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Adeno-Associated Virus (AAV) vectors are at the forefront of gene therapy, offering transformative therapeutic potential for many genetic disorders. However, the translation of this promise into accessible treatments is constrained by manufacturing challenges, including process variability, low yields, and scalability challenges. This review provides a comprehensive framework for establishing robust AAV-based gene therapy manufacturing processes by evaluating industry challenges and recent technological innovations. We studied the end-to-end AAV-based gene therapy manufacturing process, from upstream unit operations such as cell culture and transfection to downstream purification and fill-finish operations. Key upstream innovations highlighted include high-density perfusion cultures, advanced single- and dual-plasmid systems, and next-generation transfection reagents that collectively enhance the overall process quality and viral vector productivity. In the realm of downstream processing, recent advancements in serotype-agnostic affinity chromatography and ion-exchange chromatographic purifications have enhanced the critical separation of full capsids from empty capsids. The implementation of a quality-by-design framework is the heart of the AAV-based gene therapy manufacturing process. We emphasize the necessity of a rigorous process characterization, utilizing validated scale-down models and design of experiments, as a prerequisite for establishing a robust control strategy with defined proven and normal operating ranges. This data-driven approach not only mitigates process inconsistency, but it also serves as the foundation for an effective process validation and regulatory compliance. Looking ahead, the integration of artificial intelligence and continuous manufacturing methodologies will be pivotal in expediting the development of safer, more efficacious, and personalized AAV-based gene therapies.
Kowshik et al. (Wed,) studied this question.