Trying a different transfection reagent is perhaps the least involved way to achieve higher viral titers. Among the variety of reagents on the market for transfection, a subset exists that is specifically designed for high-titer virus production. Using one of these specialized transfection reagents can greatly improve results with minimal modification to your existing process (see section ‘Transfection Procedure’ below).

For example, the TransIT-VirusGEN® Transfection Reagent can be used in a diversity of adherent and suspension cell culture platforms, such as multi-well or -layered plates and fixed bed or stirred-tank bioreactors. As shown below for AAV of different serotypes, the titer and percentage of full AAV was greatly impacted by the reagent(s) used in the transfection.

TransIT-VirusGEN® Reagent and VirusGEN® AAV Kit Generate High Quality Virus across AAV Serotypes. Viral Production Cells 2.0 (Thermo Fisher) grown in Viral Production Medium (Thermo Fisher) were seeded immediately prior to transfection (2 ml, non-treated 6-well plate); AAV2 and AAV9 were produced by transfecting cells at 2×106 cells/ml, and AAV5 was produced by transfecting cells at 3×106 cells/ml. Transfections were performed according to manufacturer-recommended protocols with the indicated reagents and reagent-to-DNA ratio (vol:wt). AAV was harvested at 72 hours post-transfection. Genome copies were determined by dPCR using primers and a probe targeting the CMV promoter. Serotype-specific ELISA kits (Progen) were used to quantitate capsids. The % full capsids was calculated by dividing genome titer by capsid titer. The error bars represent the standard deviation of duplicate wells.

Transfection with TransIT-VirusGEN® Transfection Reagent yielded significantly more full AAV vectors than PEI-based Competitor A1 and A3 reagents, and further gains to yield were observed when the TransIT-VirusGEN® Transfection Reagent was used in conjunction with the VirusGEN® AAV Complex Formation Solution and Enhancer, i.e. VirusGEN® AAV Transfection Kit. Notably, no commercial license is required for use of VirusGEN® transfection reagents in further manufacturing. Thus, swapping the transfection reagent in your virus production workflow is relatively painless and may be worthwhile for improvements to titer and/or percentage of full AAV capsids.