Triple transfection is the standard production method for recombinant viral vectors — particularly AAV and lentiviral vectors — in which three distinct plasmid DNA constructs are simultaneously introduced into a producer cell line — most commonly HEK293 or HEK293T — to provide all the components required for vector assembly and packaging. In AAV manufacturing, the three plasmids typically encode the transgene expression cassette (flanked by ITRs), the Rep and Cap proteins, and the adenoviral helper functions, respectively. Triple transfection efficiency, plasmid ratio optimization, and transfection reagent selection are critical process parameters that directly influence vector titer, purity, empty/full capsid ratio, and overall manufacturing yield, making their optimization a key focus of upstream process development in CDMO operations.
