How success in gene therapy is tied to cell culture media for AAV production in HEK293 cells

A successful gene therapy combines precise vector design and biology, high-quality manufacturing, safe and efficient delivery, strong clinical benefit, and smart regulatory planning.

While all aspects are significant, many promising therapies fail in the context of manufacturing efficiency and product quality. In order to improve therapeutic potency, reducing manufacturing costs and meeting regulatory expectations, volumetric productivity (titer) and packaging efficiency (full capsids vs. partially filled/empty capsids) are essential^{1, 2}. Thus, for optimal AAV production, cell culture medium is a foundational factor. 

The medium should be:

• chemically defined and animal component-free, 
• compatible with fixed elements like viral serotype and plasmid design, 
• flexible with variable elements such as cell lines and transfection reagents, and
• capable of strong baseline performance without extensive optimization. 

Pretty high demands, I would say. 

These aspects have been systematically investigated by our R&D colleagues in the informative white paper, A flexible cell culture media for high titer production of AAV in HEK 293. 

Multiple HEK293 cell lines were cultured and transfected in TheraPEAK 293-GT^® Medium and comparator commercial media using standard triple-plasmid transfection. AAV was harvested 72 hours post-transfection, and viral genome titers were quantified by droplet-based digital PCR, while total capsids were measured by immunoassay to estimate full/empty ratios.

Results show that TheraPEAK 293-GT^® Medium robustly supports the expansion of diverse HEK293 cell lines, achieving high cell densities (>7.5 × 10⁶ cells/mL in 5 days and up to 12 × 10⁶ cells/mL by day 7) while maintaining high viability (>95%). In addition, a non-optimized screening of commercial transfection reagents and enhancers across 3 serotypes produced viral titer as high as 1 x 10¹² vg/mL and full capsid ratio up to ~60%. Both specifications were measured from crude lysate without any downstream processing.

These findings indicate that the medium provides a strong and flexible foundation for high-titer AAV production and process optimization across different cell lines and production setups.

Written by 

Isa
Senior Scientific Support Specialist
 

Figure: Genome titer of AAV2, 8 and 9 produced with leading AAV medium and TheraPEAK 293-GT^® Media System with and without commercial enhancer. Lonza Xcite^® AAV Transient Transfection Plasmids were used in both the leading AAV medium and TheraPEAK 293-GT^® Media System. Transfection reagent A was used in both cases.