From Platform Development to Scale-Up: Stable Producer Cell Line-Based Lentiviral Vector Manufacturing

Background: The development of stable producer cell lines (SCLs) represents a promising alternative for lentiviral vector (LVV) manufacturing, offering improved reproducibility and reduced manufacturing costs. In previous work, our group established an SCL generation platform based on a well-characterized packaging cell line (PCL), demonstrating successful vector production at laboratory scale. However, translation of this platform to larger scales remains necessary for industrial applications. Objective: In this study, we aimed to evaluate the scalability of the existing platform and identify key parameters that influence the production performance (e.g., cell density, harvest timing or pH). Moreover, the platform was enhanced through extensive genetic characterization and improved integration methods. Results: Our findings suggest that mildly acidic culture conditions and extended harvest periods contribute to increased LVV titers. Furthermore, we conducted a comparative analysis of transgene integration strategies to determine the most suitable method for SCL generation. The results demonstrate that transposase-mediated integration provides a precise and stable approach, minimizing concatemer formation and tandem rearrangements. Conclusions: Collectively, these findings provide evidence that our stable producer cell line constitutes a scalable, genetically characterized and consistent LVV production platform, supporting its potential for large-scale LVV manufacturing.