VIRAL SAFETY EVALUATION OF BIOTECHNOLOGY PRODUCTS: DEVELOPMENT AND OPTIMIZATIONOF RELIABLE VIRAL CLEARANCE STRATEGIES

This manuscript brings together the research activities carried out throughout my doctoral program and reflects the dual nature of the project, which developed at the interface between academic research and industrial application. The thesis is part of a broader framework dedicated to the study, optimization, and validation of viral clearance strategies within downstream processing for monoclonal antibody (mAb) production, with the overarching goal of establishing procedures that are effective, reproducible, and fully transferable to the biopharmaceutical industry. During the first year, thanks to a structured training period at the Department of Pharmaceutical Sciences (DISFARM), University of Milan, I consolidated essential skills in preparative chromatography and conducted an extensive literature analysis on viral clearance approaches. This effort culminated in a review article, entitled ‘Viral Clearance in Biopharmaceutical Manufacturing: Current Strategies, Challenges, and Future Directions’, prepared in collaboration with Prof. Altomare and recently submitted, and positively revised, for publication in Biotechnology Advances (Elsevier). Presented here as the first part of the thesis, the review provides the reader with an up-to-date overview of the principles, technologies, and challenges associated with viral removal within DSP, offering the conceptual foundation necessary to contextualize the experimental core of the project. The second part of the thesis is dedicated to the experimental work conducted jointly at DISFARM and Eurofins Biolab Srl., where I established, optimized, and subsequently transferred to an industrial setting a purification protocol based on Protein A chromatography and Ion Exchange Chromatography (IEX) for mAb manufacturing. This workflow was integrated with viral removal and inactivation studies performed at the Virology Unit of Eurofins, employing model viruses such as X-MuLV and MVM and combining chromatographic systems, filtration technologies, and advanced biological assays. This section provides an in-depth description of the methodologies adopted, the rationale behind the operational strategies, and a critical discussion of the results, with particular focus on process scalability, robustness, and industrial transferability. Overall, the thesis aims to guide the reader through a coherent path that begins with the current state of viral clearance technologies, progresses through the optimization of a laboratory-scale purification process, and culminates in its validation under industrial conditions. The overarching objective is to provide a comprehensive, structured, and forward-looking perspective on viral removal strategies for therapeutic antibody production, highlighting the scientific and technical contributions developed over the course of the PhD program.