Integrated Semi-Continuous Manufacturing of Lentiviral Vectors Using a HEK-293 Producer Cell Line

There have been considerable efforts on improving the lentiviral vector (LV) system and their production. However, there remains the persisting challenge of producing a sufficient quantity of LVs at manufacturing scale to support treatments beyond early clinical trials. Furthermore, their innately labile nature poses an equally important obstacle in LV production. As LVs lose function over time and they are sensitive to environmental factors in each unit operation in the bioprocess workflow, integrated continuous manufacturing is an attractive strategy for process intensification. This manuscript describes the implementation of nuclease treatment, clarification, and capture step in a semi-continuous mode. Combining the clarification and loading of the capture step as well as operating those steps in parallel to the purification of the capture step expedite the processing time, reducing it by 4-fold as compared to processing the same volume in batch mode using the same membrane size. This semi-continuous operation also improves the recoveries of functional vector particles and total vector particles by 26% and 18%, respectively, showing an added benefit in loading the capture membranes in series in continuous flow chromatography. Building on previously published upstream work using a scalable cell retention device in perfusion mode, this manuscript demonstrates the integration of upstream and downstream in a semi-continuous manner, reducing processing and hold times as well as showing improvements in LV product quality and recovery.