Abstract

The manufacture of recombinant AAV2 to support clinical studies has been carried out using transient transfection of HEK293 cells grown in roller bottles. While this method gives high productivity (50,000 vg/cell) relative to other methods of vector production, we have recently completed a series of studies aimed at further increasing the productivity and consistency of the transfection process. An experimental design approach was taken to initially screen for parameters that significantly affect vector yield. Sets of 7 parameters relating to the cell growth, transfection and post-transfection conditions were tested in a fractional factorial design. Results of this screening revealed that transfection efficiency is the key parameter that plays a critical role in determining AAV vector yield. Levels of certain media components such as glutamine also significantly influence vector productivity. The transfection procedure was then further optimized by examining the effects of pH, plasmid concentration, method of mixing of transfection reagents, and timing and method of addition of the transfection reagent to the cells. We observed that reduced agitation during CaPO4 buffer mixing led to an approximate 3-fold increase in average productivity to approximately 1.5 E13 vg/roller bottle (150,000 vg/cell). We further found that stabilization of the CaPO4 / plasmid precipitate following addition to cells provided a further 2-fold increase in productivity. With these optimized procedures, we are capable of producing recombinant AAV2 vectors at a level of 2-3E13 vg/roller bottle (200,000–300,000 vg/cell), making the transient transfection method more efficient and economically viable for large-scale manufacturing.

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