Production of adeno-associated virus (AAV) serotypes by transient transfection of HEK293 cell suspension cultures for gene delivery

Abstract

Adeno-associated virus (AAV) is being used successfully in gene therapy. Different serotypes of AAV target specific organs and tissues with high efficiency. There exists an increasing demand to manufacture various AAV serotypes in large quantities for pre-clinical and clinical trials. A generic and scalable method has been described in this study to efficiently produce AAV serotypes (AAV1-9) by transfection of a fully characterized cGMP HEK293SF cell line grown in suspension and serum-free medium. First, the production parameters were evaluated using AAV2 as a model serotype. Second, all nine AAV serotypes were produced successfully with yields of 10(13)Vg/L cell culture. Subsequently, AAV2 and AAV6 serotypes were produced in 3-L controlled bioreactors where productions yielded up to 10(13)Vg/L similar to the yields obtained in shake-flasks. For example, for AAV2 10(13)Vg/L cell culture (6.8×10(11)IVP/L) were measured between 48 and 64h post transfection (hpt). During this period, the average cell specific AAV2 yields of 6800Vg per cell and 460IVP per cell were obtained with a Vg to IVP ratio of less than 20. Successful operations in bioreactors demonstrated the potential for scale-up and industrialization of this generic process for manufacturing AAV serotypes efficiently.

Keywords: Bioreactor; Gene therapy; Large-scale transient transfection; Manufacturing; Process; Production.

Fig. 1

Cells at various cell densities (1, 2, 4 or 8 million cells/mL) were transfected with 1 μg plasmid DNA/mL cell culture in fresh medium. These cell densities were transfected in 20 mL cell culture volume with PEI to DNA ratio of 2:1. The cell cultures were harvested 48 h post transfection (hpt). The infective viral particles are reported as IVP/mL cell culture.

Fig. 2

Cells at various cell densities (1, 2, 4 or 8 million cells/mL) were transfected with 1 μg plasmid DNA/million cells in fresh medium. These cell densities were transfected in 20 mL cell culture volume with PEI to DNA ratio of 2:1. The cell cultures were harvested 48 h post transfection (hpt). Comparative data from Fig. 1 has been re-plotted to reflect percent GFP positive cells normalized to GFP positive cells obtained by transfecting at cell density of 1.00E+06 cells/mL.

Fig. 3

Cell density and virus yield (in terms of genomic and infective viral particle concentrations) as a function of cell density at transfection. Production and harvesting conditions is same as in Fig. 1. The genomic particles and infective viral particles are reported as Vg and IVP per mL cell culture, respectively.

Fig. 4

Cell specific genomic and infective viral particle concentrations as a function of cell density at transfection. Production and harvesting conditions is same as in Fig. 1.

Fig. 5

Effect of butyric acid (circles) and sodium butyrate (squares) on the transfection efficiency (a), GFP expression level of transfected cells (b), and AAV2 yield represented by infective viral particles (c). The infective viral particles are reported as IVP/mL cell culture.

Fig. 6

The production kinetics of AAV2 includes transfection efficiency and AAV2 yield (in terms of volumetric and cell specific) as a function of time. The genomic particles and infective viral particles are reported as Vg and IVP per mL cell culture, respectively.

Fig. 7

AAV serotypes (AAV1-9) were produced using HEK293SF cells at cell density of 1.00E+06 cells/mL in 20 mL of serum-free medium in 150-mL shake flasks in duplicates. The cell cultures were harvested 48 h post transfection (hpt). Infective viral particles were assayed using HEK293 and HeLa cells.

Fig. 8

AAV serotypes (AAV1-9) were produced using HEK293SF cells at cell density of 1.00E+06 cells/mL in 20 mL of serum-free medium in 150-mL shake flasks in duplicates. The cell cultures were harvested 48 h post transfection (hpt). The whole cell cultures and supernatant of cell cultures (by removing cell pellet by centrifugation) were purified by iodixanol protocol, and then assayed for genomic (by PCR) and infective viral particle (using HEK293 cells) concentrations.

Fig. 9

Bioreactor was inoculated at a cell density of 0.35E+06 cells/mL with 97% viability. The cells were transfected with the PEI/DNA complexes (polyplexes) with PEI to DNA ratio of 2:1 in a serum-free medium to produce AAV2. The total cell culture volume at the start of transfection was 2000 mL. Samples were taken every 8 or 12 h intervals up to 96 hpt. This figure shows the genomic particle yield (in Vg/mL whole cell culture and Vg/mL supernatant of cell culture) and Vg/IVP ratio in (a), the cell specific yields (in terms of Vg/total cell and IVP/total cell) in (b), and total cell counts/mL culture and percent of GFP positive cells in (c).