Achieving rAAV Bioreactor Titer Of >1E15 vg/L With Novel Plasmid Transient Transfection Platform

By Jin Yin, Oxford Biomedica Solutions

Transient transfection of plasmids into suspension HEK293 cells is the most common platform for producing rAAV, owing to its flexibility and speed in moving drug candidates into clinical trials. Despite this, its utility has been limited by its perceived low productivity and poor scalability when compared to other platforms (e.g., Baculovirus expression systems with insect sf9 cells, adenovirus-infected producer cell line systems, and Herpes simplex virus helper systems) [1]. Based on data from 136 ongoing clinical trials, 77% of systemically delivered rAAVs are dosed at 1E14 – 1E16 vector genomes (vg) or higher, whereas 59% of targeted delivered are dosed at 1E12 - 1E14 vg or higher [2]. The high level of rAAV dosage poses a major supply and cost challenge for manufacturers and gene therapy companies – in some cases of systemic delivery, one 200L bioreactor batch is insufficient to dose one patient.

When pursuing transient transfection, there are three key factors that can serve to inhibit a process: poor packaging, low productivity, and an inability to scale up. In conventional plasmid transfection processes, standard across the industry, bioreactor titers range from 5E13 to 2.4E14 vector genome per liter (vg/L) [4]. The pursuit of higher rAAV productivity for transient transfection has led to an increased focus on the factors that particularly influence the upstream processing. To improve bioreactor vector genome (vg) productivity, Oxford Biomedica Solutions (OXB Solutions) has developed a high cell density process with a novel transfection method. By homing in on medium additives, transfection cell density, vector and plasmid design, process optimization, plasmid transfection understanding, and equipment engineering and process control, OXB Solutions has been able to demonstrate bioreactor vg titer higher than 1E15 vg/L and successful scale-up to 2,000L bioreactor.