Establishing an in-house lentiviral vector platform and associated analytics

Abstract

Lentiviruses are used in gene therapy to deliver corrective genes to patients with genetic diseases. Evonik is interested in creating cell culture additives which improve key performance parameters of lentivirus production. Product development for this market would require a lentiviral production workflow, thus, this project aims to establish and validate a lentiviral production platform for product testing. We determined HEK293T to be the most suitable cell line for both production and testing of viruses due to a high production titre and transduction efficiency. Successful lentiviral production and transduction were validated with fluorescent microscopy, and functional viral titre and multiplicity of infection was determined with flow cytometry. Process optimisation experiments demonstrated 48 hours post-transfection to be optimal for viral harvesting, fetal bovine serum free media at transduction increased viral production titre by 28%, however plasmid ratio experiments were inconclusive. Lastly, to validate this platform for product development, we attempted to discover a novel inactivation agent. Evonik Surfactant at 0.05% and polysorbate 80 at 0.1% and 0.5% showed a decrease in functional viral titre of 87%, 9% and 50% respectively. We demonstrated a functional lentivirus production and testing platform with optimised parameters and validated its utility in a viral inactivation assay.