Development of a Serum-Free Producer Cell Line Generation Process for Scalable and Efficient rAAV Production for gene therapy applications

Abstract

1. Abstract The growing field of recombinant Adeno-Associated Virus (rAAV) gene therapy demands scalable and efficient production platforms. Manufacturing platforms based on producer cell lines (PCL) hold substantial promise due to their capacity for high productivity and scalability. However, the generation of stable cell lines for rAAV production is typically performed using serum-containing processes, which pose a risk due to safety and regulatory concerns related to animal-derived components. In this study, we established a fully serum-free process for PCL generation for rAAV production, optimizing critical stages of this process, including transfection methodologies for stable plasmid integration, isolation of highly productive cell pools, and media supplementation to support single-cell cloning. The developed serum-free process generated clonally derived PCLs with productivity and product quality on par with traditional serum-based methods. This approach significantly reduced the cell line generation timeline and eliminated the challenge related to serum supplementation, adapting it to industry requirements. The reported advances overcome a critical step in cell line development for rAAV production, facilitating the implementation of scalable and efficient production platforms to support the development of rAAV-based gene therapies.