Establishment of a suspension-based perfusion process for the effective production of lentiviruses using inducible producer cell lines

Background & Aim The field of cell and gene therapy is rapidly growing, and there is an increasing demand for stable, high-quality, high-producing lentiviral vector (LVV) processes. Process intensification has the potential of addressing these requirements, allowing for faster processing times, enhanced vector quality and reduced process costs, amongst other advantages. However, the establishment of effective process intensification strategies remains a challenge and is largely understudied. This project aims to optimise LVV production through the investigation of perfusion and the establishment of a high-throughput, scalable suspension platform. Methods, Results & Conclusion A high-productivity, inducible producer HEK293T-based cell line for LVV production has been developed by CSL Behring, and has been adapted to serum-free, suspension growth. This will serve as the basis for our platform. Perfusion mode has been demonstrated to be superior compared to the batch process, maintaining a high cell viability and a high cell productivity rate for longer periods of time and an increased process productivity.This piece of work focuses on the identification of a favourable metabolic state and bioreactor parameter optimization. To achieve this, several experiments in small scale were undertaken. Media studies and key bioreactor parameters were investigated using the small-scale, high-throughput ambr15® system to identify the optimal conditions to support a high-viability and rapid cell growth, enhance virus stability and improve process performance. The second set of experiments evaluated the impact of two main sets of compounds in a continuous process-mimic in shake flasks with the goal of increasing process productivity and viral stability. These include Viral Sensitizers (VSEs), which are designed to prevent high levels of viral auto transduction during the extended perfusion culture, and recombinant albumin. Finally, virus kinetic studies and process characterization with the identified optimal conditions were completed before scaling-up to the 5L scale in perfusion.