As AAV therapies move to broader patient populations, a greater need for highly efficient production processes has become evident. Upstream AAV production is largely driven by transient transfection of suspension HEK293 cells with multiple plasmid DNA constructs encoding essential virus proteins. Many factors are considered for transfection including, but not limited to, suspension HEK cell type, plasmid ratios, DNA dosage, transfection reagent and reagent-to-DNA ratios, culture additives, and enhancer components. Though time consuming, optimizing these factors can lead to significant increases in process yields and reduced cost per dose, effectively enabling treatment of more patients.

Mirus Bio’s TransIT-VirusGEN® GMP Transfection Reagent utilizes cationic lipid and polymers to efficiently condense and deliver nucleic acids to HEK cells for AAV production. The RevIT™ AAV Enhancer (Mirus) can be used in conjunction with TransIT-VirusGEN Transfection Reagent or conventional polymeric transfection reagents to produce 2-10X higher genome titers across a range of AAV serotypes. This combination also often enables use of less plasmid DNA which represents a key cost-saving opportunity and paves the way for driving down upstream manufacturing costs, resulting in a lower cost per therapeutic dose.

BridgeBio Gene Therapy is a commercial-stage biopharmaceutical company founded to discover, create, test, and deliver transformative medicines to treat patients who suffer from genetic diseases and cancers with clear genetic drivers. This case study explores the utilization of the VirusGEN and RevIT platform for expression of Bridge Bio’s AAV5-based therapeutic designed to treat Congenital adrenal hyperplasia (CAH) by delivering a functional copy of the 21-hydroxylase gene and restoring the patient’s delicate balance of hormone production. Previous expression platforms were sufficient for production of AAV material for early phase clinical trials. However, low titers and inconsistent scalability made the process incompatible with commercial manufacturing. Incorporation of VirusGEN and RevIT, along with other modifications to the upstream production process, enabled a 14-fold increase in patient doses without sacrificing percent full capsids, potency, or other critical quality attributes. Additionally, similar titers and product quality were observed for AAV5 batches produced at a range of scales from shake flasks to 200 L bioreactor, demonstrating robust scalability.