Comprehensive biophysical and functional study of ziv-aflibercept: characterization and forced degradation Hermosilla, Jesús Pérez Robles, Raquel Salmerón García, Antonio Casares Atienza, Salvador Cabeza, José Bones, Jonathan Navas Iglesias, Natalia Africa Health care Therapeutics This study was entirely funded by Project FIS: PI-17/00547 (Instituto Carlos III, Ministerio de Economia y Competitividad, Spain), which means that it was also partially supported by European Regional Development Funds (ERDF). Aflibercept (AFL) is an Fc fusion protein used in the treatment of colorectal cancers and different ophthalmological diseases. There are two medicines in which AFL is the active substance: Zaltrap and Eylea, referred as ziv-AFL and AFL respectively. No proper accelerated degradation studies were published on either AFL or ziv-AFL. These studies are essential during research, development and manufacturing stages. Here, we characterized ziv-AFL and submitted it to different stress conditions: light, 60 °C, freeze-thaw cycles, changes in pH, high hypertonic solution and strong denaturing conditions. We used an array of techniques to detect aggregation (SE-HPLC/DAD and DLS), changes in secondary structure (Far-UV circular dichroism), changes in conformation or tertiary structure (Intrinsic tryptophan fluorescence) and alterations in functionality (ELISA). Results indicate that aggregation is common degradation pathway. Two different types of aggregates were detected: dimers and high molecular weight aggregates attributed to β-amyloid-like structures. Secondary structure was maintained in most of the stress tests, while conformation was altered by almost all the tests except for the freeze-thaw cycles. Functionality, evaluated by its immunochemical reaction with VEGF, was found to be stable but with decrease when exposed to light and with likely partial inactivation of the drug when pH was altered. 2020-10-21T12:39:07Z 2020-10-21T12:39:07Z 2020-02-14 info:eu-repo/semantics/article Hermosilla, J., Pérez-Robles, R., Salmerón-García, A. et al. Comprehensive biophysical and functional study of ziv-aflibercept: characterization and forced degradation. Sci Rep 10, 2675 (2020). [https://doi.org/10.1038/s41598-020-59465-7] http://hdl.handle.net/10481/63848 10.1038/s41598-020-59465-7 eng http://creativecommons.org/licenses/by/3.0/es/ info:eu-repo/semantics/openAccess Atribución 3.0 España Springer Nature