under real-life light doses: a mechanistic approach.

INTRODUCTION Monoclonal antibodies (mAbs) have emerged as a prominent class of protein therapeutics for cancer treatment. However, their protein nature renders them susceptible to various stressors during manufacturing, transportation, storage, handling, and administration [1]. Notably, light exposure can induce chemico-physical changes, particularly when amino acids are located in the complementarity-determining regions (CDRs), potentially compromising efficacy and safety [2,3]. AIMS This study aimed to evaluate the chemico-physical stability and biological activity of Bevacizumab (Avastin) and Durvalumab (Imfinzi) under light doses mimicking real-life exposure. RESULTS Exposure to sunlight doses like those experienced in real-life settings did not alter the conformation of the diluted mAbs. However, light-induced aggregation was observed, with Bevacizumab exhibiting a higher extent of aggregation than Durvalumab. Durvalumab was also evaluated upon induced stress either by direct UVA/UVB exposure or by photosensitization via blue-light excitation of ruthenium(II) tris-bipyridyl dication (Rubpy²⁺)[4]. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed low levels of oxidative damage and deamidation. Chemico-physical modifications affected the target recognition ability of both mAbs (VEGF and PD-L1, respectively). Notably, no significant immunogenic potential was observed in dendritic cells derived from differentiated monocytes. CONCLUSIONS The chemico-physical changes induced by real-life light exposure did not significantly impact the overall protein structure of the tested mAbs. Minimal chemical modifications were detected in the CDRs, resulting in a marginal decrease in in vitro target recognition. While aggregation did not induce immunogenicity, it contributed to the decrease in biological activity. ACKNOWLEDGEMENTS This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement N° 101007939 (RealHOPE). The study was also supported by IOV - Veneto Institute for Oncology IRCCS with the fund “Ricerca Corrente 2024”. C.C.V gratefully acknowledges the Giulio Jori Scholarship – European Society for Photobiology for the travel grant (hosting institution Department of Pharmaceutical and Pharmacological Sciences, University of Padova). References: [1] Fayek R, Soleyman M, Jiskoot W, Crul M. Evaluation of post-production handling practices of monoclonal antibodies throughout the world. European Journal of Oncology Pharmacy 2021;4:e031. [2] Laptoš T, Omersel J. The importance of handling high-value biologicals: Physico-chemical instability and immunogenicity of monoclonal antibodies (Review). Experimental and Therapeutic Medicine, 2018. [3] Hipper E, Blech M, Hinderberger D, Garidel P, Kaiser W. Photo-Oxidation of Therapeutic Protein Formulations: From Radical Formation to Analytical Techniques. Pharmaceutics, 14:72, 2021 [4] Fancy, D. A., Denison, C., Kim, K., Xie, Y., Holdeman, T., Amini, F., & Kodadek, T. Scope, limitations and mechanistic aspects of the photo-induced cross-linking of proteins by water-soluble metal complexes. Chemistry and Biology, 7: 697-708, 2000.