Multi-Analytical approaches to assess the physicochemical stability of biotherapeutic products: application to bevacizumab

Recombinant monoclonal antibodies (rmAbs) represent the fastest-growing class of biopharmaceuticals due to their pivotal role in the treatment of chronic and life-threatening diseases, including inflammatory disorders and cancer. The expiration of patents and the increasing demand for biological therapies have further accelerated the development of biosimilars and intensified the need for robust analytical strategies to ensure their quality and stability [1]. In hospital settings, rmAbs are frequently compounded into galenical preparations to provide personalized dosing. These preparations must comply with stringent quality standards to guarantee patient safety and efficacy. In this context, we investigated the chemical and physical stability of bevacizumab (BVZ), a clinically relevant anti-angiogenic monoclonal antibody (mAb) widely used in ophthalmology, formulated as solution for intravitreal injections. A comprehensive one-month stability study was performed using a multi-analytical platform including Matrix-Assisted Laser Desorption Ionization–Time of Flight mass spectrometry (MALDI-ToF), Capillary Isoelectric Focusing (cIEF) and nano Liquid Chromatography–High Resolution Mass Spectrometry (nanoLC-HRMS, LTQ-Orbitrap). MALDI-ToF qualitative analysis of the antibody enabled rapid assessment of molecular integrity, detecting potential fragmentation or aggregation events induced by thermal, chemical, or mechanical stress. To further investigate structural features, analyses were also performed on reduced, deglycosylated, and reduced/deglycosylated forms to characterize heavy and light chains and evaluate N-glycosidic chains. Charge heterogeneity was assessed by cIEF, a gold-standard technique for qualitative and quantitative characterization of rmAbs. The cIEF protocol used for the BVZ isoelectric point determination and isoforms profile was derived from a previous study confirming its robustness as a horizontal quality control platform for rmAbs. Finally, nanoLC-HRMS peptide mapping following tryptic digestion enabled in-depth structural characterization, confirming sequence integrity and identifying potential post-translational modifications such as oxidation, deamidation, and glycosylation variants. Overall, this multi-level analytical workflow provides a comprehensive and orthogonal strategy for monitoring the stability of rmAbs, supporting hospital pharmacy practices and contributing to the safe and cost-effective management of high-value biopharmaceuticals.